International Journal of Biological Macromolecules 101 (2017) 931–957

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Review

Recent advances in enzyme extraction strategies: A comprehensive

review
Shamraja S. Nadar, Rohini G. Pawar, Virendra K. Rathod ∗
Department of Chemical Engineering, Institute of Chemical Technology, Matunga (E), Mumbai 400019, India

a r t i c l e i n f o a b s t r a c t

Article history: The increasing interest of industrial enzymes demands for development of new downstream strate-
Received 6 December 2016 gies for maximizing enzyme recovery. The significant efforts have been focused on the development of
Received in revised form 7 March 2017 newly adapted technologies to purify enzymes in catalytically active form. Recently, an aqueous two
Accepted 10 March 2017
phase system (ATPS) is emerged as powerful tools for efficient extraction and purification of enzymes
Available online 14 March 2017
due to their versatility, lower cost, process integration capability and easy scale-up. The present review
gives an overview of effect of parameters such as tie line length, pH, neutral salts, properties of polymer
Keywords:
and salt involved in traditional polymer/polymer and polymer/salt ATPS for enzyme recovery. Further,
Enzymes
Extraction
advanced ATPS have been developed based on alcohols, surfactants, micellar compounds to avoid tedious
Purification recovery steps for getting desired enzyme. In order to improve the selectivity and efficiency of ATPS,
Aqueous two phase system recent approaches of conventional ATPS combined with different techniques like affinity ligands, ionic
Three phase partitioning liquids, thermoseparating polymers and microfluidic device based ATPS have been reviewed. Moreover,
CLEAs three phase partitioning is also highlighted for enzymes enrichment as a blooming technology for effi-
Nanoflowers ciently integrated bioseparation techniques. At the end, it includes an overview of CLEAs technology and
organic–inorganic nanoflowers preparation as novel strategies for simultaneous extraction, purification
and immobilization of enzymes.
© 2017 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 932
2. Aqueous two phase system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 932
2.1. Factors affecting partitioning of enzymes in aqueous two phase partitioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933
2.1.1. Nature and concentration of polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933
2.1.2. Polymer/salt aqueous two phase partitioning system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 935
2.1.3. Polymer/polymer aqueous two phase partitioning system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 937
2.1.4. Effect of pH on partitioning of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 937
2.1.5. Effect of volume ratio and tie-lines length on partitioning of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 938
2.1.6. Effect of neutral salts on enzyme partitioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 938
3. Alcohol/salt based aqueous two phase partitioning system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 938
4. Surfactant/alcohol based aqueous two phase partitioning system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 939
5. Advanced aqueous two phase partitioning system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 939
5.1. Aqueous two-phase affinity partitioning system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 940
5.2. Ionic liquid based aqueous two phase partitioning system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942
5.3. Thermoseparating polymer-based aqueous two-phase system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942
5.4. Aqueous micellar two-phase system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 944
5.5. Microfluidic platform for ATPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945

∗ Corresponding author.
E-mail address: vk.rathod@ictmumbai.edu.in (V.K. Rathod).

http://dx.doi.org/10.1016/j.ijbiomac.2017.03.055
0141-8130/© 2017 Elsevier B.V. All rights reserved.
932 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957

6. Three phase partitioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945

7. Simultaneous purification and immobilization strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 948
8. Future perspectives and challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 949
9. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 950
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 950
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 950

1. Introduction well as they should be sustainable, efficient and biocompatible with

the enzyme conformational structure [14,15]. So there is a need
Many chemical transformation processes are carried out in to develop easy, fast, scalable and economically feasible methods
extreme conditions, high temperature and pressure involving harsh to extract enzymes either from fermentation or natural sources
and hazardous processing which led to higher consumption of (Fig. 1). In this review, we have explained various types of extrac-
chemicals and energy [1]. Also, harmful by-products are pro- tion methods with their modifications and parameters affecting the
duced due to non-specificity which have huge negative impact on extraction of enzymes for the optimization and design of efficient
the environment [2]. Hence, there is a need for development of and economic downstream processes. Also, we have mentioned
clean, bio-compatible, reliable and benign processes for industrial certain methods of simultaneous purification and immobilization
manufacturing of chemicals. Biocatalysis has emerged as a pos- of enzyme which make its application economically feasible.
sible eco-friendly and greener catalysts, meeting the demands of
green chemistry and sustainable development which is directing 2. Aqueous two phase system
more and more researchers to exploit it [3]. Mainly biocatalysts
are divided into two types: whole cells and isolated enzymes. Aqueous two phase partitioning system (ATPS), a liquid–liquid
Compared to whole cells, isolated enzymes offer several bene- fractionation method has been recognized as a superior, versa-
fits, including simpler reaction apparatus, higher productivity due tile and well-established process for separation [16]. It seems
to higher catalyst concentration and simpler product purifica- to be a promising alternative method in which clarification,
tion owing to elimination of undesirable side reaction during cell partial purification and concentration are integrated into one
growth [4,5]. Also, enzymes possess some excellent properties; step. It is very simple and easy method which is considered
high reaction rates, highly selective and specific working under as an efficient approach for purification and recovery of wide
mild conditions. Additionally, enzymatic based processes reduce range of biomolecules [17]. Generally, ATPS are composed of
adverse impact of manufacturing on our eco-system by reducing aqueous solution of two incompatible polymer/polymer and
the consumption of large amount of raw materials, energy and the polymer/salts components. When the concentration of these com-
subsequent generation of waste (as compared to chemical based ponents increases above a certain critical value, it results into
process) which is beneficial for both industries and environment formation of two phases. The extraction of enzymes with ATPS is
[6,7]. an interesting alternative to traditional separation methods mainly
Industrial enzymes originate from microorganisms which are due to retention of catalytic activity, maintenance of stability due to
fermented primarily by using renewable resources (Fig. 1) [8]. With non-denaturing conditions, high biocompatibility, lower environ-
the advancement in molecular biology and media formulation, the mental burdens, low energy consumption and relative reliability
production yield of enzyme (i.e. enzyme per mass of whole cell) in scale-up [18]. Also, these single step isolation processes allow
has increased significantly. Though there is an increasing demand simultaneous purification of target molecules and removal of con-
for enzyme production, an improvement in downstream processes taminants, therefore bringing about shorter processing time.
has been neglected. The recovery and purification of enzyme typ- Success of aqueous two-phase systems depends on the ability to
ically involves numerous process steps including; precipitation manipulate system parameters (phase composition) so as to obtain
with ammonium sulphate and/or organic solvents, filtration or cen- appropriate partition coefficients (K, the ratio of biomolecule con-
trifugation, dialysis, often followed by several chromatographic centration in top phase to that of bottom phase) and selectivity for
separation techniques such as ion exchange chromatography, size proper separation of target protein. However, the partition mech-
exclusion chromatography, hydrophobic interaction chromatogra- anisms are still not completely understood. Indeed, the extent of
phy or affinity chromatography [9,10]. At the end of the purification partitioning depends not only on biomolecule properties but also
process steps, the enzymes must be concentrated by lyophilization on system properties. The biomolecule properties include many
or ultra-filtration. Although these multistep purification processes variables such as: hydrophobicity, molecular conformation, molec-
seem to be effective, they suffer from many disadvantages such ular mass, concentration of the protein, flexible chain polymers,
as high cost, time consuming and difficulty in scale up. Also, dur- protein net electrical charge and isoelectric point (pI) [19–21]. Also,
ing such a long procedure, energy expenditure in these processes it involves the different chemical and physical interactions such as
is high especially for viscous fermentation broth. Apart from that, Van der Waals and ionic interactions of the biomolecules with the
solvent based extraction can cause considerable denaturation of surrounding phase along with hydrogen bonding and hydropho-
enzyme molecules [11]. Hence, these tedious procedures for the bic interaction too play an important role in the partitioning. In
recovery of biological products have a major impact on the final addition to it, system properties depend on molecular weight and
product cost as separation and purification accounts for a major concentration of phase forming polymer, type of phase forming salt,
fraction (70–80%) of the total bioprocess cost [12]. So, there is a concentration of salt and phase volume ratio [22–24].
demand for the development of cost-effective, mild downstream, Generally, the development of useful strategies for purifica-
more integrated separation and purification techniques with the tion of enzyme by ATPS is divided into four main steps (Fig. 2).
minimum number of steps [13]. The first step is physicochemical characterisation of the feed-
The retention of catalytic activity for enzymes is important for stock (properties of enzyme as well as contaminants), followed
the success of the extraction processes. In this context, significant by second and third steps where the type of ATPS (polymer/salt,
efforts have been focused on the development of new or adapted polymer/polymer or advanced ATPS) and system parameters (poly-
technologies for the purification of enzymes, with lower costs, as mer, tie-line length, volume ratio and pH) are selected, respectively.
 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957 933

Fig. 1. Simplified representation of the fractionation of enzymes from fermentation by aqueous two-phase processes and three phase partitioning.

Fig. 2. Practical strategy to ATPS process development and scale-up for the downstream processing of enzymes.

The final step is to evaluate the influence of process parameters
(such as sample loading, concentration, addition of neutral salts,
system geometry and recycling of chemicals) on the target product
recovery and purity [19,25]. However, the different combinations
of phase forming component have been applied for partitioning
and recovering various enzyme. Once the product of interest and
major contaminant are characterized, the bottom and top phase
forming components of ATPS must be selected. There are different
combinations of ATPS available (such as polymer/polymer ATPS,
polymer/salt ATPS and ATPS forming alternative components)
which have been reported and factors involved in the separation
of enzymes are discussed in this review.
2.1. Factors affecting partitioning of enzymes in aqueous two
phase partitioning
Although the mechanism of biomolecules partition in ATPS is
very complex, it is believed that the properties of polymer (polymer
type, concentration, molecular weight) and the other components
(salt, polymer) constituting the phase are probably the most vital
factors affecting the separation of biomolecules in different phases
[26–30]. Lee et al. conducted studies by varying a broad array of the
factors involved in ATPS. The influence of phase forming polymers
should be considered firstly, including concentration of polymer
and its molecular weight [31]. These parameters directly influence
protein partition by changing the number of chemical and physical
interactions such as van der Waals, hydrophobic, hydrogen bond
and ionic interactions. The appropriate partitioning of enzymes
(partitioning efficiency) is generally estimated in terms of purifica-
tion factor (PF), partition coefficient of enzyme (Ke ), yield, specific
activity and activity recovery.
2.1.1. Nature and concentration of polymer
Polyethylene glycol (PEG) is one of the most employed compo-
nent in ATPS as it is produced worldwide in large quantities and
in wide range of molecular weights. Molecular weight of PEG has
a strong influence on the partitioning behaviour of biomolecules
[32]. Hence, it is necessary to screen a number of different molecu-
lar weights of PEG for partitioning biomolecules in one phase. PEG
with 400–8000 Da is widely used to partition enzyme molecules
from crude fermentation broth or from natural sources. The parti-
tioning behaviour depends on the relative hydrophobicity of the
polymeric compound (i.e. the number of carbons present in an
aliphatic chain, surface hydrophobicity) [33]. As the molecular
weight of PEG increases, hydrophobic characteristic of the parti-
tioned enzyme and its partitioning coefficient linearly increase [34].
The PEG with low molecular weight (below 1000 Da) is highly sol-
uble due to shorter polymer chains with hydrophilic end group
[35]. Hence, higher concentration of polymer is required in order
to form two phases while better partitioning can be achieved due
to low interfacial tension [21,36]. It has also been reported that
higher molecular weight of PEG results in lower partition coeffi-
934 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957
cient factor on accounting of increased chain length. This further
increase the excluded volume, i.e. reduced available free volume
for the protein in the PEG rich phase which leads to partitioning of
the biomolecules to the bottom phase [37]. Therefore, for efficient
partitioning of enzyme, appropriate selection of polymer is needed
on the basis of its nature and properties along with favourable
interactions with enzyme.
From these reports and as summarized in Tables 1 and 2, it is
seen that wide range of PEG has been employed successfully for effi-
cient separation of a wide variety of enzymes. In one of the studies,
the researchers performed extractive purification of recombinant
peroxidase isozyme c from insect larvae. They observed decrease
in partition coefficient from 128 to 0.5 with increase in PEG molec-
ular weight. The shift of peroxidase from the top to the bottom
phase was observed with the increase in PEG molecular weight
[38]. Another study on the downstreaming of lipase from Rhizopus
microsporus was done by ATPS containing PEG 2000 and ammo-
nium sulphate. The lipase was partitioned into top phase with yield
and purification factor of 92.3% and 19.8-fold respectively [39].
From above examples, it can be seen that selection of appropri-
ate molecular weight of PEG help to propel the enzymes either top
or bottom phase in ATPS. In the study by Kianmehr et al. inves-
tigated the influence of PEG molecular weight (1000–8000) on
partitioning of recombinant P. fluorescens d-galactose dehydroge-
nase (GalDH) in a system composed of PEG/ammonium sulphate.
The PEG 4000 was found to be favourable for GalDH partitioning
and showed high affinity toward the top polymer phase. Further
increase in molecular weight of PEG (from 4000 to 8000) resulted
in less available space in the upper phase, which ledin the decrease
of partition coefficient (from 45 to 14) due to exclusion effect
[40]. In one of the study, simultaneous separation and purification
of ␤-galactosidase and ␤-glucosidase from barley (Hordeum vul-
gare) was carried out by employing a wide range of PEG molecular
weights (400, 1000, 1500, 4000, 6000 and 8000 Da). For low molec-
ular weight PEG (below 1500 Da), both the enzymes partitioned to
top phase whereas for high molecular weight PEG (above 6000 Da),
both the enzymes partitioned to bottom phase. PEG 1500 gave bet-
ter differential partitioning of ␤-galactosidase and ␤-glucosidase
into opposite phases in terms of activity recovery (60 and 65%) and
activity partition coefficient (2.45 and 0.47) which indicates selec-
tive partitioning of ␤-glucosidase and ␤-galactosidase between two
phase [41]. So the manipulation in PEG molecular weight can selec-
tively portioned enzyme from crude fermentation broth.
The source of enzyme also play an important role in selecting
suitable PEG molecular weight which revealed by following studies.
Yuzugullu et al. partitioned invertase from potato tuber (Solanum
tuberosum) with PEG 4000 in PEG/ammonium sulphate ATPS to
get increased the purity of 5.11-fold with the recovered activity
of 197%, whereas Yücekan et al. partitioned the same enzyme from
tomato (Lycopersicon esculentum) with PEG 3000 in PEG/sodium
sulphate system to obtain 5.5-fold purity with the recovered activ-
ity of 90%. In both cases, invertase was partitioned into the bottom
salt rich phase [30,42]. From above studies, it is clear that the prop-
erties of enzyme (i.e. molecular weight, conformation of enzyme)
play a vital role while extraction. Similarly, lipase produced by
Bacillus sp. and R. porus was purified by PEG 8000/potassium phos-
phate and PEG 2000/ammonium sulphate system at bottom phase
with best purification factor and yield [43,44]. This shows that
the distribution of targeted molecules is dependent on biochem-
ical and structural properties of proteins and molecular weight of
PEG. However, there is no clear correlation between partition coef-
ficients and molecular weight of enzyme for different molecular
weight of PEG [45,46].
The partitioning and purification of superoxide dismutase (SOD)
(small molecular weight enzyme) from P. chrysosporium was done
by using PEG 3350/potassium phosphate at pH 7.0.The total
recovery of SOD was reportes as 78.9%. During partitioning of
SOD, it was found that PEG below 1000 Da is not suitable due
to its high miscibility, and hence, it required higher concentra-
tions for inducing a two-phase system. Also, lower molecular
weight PEG may draw all proteins to the top phase that will
cause poor separation with low purification of the target pro-
tein. On the other hand, PEG above 8000 strongly increased
the viscosity which resulted into denaturation effect thereby
not suitable for ATPS [47]. Kirincic and Klofutar reported that
the increase in PEG (400–20000) molecular weight increases the
viscosity (0.959 × 103 –1.601 × 103 kg m−1 s−1 ) of an aqueous solu-
tion of PEG, ranging among 0.959 × 103 kg m−1 s−1 (PEG 400),
1.119 × 103 kg m−1 s−1 (PEG 4000) and 1.601 × 103 kg m−1 s−1 (PEG
20000) [48]. However, the polymer molecular weights influence
partitioning of enzyme by changing the polymer–enzyme interac-
tion and by altering the phase diagram. Thus, the choice of the most
suitable intermediate molecular weight of PEG is very important for
increasing the extraction efficiency of the ATPS.
Besides the molecular weight, choice of PEG concentration has
the most significant effect and provide favourable partitioning of
enzyme in ATPS. Generally, PEG concentrations are varied in the
range of 7–21% w/w to study enzyme partition. In the study carried
out by Ratanapongleka, the researchers extracted laccase (from L.
polychrous) with significantly high purification factor and yield in
top phase by ATPS, composed of low concentration of PEG 4000
and phosphate salt [49]. Similarly, serine protease was extracted
from Streblus asper leaves, the partition coefficient decreased sig-
nificantly at a low concentration of PEG 4000. This might be due
to low PEG concentration, hydrophobic interaction between PEG
and the surface of protein enhances and it leads to efficient sep-
aration of enzyme molecules [50]. On the other hand, the high
PEG concentration shows negative effect on the partition coeffi-
cient of the enzyme. In the extraction of a yeast invertase with
PEG 3000 and magnesium sulphate system, partitioning of inver-
tase was significantly affected by the high PEG concentration. The
PEG concentration (10%) resulted in the highest purification factors
(3.2-fold) with the yield of 134%, whereas at higher concentration of
PEG (from 10 to 20%), purification factors drastically decreased with
retention of lower enzyme activity [42]. The reason for this reduc-
tion in enzyme activity recovery was attributed to denaturation
due to break-down of enzyme conformation with the hydropho-
bic interaction between PEG and enzyme [39]. Additionally, high
PEG concentration in the system results in the increase of viscos-
ity and interfacial tension which resulted in more difficulties in
partition of the enzyme molecules [26]. However, in some cases,
higher concentration of PEG found to be favourable which showed
improved yield and partitioning of enzyme. In the recovery of lipase
from Penicillium cyclopium, the highest yield was obtained at higher
concentration of PEG 4000 (20%) as compared to lower concentra-
tion of PEG (with same molecular weight) in the top phase. Authors
have claimed that this was probably due to higher concentration of
PEG which led to favourable hydrophobic interactions with lipase
[51]. Similarly, relatively smaller molecular weight enzyme such as
SOD was obtained at the top phase with increasing concentration
of PEG due to the hydrophobic interactions [52]. Hence, selection of
appropriate PEG concentration is needed for good separation and
purification which is dependent on the type of ATPS and surface
enzyme properties.
In most of the cases, the effect of polymer concentration on
enzyme partitioning is straightforward. For effective partitioning
of any enzymes, both the parameters (i.e. PEG concentration and
its molecular weight) need to be selected appropriately. However,
the molecular weights of polymer which influences the partition by
altering the phase diagram and by changing the polymer–protein
interaction; thereby, section of both concentration and molecular
weight of polymer becomes complicated. Moreover, researchers
 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957 935

