THE EXTRACTION AND PURIFICATION OF LIPASE ENZYME FROM BACTERIA

Introduction
Lipases are a group of enzymes which catalyze the hydrolysis of triglycerides to diglycerides and
monoglycerides which are further hydrolyzed to glycerol and free fatty acids.

Lipases are ubiquitous in nature and are produced by various plants, animals and
microorganisms. Lipases of microbial origin, mainly bacterial and fungal, represent the most
widely used class of enzymes in biotechnological applications and organic chemistry
Some of the applications of lipase enzymes are;
- In leather and cosmetics processing
- Food processing
- Pulp and paper processing
- Animal feed
- Textiles
- Pharmaceuticals
- Detergents
This and their ability to catalyze a plethora of reactions in aqueous and non-aqueous media i.e.
esterification, alcoholysis and acidolysis, the focus on lipase has been increasing and thus they
emerge as a key class of enzymes. The downstream processing of lipases is equally difficult and
expensive since this step is crucial to obtain the enzymes with a high purity level while
maintaining their enzymatic activity and stability behavior. This and the lipase’s chemo-, Regio-
and enantio-specific behavior makes them of great interest.
The most conventional extraction technologies are still being extensively used for lipase
purification, however, these methodologies are characterized by a significant number of
drawbacks namely;
- Difficulties in scale-up
- High production costs and the lack of suitable biocompatible solvents.
These issues raise concerns not only from scientist working in the field, but also from the
industries responsible for lipase production and the companies that actually use lipase in their
processes, due to the consequent cost of this raw materials. Therefore, cost effective methods
that can continuously separate concentrate and purity proteins are of great commercial interest.
Because lipases are of microbial origin, their production is done by means of microbial
fermentation processes. Most commercial applications don’t require homogeneous lipase
preparation. However, a certain degree of purity can enable their efficient and successful usage.
Despite the versatility of the lipase production and purification conditions, these steps were not
studied and optimized until recently
The search for inexpensive production systems capable of producing large quantities of lipases
has resulted in the development of new technology using steps of purification based on diverse
extraction methodologies.

The extracellular bacterial lipases are of considerable commercial importance, as their bulk
production is much easier. Although a number of lipase-producing bacterial sources are available,
only a few are commercially exploited as wild or recombinant strains

PRODUCTION
Lipases are by and large produced from microbes and specifically bacterial lipases play a vital
role in commercial ventures. Some important lipase-producing bacterial generally include;

- Bacillus
- Pseudomonas
- Burkholderia
- Achromobacter
- Alcaligens
- Arthrobacter
- Chromobacterium

Bacterial lipases are mostly extracellular and are produced by submerged fermentation

Submerged fermentation is the cultivation of microorganisms in a liquid nutrient broth.

Industrial enzymes are produced using this process. Extracellular lipases have been produced
from microorganisms such as fungi, yeasts and bacteria
In Submerged fermentation, the substrate used for fermentation is always in liquid state which
contains the nutrients needed for growth. It is a method of manufacturing biomolecules in which
the enzymes and other reactive compounds are submerged in a liquid such as alcohol, oil or
nutrient broth
Lipases are generally produced on lepidic carbon such as oils, fatty acids, glycerol or tweens in
the presence of an organic nitrogen source
fermentation conditions.
Bacterial lipases are mostly extracellular and are greatly influenced by nutritional and physio-
chemical factors such as;

- Temperature
- pH
- nitrogen and carbon sources,
- presence of lipids, inorganic salts, agitation and dissolved oxygen concentration

The major factor for the expression of lipase activity has always been carbon, since lipases are
by and large inducible enzymes and are thus generally produced in the presence of a lipid source
such as an oil or any other inducer, such as triacyclglycerols, fatty acids, hydrolysable esters,
tweens, bile salts and glycerol. However, their production is significantly influenced by other
carbon sources, such as sugars, sugar alcohol, polysaccharides, whey, casamino acids and other
complex sources. Besides carbon source, the type of nitrogen source in the medium also
influences the lipase titers in production broth. Generally, organic nitrogen is preferred, such as
peptone and yeast extract, which have been used as nitrogen source for lipase production.

Divalent cations such as Ca2+ and Mg2+ stimulate the production of lipase from Burkholderia sp.
and Iron was found to play a critical role in the production of lipase by Pseudomonas sp. However,
most other metal ion salts are inhibitory to lipase production.

In addition to the various chemical constituents of a production medium, physiological

parameters such as pH, temperature, agitation, aeration and incubation period also play an
important role in influencing production by different microorganisms.

