Ref.

Ares(2019)868281 - 13/02/2019

D4.3 A downstream processing protocol of enzymes

purification
Algae4A-B - Algae for Aquaculture and Beauty
Development of microalgae-based novel high added-value products for the cosmetic and aquaculture industry
Project nr: 691102 Call reference: H2020-MSCA-RISE-15
st
Start date: 1 January 2016 Duration: 48 months

Deliverable identification
Leading beneficiary: AUA
Related WP: 4 Planned delivery date: M36
Related period: Actual delivery date: M38
Dissemination level: PU Delivery status: Submitted

Contributors:
Beneficiary Name(s)

Agricultural University of Athens Nikolaos Labrou, Evaggelia Chronopoulou

Fitoplancton Marino, S.L. Carlos Unamunzaga

Reviews
Version Reviewer Date
v1 Nikolaos Labrou 06/02/2019

v2 Carlos Unamunzaga 07/02/2019

V3 William Helbert 13/02/2019

 PUBLIC

Table of content
1.  Context and objectives ............................................................................................................................... 2 
2.  Description of the performed tasks and obtained results ........................................................................... 2 
3.  Conclusion .................................................................................................................................................. 6 
4.  References ................................................................................................................................................. 6 

1. Context and objectives

Downstream processing is the recovery and purification of biological products. It is an essential step in the
manufacture of enzymes and involves several processing steps of the materials collected during the
upstream stage to transform it into a finished product. This step tends to be one of the most expensive
stages of modern bioprocessing (Labrou, 2014). The downstream processing of enzymes requires
purification techniques that are friendly and soft enough to preserve their biological activity (Platis and
Labrou, 2009; Labrou, 2014; Maltezos et al., 2014;). Current purification techniques for enzymes involve a
series of low-efficiency purification steps, employing several chromatographic steps (ion-exchange, gel-
filtration etc). However, the introduction of multiple processing steps results in higher product loss and
product inactivation. On the other hand, alternative, non-chromatographic purification technologies, such as
aqueous two-phase partition systems (ATPS), aim at high throughput and seek to circumvent problems
associated with diffusion limitations experienced with most chromatographic methods, can handle large
volumes, are easily scalable and are also able to hold high biomass load in comparison to other separation
techniques (Platis and Labrou, 2009; Labrou, 2014; Maltezos et al., 2014). ATPS have been widely used as
a mild separation method in biochemistry, cell biology and biotechnology. An ATPS is formed when two
water-soluble polymers, such as a polymer and a salt are dissolved in water beyond a critical concentration
at which two immiscible phases form.
The objective of the work was the development of a downstream processing protocol for enzymes
purification. Protein isolation and purification from algae is a difficult task, owing to the complexity of the
microalgae system and the presence of polysaccharides, pigments, chlorophylls, etc. To characterize
selected enzyme activities, the initial downstream processing strategy was based on chromatographic and
non-chromatographic methods. Furthermore, a detailed study was performed to analyse the influence of
several parameters (pH, ionic composition, types of polymers, their molecular weight and concentration) on
ATPS performance and selectivity for the target enzyme purification.

2. Description of the performed tasks and obtained results

Two different anion-exchange chromatographic matrixes were used: DEAD-Sepharose and CM-Sepharose.
Optimization of chromatographic conditions (buffer, pH) was achieved and most appropriate conditions were
selected. The chromatographic procedure was as follows: microalgae lyophilized cells were resuspended in
potassium phosphate buffer (0.02 M potassium phosphate buffer, pH 6.5), sonicated, and centrifuged at
13000 g for 10 min. The supernatant was collected and dialysed (4 ºC) against 1000-volumes of potassium
phosphate buffer, pH 6.5 containing 1 mM EDTA. The dialyzed cell-free extract was applied to the ion-
exchanger previously equilibrated with 0.02 M potassium phosphate buffer, pH 6.5. Non-adsorbed protein
was washed off with equilibration buffer. Proteins were eluted with a step-wish gradient of 50-300 mM KCI in
the equilibration buffer. Collected fractions were finally assayed for enzyme activity and total protein content.
The performance of the chromatographic steps were judged adequate, mainly, due to high cost and low self-
life/fulling of the chromatographic materials.
Non-chromatographic methods were also employed to provide initial protein purification. These methods
included precipitation/fractionation using neutral salt such as ammonium sulphate and acid precipitation
employing citric acid. The concentration at which soluble protein precipitates varies among different
microalgae species and therefore detailed study was performed to analyse the effect of concentration
(ammonium sulphate and citric acid), pH, temperature and ionic composition of the system on the
precipitation of total protein and antioxidant enzymes. The appropriate number of precipitation steps
depends on the protein(s) of interest and must be determined empirically.

Page 2 of 6 D4.3 A downstream processing protocol of enzymes purification 13/02/2019

 PUBLIC

Fig. 1. Fractionation of total algae protein using ammonium sulphate precipitation steps.