Table 1
Extraction of enzymes by using polymer/salt system.

Enzyme Source Polymer Salt Kp/PF Yield Ref.

Luciferase Photinuspyralis PEG 1500 (14%) Ammonium sulphate (16%) 13.69-fold – [60]
D-Galactose Pseudomonas fluorescens PEG 4000 (15%) Ammonium sulphate (11.8%) 58.9% 92.8% [40]
dehydrogenase
Invertase Baker’s yeast PEG 3350 (10%) Magnesium sulphate (15%) 8.81-fold 77% [61]
Hexokinase Baker’s yeast PEG 1500 (13.5%) Sodium Citrate (17.5%) 1.87-fold – [68]
Lipase Penicillium cyclopium PEG 4000 (15%) Potassium phosphate (70%) 9-fold 95.7% [51]
Endo-mannanase Bacillus sp. CFR1601 PEG 3350 (12.0%) Sodium sulphate (12.0%) – 95.4% [57]
Lipase Rhizopusniveus PEG 4000 (32.5%) Sodium carbonate (18.4%) 19.3-fold 92.8% [44]
Lipase Rhizopusmicrosporus PEG 2000 (20%) Ammonium sulphate (12%) – 92.3% [39]
Invertase Lycopersiconesculentum PEG 3000 (15%) Ammonium sulphate (12%) 5.5-fold 88% [30]
Glutaminase Zygosaccharomyces rouxii PEG 2000 (14%) Sodium sulphate (10%) 1.95-fold 69.1% [64]
Acid Invertase Solanum Tuberosum PEG 4000 (12.5%) Sodium sulphate 5.11-fold – [42]
(15%)
Superoxide Phanerochaetechrysosporium PEG 3350 (24%) Potassium phosphate (5%) 0.17 – [47]
dismutase
Laccase Lentinuspolychrous PEG 4000 phosphate salt 88.3 99.1% [253]
(12%) (16%)
Lipase Bacillus sp. PEG 8000 (20%) Potassium phosphate (18%) 123.89-fold – [43]
Alcohol Baker’s yeast PEG 2000 (15%) Potassium phosphate (10%) 6.6-fold – [254]
dehydrogenase
Polyphenol oxidase Ananas comosus Merr. PEG 1500 (18%) Phosphate (14%) 2.7-fold – [66]
Amylase Bacillus subtilis PEG 1000 (16.7%) Potassium phosphate (14.8%) 5.4-fold – [80]
Phytase Schizophyllum commune PEG 1500 (22%) Sodium citrate (14%) 2.63 367% [255]
␤-Xylosidase Trichoderma koningii G-39 PEG 6200 (10%) Sodium phosphate (5%) 8.8-fold 100% [256]
Invertase Baker’s yeast PEG 3000 (15%) Magnesium sulphate (23%) 6.2-fold – [37]
Cyclodextrin Bacillus cereus PEG 8000 (19.0%) Sodium Citrate (11.5%) 16.3-fold 70% [257]
glycosyltransferase
Lipoxygenase Glycine Max PEG 6000 Ammonium sulphate 4.38-fold – [258]
Serine Protease Streblus asper PEG 6000 (16%) Magnesium sulphate (15%) 14.4-fold 96.7% [50]
Laccase Hericium erinaceus (Bull.:Fr) PEG 8000 (17%) Potassium phosphate (12.2%) 8.03 99% [259]
Pers.
proteases C. perfringens. PEG 10000 (22%) Sodium Citrate (8%) 4.2-fold 131% [28]
␤-Galactosidase Aspergilus niger PEG 2000 (15.24%) Ammonium sulphate (17.14%) 19.42-fold – [260]
Polyphenol Potato Peel PEG 1500 (17.62%) potassium phosphate buffer (15.11%) 3.7 77.8% [261]
Oxidase
Polyphenol oxidase Solanum tuberosum PEG 8000 (5%) Phosphate (28.5%) 15.7-fold 97.0% [262]
Proteinase Tuna spleen PEG 1000 (15%) Magnesium sulphate (20%) 6.61-fold 69.0% [27]
␣-Amylase hog pancreas PEG 2000 (11.7%) Fructose-1,6-bisphosphate trisodium 3.2-fold 79% [263]
salt (19%)
␣-Galactosidase Aspergillus oryzae PEG 4000 (12%) Phosphate (11.9%) 3.6-fold 87.7% [264]

Table 2
Extraction of enzymes by using polymer/polymer system.

Enzyme Source Polymer Polymer PF Yield Ref.

Serine protease Mango peel PEG 4000 Dextran 14.37-fold 97.3% [69]
Amyloglucosidase Aspergillus niger PEG 4000 Polyacrylate 15000 2.0-fold 86% [76]
Pectinases – PEG 4000 Crude dextran 1.66-fold – [74]
Alkaline phosphatase Bacillus licheniformis PEG 4000 Dextran T500 5.23-fold – [71]
L-glutaminase Bacillus cereus MTCC 1305 PEG 4000 (8.5%) Dextran T500 1.31-fold – [70]
Lipase Burkholderia pseudomallei PEG 8000 Dextran T500 2.44-fold 92.1% [73]

cannot isolate the effect of polymer molecular weight from the
effect of polymer concentration [14,53,54]. The design of experi-
ment is very useful methodology in finding the optimum partition
condition. Thereby, these two factors should be considered simul-
taneously for modelling of protein partitioning in ATPS. An ATPS
composed of PEG and phosphate ATPS is used for purification of
a highly thermostable and alkaline active recombinant xylanase
from B. halodurans [29]. Response surface methodology (RSM)
and artificial neural network (ANN) have been used to develop
predictive models for optimization of purification process param-
eters (PEG molecular weight and its concentrations) and both
models have been able to predict appropriate conditions for extrac-
tion. The results demonstrating partitioning behaviour with higher
prediction accuracy was obtained by ANN with high correlation
coefficient (R2 ) compared to RSM. Also, Desai et al. found that the
average percentage error for ANN and RSM models were 6.5 and
20% indicating the superiority of ANN in capturing the nonlinear
behaviour of the system. This superiority of ANN over other multi
factorial approaches could make this technique a very useful and
helpful tool for purification processes [55–57].
2.1.2. Polymer/salt aqueous two phase partitioning system
Since chemical cost is always a dominant factor in any sepa-
ration and purification process, an inexpensive polymer/salt ATPS
has been widely favoured for commercial application [58]. In
PEG/salt ATPS, the choice of a phase-forming salt has directly
facilitated the separation and extraction of the target molecules
between the phases (Fig. 3) [39,59]. To ensure the efficiency of
the extraction, the partitioning is carried out by employing vari-
ous phase-forming salts. The salts have ability to manipulate the
hydrophobic interactions between biomolecules in ATPS. The use
of different salts, change the environment around the enzymes
which lead to change in the partition behaviour [25]. The example
of enzyme extraction by polymer/salt system is shown in Table 1.
Priyanka et al. employed various phase forming salts; magnesium
sulphate, potassium phosphate, sodium sulphate and ammonium
936 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957
Fig. 3. Schematic diagram illustrating the partitioning of enzyme and contaminant proteins (impurities) in a polymer/polymer and polymer/salt ATPS.
sulphate together with PEG 1500 to arrive at the most suitable ATPS
to purify luciferase from fireflies (Photinus pyralis). It was observed
that luciferase preferentially partitioning to the top phase with
PEG 1500/ammonium sulphate system exhibited highest enzyme
activity recovery (97.30%) with 3.61-fold of purification factor [60].
In another study, the PEG/ammonium sulphate was found to
be the best in terms of activity recovery and differential partition-
ing of both the enzymes ␤-galactosidase and ␤-glucosidase from
barley [41]. Aqueous two-phase extraction of ␤-d-fructofuronoside
fructohydrolase (␤-d-FF) was performed by using PEG 3350/salt
(ammonium sulphate, sodium sulphate, sodium phosphate, potas-
sium phosphate, magnesium sulphate and trisodium citrate)
system. Amongst these salts, the highest ␤-d-FF activity of 84.7%
with 3.78-fold purification was observed in case of magnesium sul-
phate [61]. In polymer/salt ATPS, sometimes salts show activity
inhibition during the partitioning of enzymes. In the separa-
tion of endo-mannanase from Bacillus sp., magnesium sulphate
and ammonium sulphate displayed complete inhibition of endo-
mannanase as no activity was detected in either top or bottom
phases [57]. The probable reason for these results was the stronger
ionic strength and protein aggregation (due to salting out effect)
leads to protein denaturation [62].
Generally, two phases in ATPS have to be isoosmotic and elec-
trically neutral, when salt is added to polymer solution, cations
and anions distribute themselves differentially among the phases
depending on their affinity with aqueous phase [26]. Thereby,
an interfacial potential is produced differently which may help
in the partition of biomolecules (proteins). The performance of
salts in phase separation is reflected in the lyotropic series (a
classification of ions based upon salting-out or salting-in ability)
[63]. The effectiveness of salts with multi-charged anions is as per
SO4 −2 > HPO4 −2 > C2 H3 O2 −1 > Cl−1 , whereas, cations are commonly
ranked in the following order: NH4 + > K+ > Na+ > Mg2+ > Ca2+ . In gen-
eral, proteins with the negative charge tend to partition to the top
phase in PEG/salt systems while those with the positive charge usu-
ally go to the bottom phase [37]. The feasibility of utilizing ATPS
composed of PEG 2000 and ammonium sulphate for the purifi-
cation of lipase from Rhizopus microsporus fermentation culture
was investigated by Anvari. The lipase partitioning was much more
strongly affected by anion species than by cations [39]. In the case
of cationic salts, the interaction of cations with water molecules is
readily replaced by PEG-cation interactions. On the other hand, salts
with multivalent anions of high-charge density limits such inter-
actions with the polymer chain, leading to salt-depleted zones and
consequently formation of two phases. Hence use of anionic salts
for the partitioning of enzymes is more beneficial [64].
Apart from the salt type, another important factor which
influences the partitioning of enzyme molecule in ATPSs is the
concentration of utilized salt [61]. As evident from previous lit-
erature, the recovery of enzyme was increased with an increase
in concentration of salt to a maximum value and further increase
in concentration drastically affected purification factor as well
as activity recovery. In the separation of proteins (cytochrome c,
trypsin, bovine serum albumin and myoglobin) by the ATPS system,
Zhao et al. used the different concentration of ammonium sulphate
in the range of 10–18%. It was observed that as concentration of
ammonium sulphate increased, the top phase volume decreased
and all proteins retained in the bottom phase [65]. In many cases,
enzyme recovery is not affected significantly while purification
fold is more sensitive with respect to salt concentrations. Also, the
distribution of enzyme is controlled by concentration of the salt.
The partition coefficient of acid invertase increased rapidly with
an increase in salt concentration from 13% to 20%. The maximum
enzyme recovery (128%) with a purification factor of 3.89-fold was
obtained at 15% of salt concentration combined with PEG 4000 sys-
tem [42]. Moreover, PEG/potassium phosphate system is employed
for separation and purification of enzymes mixture polyphenol
oxidase (PPO) and bromelain from pineapple (Ananas comosus L.
Merr.. The results showed about 228% activity recovery and 4.0-fold
increase in purification factor in case of bromelain whereas, 90%
activity recovery and 2.7-fold increase in PPO purity. The increase
in salt concentration resulted in “salting out effect” which increased
partitioning of the both the enzymes to the top phase. However,
purity of bromelain in the top phase was low whereas purity of
polyphenol oxidase increased at higher potassium phosphate con-
centration due to precipitation of enzyme at the interface [66].
The polymer/phosphate salts and polymer/sulphate salt sys-
tems have been most commonly used for separation of enzymes
[22]. However, use of these salts in large scale led to a strong
negative impact on the environment. In order to reduce environ-
mental problems. citrate (biodegradable and nontoxic in nature)
and ammonium carbonate (volatile in nature) are favoured because
of their high selectivity, less pollution and biocompatibility [67].
In one of the studies, Oliveira et al. employed citrate in PEG/salt
 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957
ATPS to extract hexokinase from baker’s yeast (Saccharomyces cere-
visiae), to obtain high enzymatic recovery in the top phase with
high partition coefficient [68]. The choice of a phase-forming salt
and its concentration have a direct influence on the separation and
extraction of enzymes in ATPS due to its major effect on the system
environment.
2.1.3. Polymer/polymer aqueous two phase partitioning system
The another type of ATPS is polymer/polymer system in which
polymers were employed as another component which not only
provides good stability to proteins but also making them compati-
ble due to high water content (>90%). Two mutually incompatible
different polymers beyond a critical concentration have ten-
dency to form two-phase systems in water solutions (Fig. 3) [16].
PEG–dextran system is practised widely in the partitioning of
enzymes [69]. Selected examples of the use of polymer/polymer
ATPSs for recovery and purification of enzymes are presented in
Table 2. Singh et al. carried out partitioning studies of L-glutaminase
produced from Bacillus cereus MTCC 1305 in different PEG–salt and
PEG–dextran ATPS. The PEG–dextran system was found to be most
suitable for partitioning of L-glutaminase as compared to PEG–salt
system. The maximum partition coefficient (1.31) with 2.09 U activ-
ity was obtained for PEG–dextran system. Authors have claimed
that PEG-salt based ATPS causes denaturation of the biological
structureand dissociation (protein unfolding) of the ligand-protein
complex [70]. To understand the effect of different molecular
weight of dextran on enzyme extraction, Pandey et al. performed
partitioning of alkaline phosphatase from B. licheniformis by using
ATPS which composed of PEG and two different dextran (dex-
tran T40 and dextran T500). PEG 4000/dextran T 500 system was
selected as most appropriate ATPS for alkaline phosphatase pro-
duction based on greater partition coefficient [71]. Furthermore,
dextran demonstrates stabilizing effect on enzymes (due to enzyme
and polysaccharide interaction), which apparently maintains the
enzyme activity in the ATPS system. The dextran T500 is com-
monly employed for partitioning of enzymes in polymer/polymer
system which has less influence on the partition coefficient [72].
So, concentration of dextran is considered to attain higher purifi-
cation factor. This is investigated in the study carried out by Ooi
et al. They performed extractive fermentation using ATPS which
incorporated PEG 8000 and dextran T 500 (5–20%) to extract
lipase derived from Burkholderia pseudomallei. At lower concen-
tration of dextran, higher yield of 92% with highly active lipase
was obtained [73]. However, the ATPS based on PEG and dex-
tran is very expensive because of the high cost of phase forming
polymers. So, expensive dextran fraction with narrow molecular
weight distribution can be replaced by crude dextran. The use of
crude dextran with PEG 4000 in ATPS has been evaluated in parti-
tioning of pectinases. Almost complete one-sided partition of endo
and exo-pectinase was achieved with highest total endo- and exo-
pectinase yields (72.41% and 69.46%, activity recovery respectively)
with purification fold of 1.73 and 2.14, respectively. However, crude
dextran showed rather high viscosity in solution which results into
problem for phase separation [74]. This, in fact, limits the imple-
mentation of dextran based system at the large scale for separation
of biomolecules [15].
The above problem may be solved by using an economic alterna-
tive polymer that substitutes for dextran with equivalent partition
properties. So, there has been an interest in developing new poly-
mer systems which can enhance partitioning of enzymes between
the two phases based on the electrostatic interactions [24]. Pereira
et al. used polyacrylate as a polymer for partitioning and recov-
ery of lipase was derived from Leucosporidium scottii L117 which
showed 40.5 activity balance (ratio of total enzyme activity in top
and bottom phase to enzyme activity in crude sample) [75]. In
another study, Lizzy et al. extracted and purified amyloglucosidase
937
from Aspergillus niger using ATPS, PEG 4000 and sodium poly-
acrylate 15,000. The purification factor and the recovery yield of
amyloglucosidase were determined as 1.9-fold and 86% respec-
tively. The poly(acrylic acid) is harmless, relatively inexpensive,
easily handled and strong negatively charged polymer (pH above
7.0) which shows strong electrostatic interaction with charged
biomolecules. In addition to that, the phase separation in this
system is relatively fast and has low viscosity as compared to tra-
ditional PEG/dextran systems [76]. Another study involved use of
thermoseparating polymer as a one of the polymer component in
polymer/polymer system. Lysozyme and trypsin partitioned into a
two-phase formed by maltodextrin (a low-cost starch derivative)
and a copolymer of ethylene oxides and propylene oxides (ther-
moreactive). This system was more cost efficient as polymers were
readily recycled by temperature-induced phase partitioning with-
out costly ultrafiltration or chromatography steps [77]. In addition,
the polymer/polymer system can be used in affinity partition by
modifying one of the polymer in the two-phase system for selective
and enhance separation which is explained in later part of review.
2.1.4. Effect of pH on partitioning of enzymes
Principally, the partitioning of enzymes into two phases in the
ATPS depends on their isoelectric points (pI, the pH at which net
charge on enzyme is neutral). The addition of salts and the change
of pH or ionic strength have often been used to alter the partitioning
of the enzyme and improve the selectivity of the two-phase extrac-
tion. According to Albertsson’s equation, the partition coefficient of
charged biomolecules is influenced by short-range (van der Waals)
and long-range (electrostatic) molecular interactions [16]. The pH
of the system affects the net charge of protein and ion composi-
tion, surface character of contaminating materials which causes
variation in their partitioning into the top and bottom phases [37].
The influence of pH on partitioning of recombinant Bacillus
sphaericus phenylalanine dehydrogenase (PheDH) was observed in
PEG/ammonium sulphate ATPS. The pH of the system varied from
3 to 8 and the maximum activity recovery (120%) and yield (90%)
was obtained at pH 7.8. With increasing pH, net negative charge of
the protein increased simultaneously. So, the interaction between
protein and PEG-rich phase became stronger and partition coef-
ficient elevated [78]. As a general rule in polymer/salt systems,
most proteins with acidic pI and consequently negative surface
charges prefer to move to the top phase, while positively charged
proteins move to the bottom phase. Most of the biomolecules,
especially proteins and enzymes, are stable at neutral pH which is
favourable condition to conduct the ATPS partitioning. The pH value
above 7 is suitable for the PEG/phosphate system and pH below 6.5
is compatible with the PEG/sulphate system. Partition behaviour
of glutaminase (pI is around 5) from Zygosaccharomyces rouxii at
different pH (3–9) was studied in an ATPS of PEG 2000/sodium
sulphate. It was found that as pH increased towards 7, partition
coefficient decreased upto 9.44 with the corresponding yield of
90.42%. Further increment in pH led to decrease in yield upto
40% [79].
Bezerra et al. investigated the effect of pH on the purification fac-
tor of alpha amylase fermented from Bacillus subtilis. They found
that purification factor was increased from 0.3 to 1.8-fold in the
PEG 1000/potassium phosphate ATPS, when the pH rose from 5.0
to 8.0 [80]. In another study, extraction of lipase from Rhizopus
microsporus fermentation culture was carried out by Anvari. An
increase in pH of the ATPS (PEG 4000/K2 HPO4 ) from 7.0 to 9.0
reduced the partition coefficient of lipase from 7.94 to 4.45 and
activity recovered from 81.1 to 70.6% [39]. Enzymes are slightly
instable in the acidic area, but it was dramatically lost their activ-
ity at higher pH (above 9.0). The effect of pH (in the range of
4–11) on partition behaviour of a plant peroxidase extracted from
the leaves of Ipomoea palmetta was studied in ATPS composed of
938 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957
PEG 1550/ammonium sulphate. The effect of pH was found to be
different on partitioning behaviour of enzyme in different phase
systems. At extreme pH conditions (below pH 4 and above 11),
enzymes deactivate due to conformational changes. Their partition
coefficients in case of sodium sulphate, ammonium sulphate and
potassium phosphate phase systems were 8.5, 7.0 and 10.3, respec-
tively with the corresponding yield of 90, 85 and 73% [81]. Hence,
the partition of enzymes can be manipulated either by changing salt
component of the system or by altering overall pH of the system.
2.1.5. Effect of volume ratio and tie-lines length on partitioning of
enzymes
For an efficient separation in ATPS, it is essential to find high
recovery, purification of enzyme and favourable partition coeffi-
cient which is influenced by phase volume ratio (ratio of volume
of top phase to that of bottom phase). All two-phase systems with
overall composition represented by one of the tie-lines give phases
with same composition but different volumes [15]. Tie line length
(TLL) can affect biomolecule partitioning by interfacial tension and
hydrophobicity between two phases of ATPS. The ATPS becomes
more hydrophobic with increasing TLL due to reduction of water
availability [82]. An increase in the TLL causes an increase in the
protein partition coefficient that, in turn, increases the yield of pro-
teins in the top phase due to reduction of the bottom phase [62].
Increasing TLL in the PEG/salt system makes salting-out more effec-
tive, leading to shift of proteins to the top PEG-rich phase [83]. If
protein solubility in the PEG phase is insufficient, protein will pre-
cipitate at the interface. Solubility and salting-out depend on the
properties of individual proteins. Therefore, a different response
will be expected when a mixture of protein is handled [84].
In the study involving purification of luciferase using
PEG/ammonium sulphate ATPS, the phase volume ratio was varied
in the range of 0.12–2.50 with tie line of 16% (w/w). It was found that
the specific activity and degree of purification of luciferase in the
top phase increased with the decrease in volume ratio [60]. On the
other hand, in the recovery of lipase from Penicillium cyclopium by
ATPS, the phase volume ratio increased by approximately 5.8 times
to augment enzyme yield by 24% in the top phase while concen-
tration of phosphate was reduced by 4.4%. However, purification
factor of enzyme was reduced from 3.5 to 2.3-fold in the top phase.
The increase in yield of enzyme by varying phase volume ratio
along the lower tie-line, results in reduction of salt concentration in
ATPS which would be beneficial from the both environmental and
economic reasons [51]. ATPS has also been used to further concen-
trate and recover fibrinolytic crude extract by using six different
volume ratios above and below the equilibrium composition and
42.6% of TLL. The highest purification factor was 7.91-fold at 1.5
volume ratio. Above and below this volume ratio, purification was
not significant. In defined ATPS, the composition of the top phase
proportionally increases with the volume ratio along the same TLL.
Hence, target enzyme had been partitioned into top phase (polymer
rich phase) due to existence of more free volumes [85]. In contrast
to that, when the volume ratio decreases over the limit of solu-
bility of fibrinolytic enzyme in the PEG-rich phase, the fibrinolytic
enzyme precipitates and gets accumulated at the interphase. There-
fore, it leads to loss of fibrinolytic enzyme by precipitation. It could