The results showed that performing two ammonium sulphate precipitation steps lead to moderate protein
recovery (Fig. 1). The procedure involved the addition of the saturated ammonium sulphate solution to raise
the percent saturation of the microalgae sample solution initially from 0% to 40% and then in the second
step, from 40% to 70%. Moreover, two acid precipitation steps were performed, involving the addition of
concentrated citric acid to reduce the pH of the microalgae sample solution initially from pH 7 to 5.5 and then
in the second step, from 5.5 to 4. At each step of the purification procedure, the precipitate was analysed to
assess the degree of enrichment and the recovery of the protein of interest. The acid and ammonium
sulphate fractionation techniques enable several fold enrichment of the protein in crude extract.
In parallel, the objective of the work has been the development and evaluation of a purification method for
protease activity from Nannochloropsis gaditana extracts based on aqueous-two phase system (ATPS).
Proteases are being explored as components in cosmetics formulas as skin exfoliation agents since they can
soften the outer layer of skin by cleaving peptide bonds in proteins. Previous data demonstrated that
Nannochloropsis exhibits high protease activity. ATPS is a high-water content (>70 % (w/w) water) simple,
low cost and environmental friendly fractionation method. Protease activity was extracted by partitioning in
ATPS composed by phosphate (Pi) and polyethylene glycol (PEG) (Fig. 2). Stopped spectrophotometric
measurements were used for measuring protease activity in microalgae extracts using as substrate azo-
gelatine and azo-casein. Microalgae extracts were first prepared as follows: freeze-dried N. gaditana
biomass from FITMAR, was resuspended in phosphate buffer in a proportion 0.1 g biomass/mL buffer. Cell
lysis was performed using ultrasonics (4 cycles of 30 s each, 20% amplification), followed by centrifugation
for 15 min at 16,000 g, 4 ºC. Supernatants were then recovered and were subjected to ATPS. Several
factors (Fig. 3) that affect the partition behaviour of protease activity were investigated, such as molecular
weight of PEG, proportion of PEG and Pi in the system, the presence of a metal ion (Mn+2 or Zn+2) and the
effect of protein concentration. The standard method consisted of a 10 g system containing 2% of water, and
thus 1% of microalgae extract had to be included. After 1 h of incubation at 22 ºC under mild rotation, the
samples were centrifuged for 10 min at 2000 g to promote phase separation. Next, the upper PEG phase
and the bottom Pi phase were removed and placed on ice for subsequent determination of total protein
concentration using the Bradford method and protease activity measurements (Fig. 4, 5, 6). The effect of
three different ammonium sulphate concentrations was evaluated in terms of protein and protease recovery
in both phases. Moreover, protein partition between phases was measured in relation to initial protein input.
SDS-PAGE analysis and protease activity measurements were used for evaluating purity and recovery. The
method could be used in future studies for the purification of other novel proteases from additional
microalgae species.

Fig. 2. Aqueous Two Phase System (ATPS) of Nannochloropsis extract for protease purification.

Page 3 of 6 D4.3 A downstream processing protocol of enzymes purification 13/02/2019

 PUBLIC

Fig. 3. Aqueous Two Phase System (ATPS) of Nannochloropsis extract for protease purification

Fig. 4. Performance of ATPS used for protease purification from Nannochloropsis extracts.

Fig 5. ATPS protease purification from Nannochloropsis extracts in the presence of a metal ion (Mn+2 or Zn+2), using
azocasein as substrate. Where Me: the performance of ATPS in the absence of metal ion.

Fig 6. ATPS protease purification from Nanochloropsis extracts in the presence of a metal ion (Mn+2 or Zn+2), using
azogelatin as substrate. Where Me: the performance of ATPS in the absence of metal ion.

Page 4 of 6 D4.3 A downstream processing protocol of enzymes purification 13/02/2019

 PUBLIC

The ATPS system that was developed was also applied and assessed for the separation superoxide
dismutase (SOD) (Fig 7). SOD is one of the most important barriers to protect cells against oxidative stress,
and thus there is a great interest on it by the cosmetic industry. Several factors (Fig. 8) that affect the
partition behaviour of SOD (Fig. 9) were investigated, such as molecular weight of PEG, proportion of PEG
and Pi in the system and the effect of protein concentration. The standard method consisted of a 10 g
system containing 2% of water, and thus 1% of microalgae extract had to be included. After 1 h of incubation
at 22 ºC under mild rotation, the samples were centrifuged for 10 min at 2000 g to promote phase
separation. Next, the upper PEG phase and the bottom Pi phase were removed and placed on ice for
subsequent determination of total protein concentration using the Bradford method and SOD activity
measurements.

Fig 7. Aqueous Two Phase System for SOD purification.

Fig. 8. Aqueous Two Phase System (ATPS) for SOD purification.

Fig. 9. Performance of ATPS used for SOD purification.

Page 5 of 6 D4.3 A downstream processing protocol of enzymes purification 13/02/2019

 PUBLIC

3. Conclusion
Chromatographic and non-chromatographic methods were employing for the development of downstream
processing strategy for protease and SOD purification. A new purification method for protease activity and
SOD was developed based on aqueous-two phase system composed by phosphate (Pi) and polyethylene
glycol (PEG). This ATPS was proven a simple, low cost and environmental friendly fractionation method for
enzymes from microalgae. Furthermore, ATPS provides an easy scalable, safe in use and fast method.

4. References
Labrou NE. Protein purification: an overview. Methods Mol Biol. 2014;1129:3-10.
Maltezos A, Platis D, Vlachakis D, Kossida S, Marinou M, Labrou NE. Design, synthesis and application of
benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn. J
Mol Recognit. 2014 Jan;27(1):19-31.
Platis D, Labrou NE. Application of a PEG/salt aqueous two-phase partition system for the recovery of
monoclonal antibodies from unclarified transgenic tobacco extract. Biotechnol J. 2009 Sep;4(9):1320-7.

Page 6 of 6 D4.3 A downstream processing protocol of enzymes purification 13/02/2019