be minimised by higher volume ratios of respective ATPS. However,
higher volume ratio also has a drawback as it leads to the dilution
of enzyme concentrations [86].
2.1.6. Effect of neutral salts on enzyme partitioning
The desired biomolecule has to be preferentially partitioned
in one phase, whereas the interfering substances (other proteins,
polysaccharides and contaminants etc.) should be partitioned in
the other phase to achieve a significant extraction. However, the
partition of target enzyme is not as one sided as might be expected
and considered in conventional ATPS methods. Also, they are usu-
ally require long processing time and also expensive. Therefore,
in such a case, the addition of neutral salt can improve the par-
tition of target biomolecule which significantly increase process
yield as well as selectivity [63]. It is known that the presence of
ions has influence on the partition behaviour of proteins through
the “salting out” effect. The effect of adding salts to an ATPS is
dependent on the type of system and salt that is used [53]. Gen-
erally, the addition of neutral salts affects the electrical potential
difference between top and bottom phases and protein hydropho-
bicity. An increase in the hydrophobicity will decrease the amount
of bound water, which keeps the final composition of the system
constant. This may result in the exposure of hydrophobic patches of
protein surface, which will promote hydrophobic interactions with
the polymer phase [29,87]. In general, water structure making ions
(Li+ , Na+ , Ca2+ , Mg2+ , NH4+ , F− , SO4 2− , CO3 2− , PO4 2− , CH3 COO− )
favour more hydrophilic phase, while, water structure breaking
ions (K+ , Rb+ , Cs+ , I− , Cl− , Br− , SCN− , NO3− , ClO4− ) favour more
hydrophobic phase. Moreover, neutral salt addition affects enzyme
partitioning by altering the partition coefficient of proteins in rela-
tion to their charge [38]. Furthermore, it increases the ionic strength
and enhances the migration of low molecular weight compounds
towards the polymer phase, especially in PEG <4000 [15,88]. How-
ever, the addition of high concentration of neutral salts may cause
denaturation of proteins in the existing system, thus lower con-
centration range from 0.1 to 1.0 M or 1 to 8% w/v is preferred to
enhance the yield of the enzyme in ATPS [14].
The different neutral salts give different purification, activity
recovery and yields. In order to determine the effect of neutral
salts on partitioning of invertase, various salts such as; KCl, NaCl,
MnCl2 , Na2 CO3 , MgCl2 , MgSO4 in PEG/sodium sulphate ATPS were
evaluated. The researchers obtained maximum purification of 5.5-
fold and 154% activity recovery for KCl. A selective increase in
the partition coefficient for invertase is caused mostly by surface
hydrophobicity differences between the proteins [30]. Another key
factor affecting the enzyme partition in two phase systems is the
concentration of neutral salt used. In the purification of lipases pro-
duced by Bacillus sp. ITP-001 using PEG and potassium phosphate,
partition coefficient of enzyme was almost constant with NaCl addi-
tion till 6%, although the purification factor increased significantly
from 59.93 to 141.65-fold. Further addition of NaCl decreased the
partition coefficient of lipase [43]. Similar results were observed
with Aspergillus terreus lipase purification in the presence of NaCl
in ATPS system of PEG and potassium phosphate. The purification
factor with NaCl was 12.5% higher than that of ATPS system without
NaCl [89].
3. Alcohol/salt based aqueous two phase partitioning
system
In both polymer/salt and polymer/polymer systems, the target
biomolecules are accumulated in either the polymer-rich phase
or salt-rich phase. The recovery of biomolecules from phase-
forming chemicals/polymers is very convoluted [90]. Also, it has
been widely reported that it involves tedious purification oper-
ations, such as ultrafiltration and crystallization often needed to
get desired proteins from these conventional ATPS [91]. Mostly,
these systems require back-extraction of target biomolecule into
salt-rich bottom phase, or additional complicated operations (such
as ion exchange chromatography, diafiltration, crystallization or
ultrafiltration) to remove the phase-forming components from tar-
get product [92]. Also,in many cases, protein (enzyme) which has
low solubility in aqueous solutions tends to aggregate in poly-
mer/salt system which ultimately led to loss of enzyme [93].
Furthermore, recycling of bottom phase forming salt is very compli-
 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957
cated. This require large amounts of polymers/chemicals, leading to
environmental pollution and high operating costs [94]. Therefore,
advanced and improved ATPS have been developed using alco-
hols, surfactants, micellar compounds [95]. Also, ionic liquid, and
thermo-separating polymers (TSPs) have been employed to extract
enzymes from fermentation broth and natural sources [96,97].
The alcohol/salt ATPS is formed by mixing two immiscible com-
ponents which composed of alcohol and salt solutions. In this
system, the biomolecules were partitioned by the hydrophobic
interaction of organic solvent combined with the salting-out effect
of the salt-rich phase [98]. The salt-rich bottom phase also influ-
ences the hydrophobic interactions between proteins as salt ions
interact with the oppositely charged residues of the protein, result-
ing in the dehydration. Thus, the hydrophobic interaction between
the enzyme and organic solvent led to the desirable partition and
transfer of the enzyme to the top phase of the system. Addition-
ally, the target protein can be easily extracted and recovered by
removing the alcohol using evaporation [99]. However, an increase
inalcohol concentration in two phase system results in the par-
tial dehydration of the bottom phase [100]. Alcohol-salt ATPS have
also been used for the recovery of low and high molecular weight
biomolecules [101]. Additionally, the alcohol/salt ATPS has less tox-
icity impact to the environment and is economic as compared to the
conventional ATPS [14]. Also, it presents some additional advan-
tagessuch as high polarity, low viscosity, low cost, and ease of
recovery from the alcohol-rich phase [102].
Various examples of extraction of enzyme by using alcohol/salt
ATPS system have been summarized in Table 3. Pectinase from
mango (Mangifera indica) waste was purified using an aqueous
organic phase system of ethanol and potassium phosphate. The
salting-out effect resulted in the molecular exclusion of pectinase
to the upper phase and contaminants moving from organic solvent
top phase to salt-rich bottom phase. Based on the system, pecti-
nase was extracted with the purification factor of 11.7-fold, with
97.1% yield [62]. The selection of alcohol and salts play an important
role in the partitioning of enzyme. Ooi et al. employed differ-
ent alcohol-based top phase (ethanol, 1-propanol and 2-propanol)
and a salt-based bottom phase (ammonium sulphate, potassium
phosphate and sodium citrate) to evaluate for their effectiveness
in lipase recovery from Burkholderia pseudomallei. The maximum
lipase recovery was achieved in 2-propanol/potassium phosphate
ATPSwith purification factor of 13.5-fold and a yield of 99%. The
authors have claimed that the negatively charged lipase is repelled
from salt-rich bottom phase and longer hydrocarbon chain alco-
hol facilitated the interaction with lipase, and thus enhanced the
partitioning [103].
The adverse effect of alcohol based systems during purification
of enzyme have also been reported. In the recovery of superoxide
dismutase from Kluyveromyces marxianus by ethanol and potas-
sium phosphate ATPS, the SOD activity in each phase of the systems
decreased dramatically upto 50%. This loss in catalytic activity was
attributed to structural and conformational changes and subse-
quent denaturalization of the protein due to the high concentration
of ethanol in the system [104,105]. Therefore, it limits the usage of
ethanol/salt ATPS for the recovery of certain enzymes.
4. Surfactant/alcohol based aqueous two phase partitioning
system
Recently, a novel approach for ATPS based on surfactant and
alcohol (mostly sugar alcohol) have been developed. It has ability
to retain the biological activity of enzymes over other conventional
ATPS has been reported (Table 4). This system makes it possible to
create two phases; the top surfactant rich phase (containing non-
ionic and ionic surfactants such as Triton X-100, Tween 80, SDS)
939
and the bottom alcohol phase which can be recycled [15]. Non-
ionic surfactants have been employed for solubilizing membrane-
associated proteins without loss of their biological activity. During
solubilisation, the non-ionic surfactant swaps most lipid molecules
in contact with the hydrophobic domain of the protein and leads to
the formation of a soluble protein-surfactant mixed micelle [106].
The physicochemical environments micelle-rich and in the micelle-
poor phases form the basis for an effective separation and makes
surfactant and alcohol based system, a convenient and potentially
useful method for the separation and purification of biomolecules
like enzymes and proteins [107].
It is important to make a note that the effect of surfactants
on partition of enzyme depends on specific and selective chemi-
cal interactions between the enzyme molecules. It is manifested in
the work carried out by Zhong et al. The researchers used different
surfactant (Triton X-100, Tween 80, SDS)/mannitol to purify poly-
galacturonase enzyme from Durio zibethinus. Tween 80 exhibited
effective enzymatic partitioning with 11.2-fold purification factor
and yield of 93.2%. While, an anionic surfactant SDS showed the
minimal effect on the enzyme separation. It has been suspected
that ionic surfactant attached to protein might interrupt the ter-
tiary conformational which affect the enzyme activity [108,109].
It has been verified that the interaction between ionic surfactants
and proteins is a combination of electrostatic as well as hydropho-
bic forces [110]. Thus, the surfactant head (hydrophilic) group plays
a determining role in protein-surfactant interactions that preferen-
tially begins with strong ionic bonds formation between surfactant
polar groups. The protein-surfactant mechanism would greatly
inhibit the enzyme separation in two phase system and directly
reduce the purification factor [111,112].
In surfactant and alcohol (Tween 80/xylitol) based ATPS sys-
tem, different concentrations of surfactant were employed to purify
lipase from Pumpkin (Cucurbita moschata) Seeds. The lipase activ-
ity was augmented by increasing Tween 80 concentrations upto
10%, while the activity decreased beyond this concentration due to
partially inactivation of enzyme. The inactivation of enzyme was
attributed to the binding of Tween 80 surfactant to the proteins,
which disrupted the majority of the globular structures resulting
into reduction in purification factor [113,114]. The high recovery
of enzymes along with recycling of phase forming components is
advantage of surfactant/alcohol based system. The purification of
polygalacturonase from Durio zibethinus murry was successfully
done by using Triton X-100 and mannitol ATPS to get 16.1-fold
purification factor and 97.3% yield. Further, surfactant and alcohol
was successively recycled for 5 times with minor loss of phase form-
ing component. This could be explained by the selective interaction
of the surfactant toward desirable protein into the top phase after
being reused several times [115]. Therefore, this surfactant/alcohol
based system presents a promising alternative to the conventional
ATPS which includes beneficial properties like inexpensive phase
formation compounds, mildness of the recovery conditions (high
water content) and simplicity of the procedure. However, these
system cannot be suitable for the enzymes which are sensitive to
surfactant as well as alcohol. Vicente et al. observed that during the
recovery of bromelain (cysteine endopeptidases) from pineapple
stem by surfactant based ATPS, enzymatic activity was reduced by
40% due to unfavourable interaction with surfactant Triton X-114
[116].
5. Advanced aqueous two phase partitioning system
The growing demand for biotechnological fine chemicals and
biological molecules, in particular proteins has led to the develop-
ment of specific separation and purification methods. In general,
ATPS has proven to be effective for the recovery of biological prod-
940 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957
Table 3
Extraction of enzymes by using alcohol/salt-based aqueous two-phase system.
Enzyme Source Alcohol
Lipase Burkholderia pseudomallei 2-Propanol (16%)
Pectinase Mangifera indica Ethanol (19%)
SuperoxideDismutase (SOD) Kluyveromyces marxianus Ethanol (17%)
Serine protease Mangifera Indica 2-Propanol (16%)
Table 4
Extraction of enzymes by using surfactant/alcohol based aqueous two-phase system.
Enzyme Source Surfactant
Lipase Leucosporidium scottii L117 Triton X-114
Polygalacturonase Durio zibethinus Murry Tween- 80 (25%)
Lipase Cucurbita moschata Triton X-100 (24%)
ucts from different origins [19,117]. However, one major drawback
concerning the use of ATPS is their lack of specificity. Also, tra-
ditional methods for purifying and separating proteins are highly
complex, time consuming and expensive. Although the manipula-
tion of the system parameters may favour the selective partitioning
of the desired compound towards a particular phase in traditional
ATPS, specificity is not high enough to accomplish complete separa-
tion between product and contaminants [98]. Hence, the modified
ATPS was employed as down streaming strategies to enhance the
purification of biomolecules which is briefly summarized as below.
5.1. Aqueous two-phase affinity partitioning system
Affinity aqueous two phase partitioning provides a highly com-
patible system, based on the specific interaction between the
biomolecule and affinity ligands which can be manipulated in order
to develop more predictable purification strategies. Therefore, it
can bepossible to get specific partition coefficients for a defined
ATPS [104]. Generally, it consists of chemical modification of at
least one phase forming component of the ATPS by coupling affinity
ligand (Fig. 4 Left). Therefore, selective interaction between spe-
cific ligands in one of the polymeric phases of the system and
biomolecule of interest followed by their partition towards the
modified polymer rich phase is achieved [118]. Another way of
affinity partitioning ATPS can also be attained by the addition of free
ligands (chemical or biological molecules) in conventional ATPS in
order to encourage a partition shift of the desired target molecule
instead of the chemical modification of the phase forming com-
ponent (Fig. 4 Right) [53]. At preparative scale, these systems also
present important advantages such as: high ligand concentration
per unit volume of polymer solution, which renders high concen-
tration of bound target biomolecule in one of the phases of system
(process intensification) and fast equilibrium achieving [119]. As
it combines the selective affinity technique with the robust tradi-
tional liquid–liquid ATPS, it can be implemented for the specific
recovery of diverse biological molecules with the significant purifi-
cation with improved yield.
Most of the reported investigations about affinity partitioning
belong to polymer/polymer ATPS. Very few reports are available on
affinity partitioning in polymer/salt ATPS mainly due to the inter-
ference of high salt concentration with the biospecific interactions
[120]. Based on our literature, we have identified four additional
parameters usually studied for affinity ATPS; pH level for product
partitioning, ionic strength of the system, affinity ligand nature and
its concentration (or modified polymers concentration). These fac-
tors must be taken into consideration in order to optimise ATPS
as affinity interactions between enzymes and affinity ligands are
highly reliant on the ionic state of the active groups (according
to pH) and the ionic strength of the system (depending on con-
centration of salt) [121]. In addition, ligand concentration and
Salt PF Yield Ref.
Potassium phosphate (16%) 13.5-fold 99% [103]
Potassium phosphate (22%) 11.7-fold 97.1% [265]
Potassium phosphate (26%) 9.8-fold 80% [105]
Potassium phosphate (20%) 11.6-fold 96.7% [62]
Alcohol Kp Yield Ref.
McIlvainebuffer 4.77 88.94% [75]
Mannitol (22%) 16.1 97.3% [266]
Xylitol (20%) 16.4 97.4% [114]
modified polymer concentration could also enhance the recovery
and purification yield of specific enzyme in affinity ATPS. Neverthe-
less, appropriate levels of ligands should be added to the system
as their higher ligand concentration could introduce shielding
effect towards target biomolecules and also higher concentration
of modified polymer can generate steric hindrance between active
receptor sites and target molecules [98,117]. These possible alter-
natives emphasize the importance of the different parameters in
affinity ATPS systems, in order to design efficient and novel purifi-
cation strategies.
Affinity ligand provides high resolution which can be coupled
with traditional ATPS. The polymer component (PEG or dex-
tran) has to be chemically active to purify enzymes.The several
example of affinity aqueous two phase system for enzyme extrac-
tion is listed in Table 5. These strategies could be developed
using derivatives of PEG as affinity ligands such as PEG-benzoate,
PEG palmitate, PEG phenylacetamide or PEG trimethylamine
[122]. In one of the studies, modified PEG and sulphate ATPS
was utilized to purify ␤-Galactosidase and glucose oxidase from
Kluyveromyces lactis and Aspergillus niger fermentation. In this par-
ticular case PEGs were modified as: PEG-trimethylamine (TMA
ionic interaction ligand), PEG-benzoate (pseudo specific ligand),
PEG-palmitate (hydrophobic ligand) and PEG-Coomassie brilliant
blue G-250 (pseudo-specific ligand). The glucose oxidase was
partitioned toward the PEG-Coomassie at bottom phase with
19-fold purification and 80% recovery, while the contaminants pro-
pelled to the upper phase. ␤-Galactosidase was best extracted in
PEG–TMA systems (7-fold purification increase with 80% recov-
ery). Authors have claimed that it was because of repletion between
positively charged ligand TMA and negatively induced-charged ␤-
galactosidase favouring its partition towards the bottom phase. The
affinity attractions between active groups in polymer and contam-
inant proteins, which in turn provides a new strategy and open a
new research area for purifying target enzyme molecules [123]. The
interaction between affinity ligand and specific biomolecule results
in an efficient separation process. However, the extended protocol
time and relative complexity within activation process may limit
their use.
An affinity ATPS purification strategies using free ligands has
been implemented to recover various enzymes. In this methodol-
ogy, neither polymer activation nor ligand coupling are required,
which ultimately reduce the time and chemical consumption [117].
Sharma et al. investigated an affinity ATPS consisting of PEG/salt for
purification of phospholipase D in the presence of alginate (a known
macroaffinity ligand) from peanuts and carrots. Incorporation of
alginate in the PEG phase was resulted in 91 and 93% of the enzyme
activity from peanuts and carrots respectively [124]. Another study
mentions the application of affinity ATPS using free ligand addi-
tion in the partition of chitinase. The systems were formed by PEG
6000/potassium phosphate ATPS in the presence of free chitosan
 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957 941

Fig. 4. Schematic diagram illustrating the partitioning of enzyme and contaminant proteins (impurities) in aqueous two phase affinity partitioning system: addition of a free
ligand (Right) or modification of PEG with ligands (Left).

Table 5
Extraction of enzymes by using aqueous two-phase affinity partitioning system.

Enzyme System Modification Remark Ref.

␤-Galactosidase Dextran T500/PEG 8000 Dextran-benzoyl Kp decrease: 0.036–0.018 [267]

Glucoamylase PEG 300/Phosphate Starch as free ligand Kp decrease from 6.6 to 0.73 [268]
Chitinase PEG 6000/Phosphate Chitosan as free ligand 90% recovery in UP after final elution [125]
Penicillin acylase PEG 4000/Phosphate PEG-benzoate 14-fold increase of Kp (0.1–1.4) and 60% [119]
recovery in a single
Cutinase PEG 1500/Phosphate Butyrate as free ligand 98% recovery in first extraction and Kp of 135 [269]
Penicillin acylase PEG 4000/phosphate PEG-trimethylamine Kp of 2.1 with 70% recovery in single step [119]
extraction
Phospholipase D PEG 6000/phosphate Alginate as free ligand 85% recovery in UP and 78-fold purification [270]
factor
Penicillin acylase PEG 4000/Phosphate PEG-palmitate Kp of 3.22 and 0.56 for contaminant proteins; [119]
75% recovery
␤-Mannanase Guar Gum/PES 200 Galactomannan as FL Kp increase from 1.37 to 7.06 [271]
Glucose-6-phosphate dehydrogenase PEG 6000/PES 100 Cibacron Blue F3GA (FL) ATPS with dye induced an increase in [117]
Kp from 0.73 to 1.59
Cellulase PEO/Dextran T500 Maleic acid-PEO 2-Fold increase in Kp for cellulose with [117]
40% modification rate at PEO phase

in solution as affinity ligand. Chitinase was extracted from differ-
ent sources such as puffballs (Lycoperdon umbrinum), Neurospora
crassa and cabbage (Brassica oleracea). At lower concentration of
chitosan (0.1%), chitinase recovered from N. crassa exhibited higher
activity recovery (86%) with 34-fold purification increase in top
phase, while, extraction from puffballs and cabbage exhibited a
38-fold and 20-fold of purification with total activity recovery of
88 and 80% respectively in the PEG rich phase. On the other hand,
as the concentration of ligands increased, purification factor and
yield of enzyme reduced drastically. The authors suggested that
high concentration of free ligands caused the non-specific interac-
tions between the chitinase and the affinity ligand, which lowering
the overall binding capacity probably due to “crowding effect”.
Based on the results, authors demonstrated the potential of affin-
ity partitioning of enzymes, emphasizing the effect of polymer
characteristics (molecular weight and concentration), on ligand
distribution through the system, hence those critical parameters
should be considered in order to develop efficient recovery and
purification strategies [125].
Metal affinity partitioning ATPS exploits the affinity of tran-
sition metal ions for electron-rich amino acid residues (cysteine
and histidine) present in enzymes. The metal ions are partially
chelated and coupled to a linear polymer (PEGor dextran), the
resulting polymer-bound metal can be used to augment the par-
titioning of metal binding proteins into the polymer-rich phase in
PEG/salt or PEG/polymer ATPS [126]. In one of the studies, affin-
ity of papain towards metal ions (Cd, Co, Zn and Cu ions) was
investigated. The papain enzyme was extracted from papaya fruit
(Carica papaya) by traditional ATPS composed of PEG/ammonium
sulphate and metal affinity ATPS. The yield was found to be 74.5,
72.3, 53.3 and 53.5% for Cu2+ , Zn2+ , Co2+ and Cd2+ ion respectively
whereas for conventional ATPS, the yield was found to be 33.3%.
This showed that ATPS coupled with metal ion enhanced the extrac-
tion of enzyme due to affinity of enzyme towards metal ions [127].
Another comparative experiments determined that, lactate dehy-
drogenase was extracted efficiently by using PEG/hydroxypropyl
ATPS in the presence of copper ions (Cu2+ ) and authors confirm that
a strong interaction between the copper ion and enzyme allows
to concentrate in a specific phase [122]. Freire et al. evaluated
the affinity dye based ATPS for papain partitioning from crude
latex using tri-azine dyes (CIBACRON Blue F3GA and PROCION Red
HE3G). These dyes have highly specific functional groups and act
as affinity factors in most of chromatographic applications. It was
found that initial increase in PEG molecular mass and dye concen-
tration resulted in high partition coefficient value while further
increase in dye concentration reduced the partition coefficient of
942 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957
papain. In addition to that when polymer concentration increased
to maximum, partition coefficient of papain enzyme reduced along
with the purity factor. The dyes CIBACRON Blue F3GA and PROCION
Red HE3G combined with PEG/ammonium salt resulted into 11.6
and 13.4-fold purification factor [128]. Affinity partitioning ATPS
has a wide scope for enzyme purification, however the application
of aqueous two-phase affinity partitioning system for recovery of
high commercial value biologicals products is expected.
5.2. Ionic liquid based aqueous two phase partitioning system
In last decade, ionic liquids (ILs) have emerged as an alternative
phase forming component for ATPS particularly when proteins are
easily denatured and lose their biological activity in contact with
organic solvents [129]. They have unique properties such as neg-
ligible vapour pressure, non-flammability and high thermal and
chemical stability with wide electrochemical window [129,130].
Most water-soluble ILs are salting-in inducing electrolytes (known
as “chaotropic” salts) in the presence of aqueous solutions and
salting-out inorganic salts (known as “kosmotropic” salts) result-
ing in the formation of two phases in ATPS [131–133]. The driving
force for phase separation in IL ATPS is the competition between
the salt and IL for water molecules. The higher affinity of the inor-
ganic salt for water induces a migration of water away from the IL
ions which decrease their hydration and reducing the solubility of
ILs in aqueous solution [134]. The commonly employed inorganic
salts are ammonium, sodium or potassium based salts of multiple
charged anions, such as carbonate, citrate phosphate or sulphate
(strong salting-out inducing anions) [135,136]. Recently, the possi-
bility of extracting some macromolecules (proteins and enzymes)
using ILs has been explored as ILs have the potential to retain high
enzyme activity and stability with significant extraction efficiency
[137]. The ATPS formed by ILs and salts are reported to give higher
recoveries of the proteins as compared to conventional ATPS com-
posing PEG and inorganic salt [138]. In addition, IL-based ATPS also
exhibit better process properties, such as little emulsion formation,
lower viscosity and fast phase separation [139]. On the whole, it
seems that water-soluble ILs are likely to be more appropriate for
the isolation of enzymes which can be considered as an economi-
cal and efficient extraction approach for those separation systems
[129]. Therefore, IL based ATPS offer the opportunity to combine the
extraction and concentration of biocatalysts in active form [140].
The enzyme extraction by using IL is summarized in Table 6.
Deive et al. have compared IL based system with conventional
PEG based system for extraction of three different lipases; CalB,
CalA and Thermomyces lanuginosus lipase. The partition coefficients
in the ILs system was two to three folds higher than those obtained
with PEG based systems. The author attributed this favourable par-
tition ratio to the large difference in polarity between the two
phases in the presence of IL [141–143]. In another study, the sepa-
ration and purification of lipolytic enzyme produced by Bacillus sp.
was done from the fermentation broth by using IL based ATPS. At
the end of the production phase, improved purification factors and
enzyme recovery efficiencies was observed[140]. In many cases,
imidazolium-based ionic liquids have proven to be the most effi-
cient cations, and [C4 SO4 ], [C2 SO4 ] and Cl− were chosen as the
optimal anions [129]. Other works have focused on the extraction of
a peroxidase and two different alcohol dehydrogenases, using imi-
dazolium and ammonium based ionic liquids. In the latter work,
apart from the increased specific activity for two different alco-
hol dehydrogenases, it also manifested that the presence of an IL
provides the opportunity to combine an extraction process with
efficiencies of 90% and the increased performance in the biocat-
alytic reaction [144].
The extraction of enzyme also depends on the chemical nature
of ILi.e. cation alkyl chain length and the anion and cation core.
This has been demonstrated in the study carried out by Ventura
et al. The researchers purified lipase B from Candida Antarctica using
IL/potassium salts [142]. The results suggested that the increase in
the alkyl chain length induces drastic increase in partition coef-
ficient of other protein than partition coefficient of enzyme which
remains consistently very low. The increase in the alkyl chain length
makes the ionic liquid more hydrophobic, decreasing the Coulom-
bic interactions and increasing the dispersive forces that occur
between the enzyme molecules and the ionic liquids at the IL-rich
phase. These factors seem to partition the contaminant proteins
into upper IL-rich phase. Due to its low isoelectric point (pI 6.0),
the lipase is negatively charged at these pH conditions (pH 7.0)
which resulted in the increase of its hydrophilic character, creating
stronger affinity of enzyme for the bottom salt-rich phase [145].
The difference observed in the partitioning behaviour between the
contaminant proteins and enzyme, must result from differences
in their hydrophilicity/hydrophobicity that will allow the parti-
tion of contaminant proteins and enzyme into different phases. The
enzyme recovery obtained were consistently above 96% with purifi-
cation factor of 2.6-fold for 1-methyl-3-octylimidazolium chloride
([C8 mim]Cl). The presence of IL in the medium is reported to have a
harmful effect on the enzyme activity, though not very significant at
low concentration level [146]. The increase in the purification fac-
tor must be the result of elimination of the contaminant proteins,
which are acting as enzymatic inhibitors.
Aforementioned studies involving ILs in extraction, purification
and recovery of enzymes depict that, in many cases, the substitution
of toxic and flammable organic solvents is not only beneficial for the
environment but also results in higher yields in catalytically active
form compared to the classic ATPS. Even though ILs can perform
better than organic solvents in biotechnological applications, much
research still needs to be done, especially concerning synthesis and
the recovery of the IL due to its high cost. In addition, up-scaling
and integration of the newly advanced processes to larger scale
needs further exploring. Nevertheless, the research in the field of
ILs for extraction and biocatalysis (in synthesis) is very interesting
and found to be very promising.
5.3. Thermoseparating polymer-based aqueous two-phase system
An advanced and improved ATPS have been developed by using
thermoseparating polymers (TSPs) consisting of a high water con-
tent (70–90% w/w) that provides a mild environment for separation
of sensitive biomolecules (mostly enzymes). It requires a lower
amount of phase forming components for two phases formation
with thermoseparating ATPS, thereby, minimizing the risks of both
salting out and precipitation of proteins and preventing enzyme
denaturation [113,147]. Moreover, phase separation can be done
more rapidly and selectively at room temperature due to efficient
mass transfer facilitated by high interfacial contact area. Addition-
ally, the phase-forming components of thermoseparating ATPS are
generally more safe, non-flammable, non-toxic and relatively envi-
ronmentally friendly in nature [148,149]. Along with ease of phase
separation and biomolecule partitioning, it has the ability to han-
dle high capacity and reduce the volume of subsequent purification
steps, large-scale purification using thermoseparating ATPS can
be easily and reliably predicted from laboratory data [150,151].
Thus, thermoseparating ATPS is a potential solution for industrial
demand of highly-efficient large-scale and cost-effective separa-
tion technology with short processing time [152]. Various examples
of extraction of enzymes by using ATPS based on TSP are mentioned
in Table 7.
The temperature-induced ATPSs give rise to development of
TSPs with distinctive thermoseparating properties and easy recy-
clability (Fig. 5) [153]. The common employed TSPs are random,
diblock and triblock copolymers of hydrophilic ethylene oxide (EO)
 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957 943

Table 6
Extraction of enzymes by using ionic liquid based aqueous two-phase system.

Enzyme Source Ionic liquid Salt Remark Ref.

Lipase B Candida antarctica ([C2 C1 im]Cl, Dipotassium Maximum [142]

[C7 H7 C1 im]Cl, phosphate and purification and recovery were
[C4 C1 im][CF3 SO3 ], Potassium dihydrogen obtained for cations with a C8
[C4 C1 im][N(CN)2 ], phosphate side alkyl chain, the [N(CN)2]
[C4 C1 im][C1 SO3 ], anion which belonging to the
[C4 C1 pyr]Cl, pyridinium family
[C4 C1 py]Cl,
[C4 C1 pip]Cl,
[C8 C1 py][N(CN)2 ])
Lipase Bacillus [C4 C1 im]Cl Dipotassium IL-based ATPS was superior to [272]
sp. ITP-001 phosphate and PEG-based ATPS for the
Potassium dihydrogen purification
phosphate
Alcohol Lactobacillus brevis Ammoeng100TM , Dipotassium The presence of IL exhibited [273]
dehydrogenases and thermophilic Ammoeng101TM , phosphate and positive stability-enhancement
bacterium Ammoeng110TM Potassium dihydrogen effect on enzymes during
phosphate purification.
Horseradish [C4 C1 im]Cl Dipotassium The lyotropy is beneficial to [274]
Peroxidase phosphate enzyme activity maintaining
which should be consider
while choosing proper IL-ABS
as extraction system
Lipase A Candida antarctica [C2 C1 im][C4 SO4 ] Ammonium sulphate High charge–density salts and [143]
amino acids have been studied
in order to evaluate their
potential as phase forming
agents and as stabilizers of
enzyme.

Table 7
Extraction of enzymes by using thermoseparating aqueous two-phase system.

Enzyme Source Thermo- Salt/ Recovery Polymer recycling Ref.

separating alcohol efficiency
Polymer

␣-Amylase Leucosporidium EOPO 2500 Sulphate 91% yield and – [147]

scottii L117 3.31-fold PF
CGTase Bacillus cereus EOPO 3900 Phosphate 87% yield 2 times [156]
and 13.1-fold PF
Endo- Kluyveromyces UCON Sulphate Up to 99% yield and – [275]
Polygalacturonase marxianus 10-fold PF
Laccase Peniophora cinerea UCON Potassium 134% yield and – [276]
dihydrogen 1.31-fold PF
phosphate
Lipase Burkholderia EOPO 3900 Phosphate 99% yield and 3 times [90]
cenocepacia strain 14-fold PF
ST8
Laccase T. versicolor UCON Dipotassium Kp = 0.272 – [97]
phosphate
Lipase Burkholderia EOPO 3900 – 99% yield and – [157]
cepacia 14-fold PF
Lysozyme Hen egg white EO50 PO50 Dipotassium 85% yield and 3 times [159]
phosphate 16.9-fold PF
Thermostable Methanococcus PEO–PPO Ammonium Up to 90% and – [277]
␣-amylase jannaschii sulphate 3.31-fold PF
L-asparaginase E. coli 11303 EO40 PO60 -870 Potassium 73.3% yield 2 times [158]
dihydrogen
phosphate and
Dipotassium
phosphate
Thermo-acidic Hylocereus EOPO 2500 2-propanol 96.6% yield and 5 times [278]
amylase polyrhizus 14.3-fold PF

and hydrophobic propylene oxide (PO). The EOPO copolymers have
been usedas replacement of PEG with different type of salts for
partitioning. These polymers have a wide range of application as
their EO/PO ratio and molecular weight can be greatly varied which
shows significant influence on the separation of biomolecules
[154]. Other examples of TSPs include poly ethyl hydroxyethyl
cellulose, poly (vinylcaprolactam), poly (N-isopropylacrylamide)
and non-ionic surfactants [155]. Peeples et al. purified a recom-
binant, thermostable ␣-amylase from Methanococcus jannaschii by
employing random copolymer of EOPO and compared it with tra-
ditional PEG/ammonium sulphate ATPS. The 3.33-fold purification
factor was observed for copolymer/salt system, whereas it was
2.90-fold for PEG/salt system [150]. In another extraction studies,
Ng et al. purified cyclodextrin glycosyltransferase from B. cereus
upto 13.1-fold with a yield of 87% and the recovery upto 80% by
ATPS composed of EOPO 3900 and potassium phosphate salt [156].
The concentration and molecular weight of EOPO also shows
a significant influence on the partitioning of protein due to
944 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957
Fig. 5. Schematic diagram illustrating the principles of a temperature-separating polymer based ATPS.
change in hydrophilic to hydrophobic ratio. The hydrophobicity
of the polymer increase with molecular weight due to increase
in hydrophobic area to hydrophilic group ratio. The extraction of
lipase derived from Burkholderia cepaci was carried out withdiffer-
ent molecular weights (970, 2500, 3900 and 12000 Da) by varying
concentrations (2–20%) of EOPO. The purification factor increased
with increase in molecular weight till EOPO 3900 and further
increase in molecular weight resulted into lowering of purification
factor [157]. In partitioning of L-asparaginase by PEO–PPO–PEO
(triblock copolymers)/phosphate system, the purification factor
showed an increasing trend for increasing triblock copolymer
molar mass. Further, the yield of L-asparaginase obtained by
triblock copolymer/phosphate system was 73.3%, while for con-
ventional (PEG/salt) ATPS, it was 52.1% [158].
System pH plays a crucial role in TSPs based ATPS as it modifies
the net charge on protein molecules and thus, influence partitioning
of the target product. The influence of pH (7–10) on lysozyme par-
titioning in EO50 PO50 /potassium phosphate ATPS was studied by
Dembczyński et al. The pH 9 was found to be effective for extraction
of lysozyme (pI 10.9) [159]. Some enzymes such as fungal laccase
(pI 6) and l-asparaginase (pI 4.9) have been extracted within range
of acidic pH. The hydrophobic interaction dominates at pH around
pI of biomolecules due to zero net charge of proteins, while electro-
static forces show greater effect at other pH. Besides that, system
pH generally has no effect on the cloud point (CP) of TSPs, with
the exception of cationic hydrophobically modified ethylene oxide
polymer for which CP increased rapidly at pH <9 [160].
Apart from outstanding advantages of temperature-induced
separation, TSPs can be recycled and reutilized for multiple times.
After heating beyond a critical temperature known as lower criti-
cal solution temperature (LCST) or cloud point (CP), the solubility of
TSPs decreases and solution turns into turbid and hazy to form two
phases eventually [161]. In these systems, the top phase has almost
100% water content, while the bottom phase contains copolymer
[162]. This feature helps in easy extraction of target product and
helps to recycle and reutilization of polymers possible without the
need for additional third component or extra separation processes.
This makes the separation process not only a greener and economic
downstreaming technique, but also makes the recovery more effi-
cient. The commonly used PEG polymer in two phase partitioning is
also a TSP. However, LCST above 100 ◦ C (low-molecular-mass PEG
has a LCST as high as 180 ◦ C) is unsuitable in most case of thermo
separation of biomaterials [154]. On the other hand, hydrophobi-
cally modified EOPO has low LCST around 12 ◦ C at 3% w/w, which
means that thermo separation can be done near room tempera-
ture [163]. LCST of thermoseparating polymers depends on the
EO/PO ratio, as well as block size and molecular mass [93,148].
Also, the LCST of TSPs can be lowered by making the polymer
more hydrophobic (i.e. increase of PO content) or by addition of
hydrophilic salt (e.g. sodium sulphate), acids/bases or ionic sur-
factants (e.g. SDS) which promote partitioning of target products
[147,162,164].
In one of the studies conducted by Ng et al., employed ATPS com-
posed of TSP and phosphate salt for the recovery of cyclodextrin
glycosyltransferase from Bacillus cereus and recycled phase com-
ponents over four successive extractions. The purification factor
and the yield of lipase were obtained 14 and 99%, respectively even
after four successive purification cycles[156]. In most of the recy-
cling studies, the yield and purity fall following the recycling cycles
due to increasing contaminants in bottom phase after each cycle,
thus, recommending that the EOPO copolymer can only be recy-
cled and reused for few times. However, there are exceptions in
both lysozyme and lipase extraction studies which showed that
the parameters such as purity, yield and volume ratio are quite
stable, regardless of high accumulation of contaminant protein in
the bottom phase throughout four consecutive recoveries [90,165].
Thermo-acidic amylase from red pitaya (Hylocereus polyrhizus) peel
was satisfactorily partitioned into the polymer-rich top phase in
the system composed of 30% (w/w) EOPO 2500 and 15% (w/w) 2-
propanol at pH 5.0. The amylase was successfully recovered with a
high purification factor of 14.3-fold along with the yield of 96.6%
and copolymer was also recovered and recycled above 97%, making
the method more economical than the traditional ATPS method.
In thermoseparating based ATPS, simplicity of product recovery
and economic chemical cost provide a huge scope in extrac-
tion of enzymes. However, novel TSPs should be developed with
unique characteristics and also, different combinations of two
phase system needs to be explored. It is necessary to find corre-
lations between different parameters and predict the partitioning
behaviour by using mathematical models. Additionally, kinetics of
bioseparation and underlying mechanisms need be further studied
and simplified into simpler concepts.
5.4. Aqueous micellar two-phase system
The aqueous micellar two-phase system (AMTPS) is another
variation in ATPS, which is also known as surfactant-mediated
phase separation. It is a more efficient method for purification of
biomolecules. AMTPS utilizes a binary phase micellar system with
the help of surfactants at low concentrations in aqueous solutions to
induce micelles [166]. Once the temperature of the surfactant solu-
tion reaches a CP temperature, two phases are formed in AMTPS due
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to difference between the physicochemical environments [167].
When the temperature is above the CP, the surfactant molecules
self-aggregate to maintain an aqueous (surfactant-depleted) phase
and results into two phase formation i.e. in a bottom, micelle-
rich phase and a top, micelle-poor phase [168]. The most common
phase-forming nonionic surfactants used are Triton X-114 and
other alkyl polyoxyethylenes (Cm EOn ) such as n-decyltetraethylene
oxide. The partitioning of proteins (enzymes) are governed primar-
ily by steric, repulsive and excluded-volume interactions between
the globular hydrophilic proteins and the non-charged cylindrical
micelles, which drive desired biomolecules preferential into the
bottom micelle-rich phase while simultaneously eliminating most
of the contaminants (proteins) to the micellar poor top phase, due
to excluded-volume interactions between the contaminates and
micelles. The formation of micelles represent a delicate balance of
several intermolecular forces including those of the hydrophobic,
van der Waals, steric and electrostatic types. The resulting micelles
are labile microstructures formed by the non-covalent binding of
individual surfactant molecules, contrary to polymers, which are
formed by the covalent binding of individual monomers [169]. Also,
micellar size and shape, as well as the associated micellar solution
properties can be tuned in situ by varying solution conditions, such
as temperature, surfactant concentration, salt type and concentra-
tion.
Jaramillo et al. carried out extraction of the pectinase (from
Aspergillus oryzae) by using a micellar Triton X-114/sodium phos-
phate AMTPS system. This system gave highest yield of 375.5% in
the top phase [170]. In another studies, Duarte et al. extracted lipase
produced by psychrotrophic yeast Leucosporidium scottii using
AMTPS composed of Triton X-114 (TX-114)/McIlvaine buffer pH
7.0 and compared it with PEG/phosphate salts and PEG/polyacrylic
acid. Lipase preferentially partitioned into the micelle-rich phase
with the purification factor 1.32-fold which is much higher than
traditional ATPS (compose of PEG/phosphate) systems [75]. Amid
et al. investigated the influence of different non-ionic surfactants
(Pluronic L31, Pluronic L81, Pluronic L61, Pluronic L121, TX-114)
and different types of salts (K2 SO4 , KNO3 KH2 PO4 , and KCl) on the
purification of serine protease from kesinai leaves. The protease was
partitioned into bottom micellar phase with high purification fac-
tor (10.3-fold) and yield of 92% in the AMTPS composed of Pluronic
L61/KNO3 . [112]. In above all cases, the enhanced purification was
observed due to improve protein solubility in bottom phase by the
salt addition and increase the hydrophobic interaction between
the surfactant phases. In addition to that, the protease from the bot-
tom phase was re-extracted to a new aqueous phaseand recycle the
surfactant. In another studies for easy recycling, thermoseparating
polymer triblock copolymer (PEOn –PPOm –PEOn ) was incorporated
with Triton X-114 to extract lipase (from Burkholderia sp. ST8).
Lipase recovery was achieve with purification factor of 7.2-fold and
0.032 partition coefficient [171].
5.5. Microfluidic platform for ATPS
In conventional ATPS, separation of two phases usually takes
few hours, and in some cases agitation is required to mix the two
phases. The partitioning of two phase system typically depends
on the diffusion coefficient. Recently, the microfluidic technology
has emerged as a promising tool which offers a new approach
of integration and miniaturization to address issues associated
with traditional APTS. The microfluidic devices contain a num-
ber of co-flowing streams of immiscible phases guided through a
microchannel [172,173]. The length of the diffusion path is adjusted
by geometric constraints or hydrodynamic means which speeds
up the mass transfer. Two-phase flow of ATPS in microfluidic
chips allows the low interfacial tension resulting in high capil-
lary number even at low flow rates, making it straightforward to
945
establish a stable interface without surfactants or special surface
treatment. In recent years, the parallel laminar flow of ATPS in
microfluidic devices is widely studied, serving as an ideal plat-
form for rapidly extracting and separating biomolecules [174,175].
Compared with conventional batch processes, microfluidic proto-
cols offer a number of advantages. Microfluidic systems can easily
manage microliters to picoliters of fluid volumes, which reduce
sample volumes and reagent costs. Also, short diffusion distances
of microfluidic systems translate into faster and more efficient sep-
aration without further need of mixing [176,177]. Moreover, two
phases are easily recovered from different exit branches by using
designed channel architectures, whereas in the standard batch
processes, the phases are separated in a settler which usually con-
sumes a significant amount of time. Further, separation processes
by microfluidic device can allow real time monitoring of the status
of a separation process by using optical detection techniques and
performing multidimensional separations on the basis of affinity
partitioning in ATPS [178].
In the past few years, a number of groups around the globe have
discovered the opportunities related to ATPS in microfluidic devices
for enzyme separation. Novak et al. used a three-stream ATPS with
PEG in the outer stream and potassium phosphate in the middle
stream to extract the enzyme ␣-amylase. They demonstrated a sig-
nificant improvement in separation time. The microfluidic system
performed extraction within 10 s, with extraction efficiency (i.e.
the difference in concentration between the inlet and outlet of the
microchannel of enzyme) of 52%. On the other hand, conventional
macro-scale ATPS (composed of PEG 4000 and potassium phos-
phate) extracted ␣-amylase with an extraction efficiency of 74% in
2.5 h. Further, the researchers have claimed that during continuous
parallel flow extraction processing, simultaneous phase separation
occurred at the exit of the microchannel system, which resulted
in shortening of the process time as compared to the conventional
batch extraction [179]. In another study, Hahn et al. separated car-
bonic anhydrase and beta-galactosidase using a microfluidic ATPS.
The dextran phase containing enzyme was sandwiched between
two PEG phases and electric field was applied perpendicular to
the interface in a channel. They demonstrated that separation of
protein can be achieved through differences in electrophoretic
mobility and/or partition coefficients in microfluidic ATPS [180].
Undoubtedly, ATPS combined with microfluidic system can
provide a platform for continuous separation/purification of
macromolecules like proteins/enzymes. However, in order to
achieve higher extraction efficiency by using microfluidic sys-
tem, it is necessary to screen different combinations of number
of inlet/outlet of the device to generate stable vertical interface
between the two phases, enabling better partitioning of desired
biomolecules. Also, integration of multi-dimensional separations
(i.e. electrochemical potentials) with ATPS along or across the
microchannel will improve or shift the selectivity in microfluidic
ATPS [172].
6. Three phase partitioning
In recent years, three phase partitioning (TPP) has emerged
as a simple, rapid, efficient, modest, inexpensive and integrated
bioseparation technique for recovery, purification and enrichment
of biomolecules such as enzymes from complex mixture [181]. It is
easily scalable and can be employed directly with crude slurry and
is performed at room temperature. It does not involve any addi-
tion of polymer in the system to be removed later [96,182,183].
It consists of the sequential addition of a sufficient amount of
salt (ammonium sulphate, potassium phosphate, and sodium cit-
rate etc.) and an organic solvent (t-butanol, 2-butanol, 1-propanol,
and 2-propanol etc.) in the crude extract. The reaction mixture
946 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957

Table 8
a Extraction of enzymes from fermentation broth by using three phase partitioning (TPP).

Enzyme Source Salt Organic solvent pH Purification factor Ref.

a-Galactosidase and invertase Aspergillus oryzae Ammonium tert-butanol 4.3 15 and 12-fold respectively [206]
sulphate
Serratiopeptidase Serratia marcescens Ammonium tert-butanol 7 9.4-fold [191]
sulphate
Laccase Coriolopsis trogii Ammonium tert-butanol 5 15.6-fold [213]
sulphate
Fibrinolytic enzyme Bacillus sphaericus MTCC 3672. Ammonium tert-butanol 9 16.15-fold [220]
sulphate
Aryl Alcohol Oxidase Pleurotus ostreatus Ammonium tert-butanol 6 10.19-fold [185]
sulphate
Laccase Pleurotus ostreatus Ammonium tert-butanol – 7.22-fold [279]
sulphate
Exo-inulinase Aspergillus niger Ammonium tert-butanol 4 10.2-fold [212]
sulphate
L-asparaginase Capsicum annuum L. Ammonium tert-butanol 8.5 6.83-fold [280]
sulphate
Naringinase Aspergillus brasiliensis Ammonium tert-butanol 5.5 4.4-fold [209]
sulphate
Pecinase Aspergillus niger Ammonium tert-butanol – 10-fold [199]
sulphate
␤-galactosidase Lactobacillus acidophilus Ammonium tert-butanol 7 7.5-fold [281]
sulphate
Laccase Ganoderma sp. WR-1 Ammonium tert-butanol 7 13.2-fold [282]
sulphate
Invertase Saccharomyces cerevisiae Ammonium tert-butanol 4 15-fold [204]
sulphate

Table 8b Extraction of enzyme from natural sources by using three phase partitioning (TPP).
Enzyme Source Salt Organic solvent pH Purification factor Ref.

␣-Galactosidase Citrullus vulgaris (watermelon) Ammonium tert-butanol 5.5 2.7-fold [211]

sulphate
Ipomoea peroxidase Ipomoea palmate (leaves) Ammonium tert-butanol – 18-fold [200]
sulphate
Proteases Papaya peels Ammonium tert-butanol 7 10.1-fold [208]
sulphate
Invertase Solanum tuberosum (Potato tubers) Ammonium tert-butanol – 5.67-fold [283]
sulphate
Polyphenol oxidase Malus domestica (Fuji apple) Ammonium tert-butanol – 54.41-fold [284]
sulphate
Pectinase Lycopersicon esculentum (Tomato) Ammonium tert-butanol 6.5 9-fold [199]
sulphate
␣-Galactosidase Solanum muricatum (Pepino) Ammonium tert-butanol 5.5 6.2-fold [192]
sulphate
␣-Galactosidase Lycopersicon esculentum (Tomato) Ammonium tert-butanol 4 4.3-fold [205]
sulphate
␤-Galactosidase Cicer arietinum (Chick pea) Ammonium tert-butanol 6.8 10.1-fold [195]
sulphate
Peroxidase Citrus sinenses (orange peels) Ammonium tert-butanol 6 18.20-fold [190]
sulphate
Catalase Solanum tuberosum (Sweet Potato) Ammonium tert-butanol 7 14.1-fold [203]
sulphate
Invertase Lycopersicon esculentum (tomato) Ammonium tert-butanol 4.5 8.6-fold [197]
sulphate

was mixed well and decantation was carried out. The mixture
separates into three distinct phases: a t-butanol rich upper layer
and an aqueous lower layer which are formed [184]. The third
phase is formed by a protein-enriched in between the polar and
non-polar solvent. During the separation, some small molecular
weight and non-polar compound impurities (such as lipids, phe-
nolics and some detergents lipids and pigments) are accumulated
in upper t-butanol phase while polar compound impurities (like
protein contaminant, saccharides and cell debris) get concentrated
in the lower aqueous phase (Fig. 6) [96,185]. The probable reason
behind this protein isolation is that organic solvent binds to the
precipitated enzymes, thereby increasing their buoyancy and caus-
ing them to float above the denser aqueous salt layer and forms
the third phase (interphase). Additionally, combination of salting
out, isotonic precipitation, co-solvent precipitation, osmolytic and
kosmotropic cause enzyme hydration shifts which have been pos-
tulated as key contributors for the partitioning of the target enzyme
[185,186]. The TPP process is affected by physiological parameters
like temperature, pH salt type, co-solvent and their concentration.
The excellent results can be obtained by streaming these parame-
ters. Various enzymes were extracted from fermentation broth as
well as from natural sources which are summarized in Table 8a and
8b. Gu et al. showed the effectiveness of TPP for the extraction of
␣-galactosidase from Aspergillus niger by comparing it with ATPS
(which are considered as efficient and economical strategies). For
ATPS comprising PEG/phosphate, highest purification factor upto
3.25-fold with 84.41% activity recovery were obtained. On the other
hand, in TPP system made up of ammonium sulphate and t-butanol
was exhbited excellent purification (6.27-fold) with 97.21% activity
recovery [187].
The solubility of proteins/enzymes is mainly dependent on the
effect of ionic strength of the solution [188]. Hence, it is essential to
 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957
Fig. 6. Schematic diagram illustrating the partitioning of enzyme and (polar and
non-polar) contaminant impurities in three phase partitioning.
observe the effect of various salts and salt concentration on parti-
tioning of enzyme in TPP system. Salts such as ammonium sulphate,
magnesium sulphate, and sodium sulphate have been mostly used
in TPP process [189]. At lower salt concentration it may not able
to alter the hydrophobic surface of enzyme leading to insufficient
precipitation of desired enzyme. On the other hand, at higher salt
concentration, water molecules are attracted by salt ions result-
ing in enhanced protein–protein interactions and the coagulation
of protein molecules through hydrophobic interactions [190]. Sul-
phate ions being early member of the hofmeister series interact
well with water, forming hydrogen bonds and dehydrating pro-
teins [96]. Ammonium sulphate showed the maximum yield and
recovery in most of the cases of TPP. This could be due to ammo-
nium sulphate, as it has a high hydration capacity and affinity for
water compared to other salts. Large proportion of water molecules
are involved in hydration of sulphate ions thereby increasing their
effective radius and acting as pushing kosmotrope through its sig-
nificant hydration. It also promotes protein stability by favouring
hydrophobic interactions [184]. In addition to that, various other
researchers have reported that, the salting out effect in TPP is asso-
ciated with kosmotropy, ionic strength effects and the binding of
sulphate to cationic sites of a protein [191,192]. Additionally, Rao
et al. claimed that large sulphate ions crowd together in aqueous
phase and alter the solubility of protein in bottom phase, finally
pushing it into the organic phase due to increase in the interfa-
cial tension. Higher interfacial tension generates a greater polarity
difference and a higher density difference between both phases
in the salt system [193]. However, with an increase in concentra-
tion of ammonium sulphate, the degree of purification factor and
activity recovery decreased significantly [194,195]. The ammonium
sulphate saturation (40–60%) resulted in the 16.2% yield of nattok-
inase from Bacillus natto NRRL-3666 with a 1.46-fold purification.
Further increase in ammonium sulphate saturation (80–95%) dras-
tically reduced the yield (0.9%) and purity (0.23-fold) [196]. Apart
from ammonium sulphate salt, various researcher used different
type of salts in TPP. The salts such as sodium citrate, potassium
phosphate were used to get the maximum recovery of alkaline pro-
teases from farmed giant catfish viscera. Sodium citrate provided
the highest recovery (142%) with purification (4.65-fold) followed
by ammonium sulphate and potassium phosphate. In some cases,
It has been often observed that three-phase partitioning leads to
simultaneous activation of the enzyme molecules which (if the
enzyme recovery is high) resultsinto an apparently observed higher
yield (more than 250%) [189]. During TPP, enzymes were undergo
947
not only the overall conformation changes but also alter the sec-
ondary structure content of the enzyme resulted into increased
flexibility in activated enzyme molecule [197,198]. A remarkable
increase in the catalytic activity and yield of pectinase, peroxidase,
proteinase K and trypsin inhibitor obtained from TPP have been
reported [199–202]. Hence, it is necessary to select appropriate salt
and its concentration during enzyme extraction by TPP system.
Another important parameter is pH value of partitioning
medium and isoelectric point (pI) of target protein which directly
influences the recovery and purification factor of proteins. Distri-
bution and partitioning of biomolecules in TPP systems alternated
with pH due to electrostatic interactions between charged pro-
tein (enzyme) and phases [203]. Electrostatic forces and binding of
anionic salts (i.e. SO4 2− , HPO4 2− , and C3 H5 O(COO)3 3− ) to cationic
protein molecules, which promote conformational shrinkage and
macromolecular contraction, are the main causes of the strong salt
anion-pH dependency in salting out [189,204]. Proteins tend to pre-
cipitate most readily at their pI. When system pH is above the p I of
protein, it will attain net negative charge and would be propelled
to the bottom aqueous phase. On the other hand, if system pH for
the TPP process is lower than the pI of the protein, enzymes get
precipitated or accumulated between the two phases in TPP. This
might be due to the electrostatic component of the reactions when
anion/cation of the salt binding to oppositely charged proteins
leading to the partition of most contaminant proteins into aque-
ous bottom phase above isoelectric point. Akardere et al. purified
invertase from Saccharomyces cerevisiae upto 15-fold purity with
363% activity recovery at pH 4.5 while at pH 5.0 provided 8.5-fold
purification with 300% activity recovery [204]. Similarly, Çalci et al.
reported that at pH 4.5, ␣-galactosidase from tomato was extracted
upto 4.3-fold purification with 80% yield in the system composed
of ammonium sulphate and t-butanol which was increased by two
folds at pH 6.0 [205]. Dhananjay et al. reported the extraction of
␣-galactosidase and invertase from Aspergillus oryzae which were
accumulated in a coherent middle phase in a TPP system under dif-
ferent pH conditions. The pH of the TPP system was adjusted at 4.3
for ␣-galactosidase to get 50% activity recovery with 15-fold purity
whereas invertase were extracted at 5.0 pH with 12-fold purity and
54% activity recovery [206].
In TPP system, the solvent plays a vital role in the formation of
phases. Mostly tertiary butanol (t-butanol) is employed as organic
solvent as it acts as a kosmotrope in synergism with salt kos-
motropy [200]. The phase formation so far dependent on t-butanol
and salt, a C4 alcohol which is a differentiating solvent and ammo-
nium sulphate as effective kosmotropic agent. t-Butanol binds to
the precipitated proteins, thereby increasing their buoyancy and
causing the precipitates to float above the denser aqueous salt
layer. t-Butanol appears to be kosmotropic and crowding agent
at room temperature, resulting in “enzyme–t-butanol coprecipi-
tate” which float above denser aqueous salt layer [203,207]. It is
believed that because of its size and branched structure, t-butanol
does not easily penetrate interior to the enzyme molecules and
hence does not cause denaturation [184]. t-Butanol is not univer-
sal solvent to be always used for TPP, other C3 , C4 and co-solvents
may also serve because they too act as a differentiating co-solvents
[208,209]. Ketnawa et al. used t-butanol, 1-butanol, 1-propanol and
2-propanol to purify alkaline proteases derived from fish viscera.
2-propanol provided higher purification as compared to other sol-
vents, whereas, t-butanol provided higher activity recovery for the
same proteases [189]. Similarly, in the extraction of ␣-galactosidase
from fermented media of A. oryzae, t-butanol gave the 12-fold
purification factor and 92% activity recovery while 1-butanol, 2-
propanol and 1-propanol gave 69, 35 and 40% recovery, respectively
[188]. In this TPP based protocol for enzyme partitioning, t-butanol
has been found to consistently perform better than all other organic
solvents. The concentration of t-butanol need to be optimum to get
948 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957
synergic kosmotrope effect with salt. This is because higher solvent
quantities attract more water from aqueous phase which leads to
increased salt concentration in aqueous phase and precipitation of
enzyme at interface [210]. Besides, t-butanol can be recycled by
easy evaporation at 82.4 ◦ C (boiling point) [96].
Apart from pH, temperature is an important operation param-
eter affecting the enzyme configuration and overall stability [124].
The use of low temperatures in solvent or salt precipitation
prevents the heat generation, thus ensures minimal protein denat-
uration [211,212]. At lower temperature (4 ◦ C) the recovery was
same as at the ambient temperature which might be due to the
low movement of particle solvent at lower temperature. However
at 25 ◦ C, the conformation of enzyme changed into highly active
form.While, enzyme activity recovery decreased at higher temper-
ature (131% activity recovery at 45 ◦ C) in comparison to ambient
temperature (154%, 31 ◦ C) but considerably increase in the purifi-
cation fold of the enzyme in the precipitate. This may be due to the
synergistic effects of the phase components along with tempera-
ture, which decreases the selectivity of extraction, thus reducing
the yield as well as the degree of purification [213].
The partitioning of enzyme is always restricted due to mass
transfer driven phenomenon and any improvement in mass trans-
fer leads to enhanced partitioning and subsequent purity of the
target enzyme [214]. Hence, ultrasound is employed as the pro-
cess intensification tool in the extraction of enzymes in recent
times [215]. An ultrasonic wave leads to creation of small rapidly
growing bubbles; collapse of such bubbles is accompanied by cre-
ating shock waves that cause strong shear formation of gas filled
bubbles [216]. The subsequent collapse of these bubbles imparts
shock wave and mechanical shear to the surrounding environ-
ment. The change in local energy densities and mechanical shears
incurred due to cavitational phenomenon accelerates the mass
transfer across the different phases [217]. In ultrasound assisted
TPP (UATPP), ultrasound power, duty cycle and irradiation time
are critical operating parameters to be considered while extrac-
tion of biomolecules [218,219]. Avhad et al. extracted fibrinolytic
enzyme from Bacillus sphaericus by using ultrasound coupled TPP
system. UATPP showed increase in purity and activity recovery of
fibrinolytic enzyme by 7-fold and 4-fold respectively, as compared
to conventional TPP [220]. Pakhale et al. purified serratiopeptidase
from Serratia marcescens NRRL B 23112 with UATPP. The process
time for UATPP was significantly reduced to 5 min from 60 min
as compared to conventional TPP. The overall results showed that
the UATPP is an effective technique for the purification of serra-
tiopeptidase with maximum purity, activity recovery and reduced
processing time [191]. These stories of success highlight the advan-
tage of ultrasound technique with TPP for the establishment of easy
and simple processes to implement which is attractive from an
economic viewpoint.
7. Simultaneous purification and immobilization strategies
Apart from the inherent advantages of enzyme, the industrial
applicability of enzyme is always obstructed due to insufficient
stability in operational conditions, difficulties in recovery from
reaction mixture and reusability for multiple cycles [6,221,222].
Also, the use of commercial enzymes in soluble form not only
leads to the waste but also it contaminates the product, ultimately
increasing the purification cost of desired product. These hur-
dles can be prevailed by the enzyme immobilization tools [223].
The immobilization greatly enhances the properties of enzymes
in terms of thermal stability, tolerance to extremely high pH and
organic solvents, selectivity and activity to meet the demands of
practical uses. It also facilitates the efficient recovery and re-use of
the enzyme, thus enabling its cost-effective use and thereby mak-
ing their industrial applications economically feasible [224]. One of
the effective means to decrease the cost of an enzyme is the reduc-
tion of separation and purification cost, which can be achieved by
either reducing the number of purification steps and/or increasing
recovery rates or developing simultaneous purification and immo-
bilization [225]. There are few strategies in which the enzymes are
purified and immobilized parallelly. It not only reduces the steps
of biocatalyst preparation but also makes application of biocatalyst
economically feasible [226–228].
From past decades, cross linked enzyme aggregates (CLEAs)
have emerged as a versatile and novel carrier free immobilization
technique [229]. The enzyme immobilization by CLEAs involves
two steps: precipitation of an enzyme from aqueous solution fol-
lowed by cross linking with a bi-functional reagent [230]. In the first
step the soluble enzyme is aggregated by the addition of precipi-
tating agents such as salts (ammonium sulphate), water-miscible
organic solvents (methanol, ethanol, propanol, DMSO, etc.) and
non-ionic polymers (PEG) to an aqueous solution of enzyme. In
the aggregate, the enzyme molecules are held together by non-
covalent bonding without perturbation of their tertiary structure,
i.e. without denaturation. In the second step, the formed aggregates
are chemically cross linked to each other by a bifunctional reagent
(mostly glutaraldehyde) via the reaction of amino groups of Lys
residues on the external surface of the enzyme (Fig. 7) [231,232].
After chemical cross linking, the aggregates become permanently
locked into insoluble pre-organized superstructure with the reten-
tion of their catalytic activity [233,234]. Immobilization using the
CLEA technique combines purification and immobilization into sin-
gle unit operation. Therefore it is possible to isolate an enzyme in
its immobilized form directly from a fermentation broth or any nat-
ural sources [229,230]. During the CLEAs preparation, the effects of
precipitating agent, concentration, cross-linkers concentration and
cross-linking time are need to be studied to get highly active, robust
and stable biocatalyst [235]. Yusof et al. prepared CLEAs of seven
hydrolase enzymes (amylase, mannanase, glucosidase, glucanase,
protease, lipase and fructosyltransferase) obtained from cocoa pod
husk [236]. Fazary et al. prepared cross-linked feruloyl esterase
aggregate (CLEA) from Aspergillus awamori which were thermally
stable in the range of 25–85 ◦ C as compared to native form [237].
Most of the cases, glutaraldehyde has been used as a cross-linker
in the preparation of CLEAs. However, it can’t be employed for all
enzymes, especially sensitive to it. The small size of glutaraldehyde
cross-linker gets easily penetrated into the enzyme active site and
reacting with catalytically active amino acids residues leading to
enzyme inactivation. Moreover, cross linking of enzyme aggregates
by glutaraldehyde forms compact and supramolecular structures
hindering the active sites of enzyme [238,239]. To overcome these,
Nadar et al. prepared CLEAs by precipitating crude enzyme and
subsequently cross-linked them by biocompatible, biodegradable,
non-toxic, renewable and macromolecular polysaccharide based
cross-linker viz. agar, chitosan, dextran, pectin and gum arabic to
get highly active CLEAs [240,241]. However, the CLEAs preparation
is inadequate for all enzymes since crosslinking largely requires
amino groups in the form of Lys residues on the external surface of
the enzyme. Hence, it may lead to low mechanical stability or even
releasing enzyme into the reaction media during reaction [242].
Recently, another approach was used to prepare CLEAs in pres-
ence of magnetic nanoparticle called as a magnetic CLEAs. It
was simply prepared by addition of amino functionalized mag-
netite nanoparticles as an additive into enzyme (with low Lys
residue content) solution, which further precipitate enzyme to
form aggregates and then cross-linking of enzyme aggregates and
nanoparticles [243]. The prepared magnetic CLEAs showed high
thermal, mechanical stable and non-leachable CLEAs due to suf-
ficient cross linking of enzyme aggregates [244]. These magnetic
nanoparticles not only provide the large surface area for enzyme
 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957
Fig. 7. Schematic diagram illustrating preparation of cross-linked enzyme aggregates.
anchoring but also provide higher operational stability. Addition-
ally, magnetic nanoparticles help to ease recovery from reaction
mixture by using the external magnetic field, instead of using tradi-
tional filtration and centrifugation [245,246]. Talekar et al. prepared
the magnetic CLEAs of crude alpha amylase which retained almost
100% residual activity even after six cycles of reusability in batch
mode [242]. In addition to that, CLEAs have also been cross-linked
to magnetic mesoporous silica particles and it maintained its initial
activity even after 35 days of storage [247].
Recently, construction of organic–inorganic hybrid material is
a rapidly expanding field of material chemistry for its advanced
designs with a specific structure and functionality [248,249]. When
crude enzyme is mixed with metal ion solution, it binds specifically
with enzyme which initiate synthesis of hybrid nanoflowers of that
enzyme. This would result in simultaneous purification and immo-
bilization of enzyme into hybrid organic–inorganic nanoflowers.
The binding of a ligand to one site (which we designate as the
allosteric site) on a protein molecule indirectly manipulates the
properties of another specific site (the functional site and in
enzymes called the active site) on the same enzyme as a con-
sequence of conformational changes. Organic–inorganic hybrid
nanoflowers were prepared by using copper ions as the inor-
ganic component and enzyme as the organic component (Fig. 8).
Recently, in the work of Zare et al. a simple approach was devel-
oped for fabrication of enzyme–inorganic hybrid nanoflowers by
using HRP as the organic component and Cu3 (PO4 )2 ·3H2 O as the
inorganic component [250]. In a typical experiment, the aqueous
CuSO4 solution was added to phosphate buffered saline (PBS) con-
taining enzyme. The copper phosphate nano-crystals and enzymes
were self-assembled to form the high surface area hierarchical
nanoflowers embedded with enzymes [251]. Nadar et al. synthe-
sized hybrid glucoamylase nanoflowers which exhibited excellent
reusability and reproducibility in cycle analysis [252]. In another
study, lactoperoxidase was purified from bovine milk and simul-
taneously immobilized by copper metal ions (Cu2+ ) via a greener
approach to form nanoflowers. This resulted in enhancement in
catalytic activity and stability [226].
In a nutshell, the advantages provide by CLEAs technology and
hybrid nanoflowers, the simultaneous purification and immobiliza-
tion strategies exhibit a great potential in industrial application,
following greener approach.
8. Future perspectives and challenges
To fulfil the demand of a growing market of industrial enzymes,
the development of new and innovative downstream methods for
purification of catalytically active enzymes is of great importance.
The main evaluation between these techniques determined their
efficiency in terms of purity and yield for a specific product, and the
economic aspects of each. The well-established chromatographic
949
techniques applied for the selective recovery and purification of
biomolecules could possibly hinder ATPS application at industrial
levels. The development and application of ATPS is limited due
to two major restrictions. Firstly, the limited predictive process
design due to the poor understanding and lack of the knowledge of
the responsible mechanisms for the behaviour of enzymes in ATPS
and interactions solute–solvent between target product and con-
taminants. Secondly, monitoring the characteristics of proteins is
the basic requirement for assessment of bioprocesses, which are
affected frequently by the presence of different polymers or salts.
Considerable time is needed to build first recovery process of the
experiment, while big budget is needed for installation and limited
output of the purification units. Compared with a novel alcohol/salt
ATPS, polymer/polymer, polymer/salt and surfactant/alcohol ATPS
systems have some disadvantages such as the high cost, viscos-
ity, ionic strength as well as slow segregation (Table 9). There are
difficulties in isolating the extracted enzymes from the polymer
phase as well as from micellar phase by re-extraction and envi-
ronmental pollution resulting from the recycling of phase-forming
polymers. Hence, the ATPS is expected to overcome the drawbacks
and challenges by rapid evolution theoretically and experimentally.
The modification and improvement in ATPSs such as free
affinity ligands or using modified or activated copolymers are
more effective due to the high efficiency and selective separa-
tion and purification which ultimately increasing recovery yields
of biomolecules. The novel type of ATPS combined with other tech-
niques such as chromatography which might help intensifying the
capability of existing ATPS for recovering biocatalyst at commercial
scale. Also, the use of 3-D mapping for analysing protein pro-
files allows the identification of the molecular properties from the
main protein contaminants. This information will facilitate estab-
lishment of concentration and specific purification conditions for
enzymes. Further, experimental design can be used as a strategy
to screen and optimise the purification process conditions, allow-
ing a fast assessment of the different experimental parameters
involved as well as their possible interactions in the optimal ATPS
system for partitioning of desired biocatalyst. In the last few years,
some studies focused on the development of excellent polymers
forming ATPS for higher recovering. The development of smart
polymers which by responding to a change in temperature (PNB ) or
pH (PADB ) or light (PNBC ) are able to change conformation, thereby
allowing the recovery of enzyme of interest along with the poly-
mer in opposite phases. Another breakthrough could arise from
the integration of functional magnetic particles into ATPS, which
could speed up phase separation and enhance process throughput.
Extractive fermentation and purification is one of the emerging
continues extraction strategies containing in situ product recov-
ery and simultaneously performed fermentation, separation and
purification of enzymes instantaneously. Moreover, the simulta-
950 S.S. Nadar et al. / International Journal of Biological Macromolecules 101 (2017) 931–957

Fig. 8. Schematic diagram illustrating preparation of organic-inorganic hybrid nanoflowers.

Table 9
comparison of different types of ATPS.

Type Advantages Disadvantages

Polymer/polymer ATPS Easily amenable and modifiable High viscosity

Polymer/salt ATPS Lower viscosity and less time consuming High ionic strength
Alcohol/salt ATPS Low cost, lower viscosity, less time consuming Denaturation of labile enzyme, high volatility
and recycling of alcohol of alcohol
Surfactant/alcohol ATPS Recycling of alcohol, low viscosity Denaturation of labile enzyme
Aqueous affinity two phase system High resolution technique, high selectivity Time consuming, relative complexity within
activation process
Ionic liquid based ATPS Low viscosity, Non-flammability, strong High cost
solubilizing power, high thermal stability
Thermo separating polymer based ATPS Simplicity of product recovery, cheap chemical Denaturation of heat labile enzyme
cost and environmental friendliness, easy
recycling of polymers
Aqueous micellar two phase system High selectivity and recycling of surfactant High cost and not compatible to thermo
sensitive enzyme
Aqueous two-phase flotation High concentration coefficient, soft separation, Denaturation of heat labile and sheer sensitive
low dosage of organic solvent, simple enzyme
operation and low environmental impact

neous immobilization and purification of enzymes make it a great CLEAs and organic–inorganic nanoflowers preparation are of high
impact on down streaming and various industrial applications. potential.

9. Conclusion
An efficient extraction/purification technology needs to offer
several benefits includes less time-consuming operations, lower
consumption of energy and resources and lower labour costs. Over
the last decades, ATPS has been reported as an attractive alterna-
tive or platform in downstream processing that can be adopted by
industries due to its high capacity and easy scalability. This review
presented parameters that contribute to the establishment of effi-
cient ATPS processes to get highly purified enzyme in active forms.
Also, the modifications of ATPS and process integration for aug-
menting recovery and purification of enzymes were highlighted.
Although, the potential of ATPS for the recovery of catalytically
active enzyme has been proven, the technique has not been widely
exploited at a commercial scale. Another attempt has been made
to develop a simple, more efficient and economical method for
separation and purification of enzyme molecules by three-phase
partitioning. The rapid, economic and single pot purification meth-
ods have good potential to purify enzymes with high catalytic
activity. An effective means to decrease the enzyme cost is reduc-
tion of its separation and purification cost, which can be achieved
by integrated purification and immobilization techniques. So as
discussed in this review article, there are few strategies such as
Acknowledgement
The authors would like to acknowledge the University Grants
Commission (UGC) of India for financial assistance to the research
work.
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