The initial pH of the growth medium is important for lipase production. Largely, bacteria prefer
pH around 7.0 for best growth and lipase production, such as in the case of Bacillus sp.

The optimum temperature for lipase production corresponds with the growth temperature of
the respective microorganism. For example, the best temperature for growth and lipase
production in the case of Bacillus sp. RSJ1 is 50°C. It has been observed that, in general, lipases
are produced in the temperature range 20–45°C. Incubation periods ranging from few hours to
several days have been found to be best suited for maximum lipase production by bacteria

Thus, bacterial lipases are generally produced in the presence of oil or any other lepidic substrate
(viz. fatty acid esters, fatty acids, glycerol) as carbon in the presence of any complex nitrogen
source. The requirement for metal ions varies with the organism. However, physical parameters
such as pH, temperature, agitation and aeration influence lipase production via modulating the
growth of the bacterium. Lipases are produced throughout bacterial growth, with peak
production being obtained by the late log phase. The production period for lipases varies from a
few hours to a few days.
OUTLINE OF THE FLOW CHART SHOWING THE PRODUCTION OF LIPASE ENZYME FROM BACTERIA

PRODUCTION
Intracellular production
Cell lysis
Fermentation Solid – liquid separation (e.g.
Fermentation centrifugation, filtration,
Microorganism
broth sedimentation, flocculation)
Product
Extracellular production

Bio mass

PRE-PURIFICATION/PRODUCT CONCENTRATION
(NH4)2SO4

Low size Dialysis Lipase-rich Salt Liquid

contaminants supernatant preparation extract
(e.g. salts)

Precipitated (contaminated protein)

PURIFICATION
High resolution purification
Low resolution purification techniques
Lipase-rich techniques (e.g. crystallization,
(e.g. solvent extraction, liquid-liquid
dialysate chromatography, fractional
extraction)
precipitation, fractional liquid-
liquid extraction

contaminants

ISOLATION/PRODUCT POLISHING

Determination of purity level and Product

Final stability/activity of the target (high purity)
product product
 - Most lipases produced by microorganisms are extracellular implying that the process of
cell lysis is avoided and only the first step of cell separation is required which is included
in the pre-treatment/pre-purification of the fermentation broth. This step is normally
carried-out in low resolution processes such as centrifugation or filtration to eliminate
the biomass.
- Following the removal of the cells, a precipitate is applied to the liquid extract by
saturation with salts normally ammonium sulfate or organic solvents such as alcohol or
acetone aiming at the removal of the majority fraction of contaminants in particular
proteins.
- The lipase rich supernatant is then introduced into the dialysis system to eliminate
small particle contaminants such as cell debris or salts left over from the fermentation
process and or the precipitation of proteins.
- The final output of the pre-treatment task, a dialysate is obtained for posterior use in
the purification step, richer in lipase and free from the most common and highly
concentrated contaminants, which will then be used in the purification stage.
- The extent of the separation/purification process varies with the order and the
resolution of each purification step. The most common purification methods employed
are chromatographic processes, membrane and immune-purification.

PURIFICATION
A large number of lipases from fungal and bacterial sources have been isolated and purified to
homogeneity. This success is attributed to the development of both conventional and novel
purification techniques. This review highlights the use of these techniques in lipase purification,
including conventional techniques such as;
- ammonium sulfate fractionation
- ion-exchange
- gel filtration
- affinity chromatography
As well as novel techniques such as;
- reverse micellar system
- membrane processes
- immune-purification
- aqueous two-phase system
- aqueous two-phase floatation
In most lipase purification plants, chromatography is used to achieve the level of purity required
which is entirely dependent on the final application, and thus the use and combination of
different chromatographic processes may be considered. The 3 most commonly used and known
chromatographic methods are;
 - Ion exchange method
- The gel filtration method
- Affinity chromatography
The ion exchange method is widely used due to it’s high applicability.
The chromatographic methods mainly used according to their main properties are;

PROPERTIES TECHNIQUES
Size Gel filtration chromatography
Charge Ion exchange chromatography
Hydrophobicity Hydrophobic interaction chromatography
Ligand Specificity Affinity chromatography

ION EXCHANGE CHROMATOGRAPHY

This separates compounds according to the nature and degree of their charge. The column to be
used is selected according to it’s type, strength and charge. Resins which have negative charge
are used to retain and separate positively charged compounds and resins with a positive charge
are used to retain and separate negatively charged compounds. Before separation begins, a
buffer is pumped into the column to equilibrate the opposing charged ions. Upon injection of the
sample, solute ions will exchange with the buffer ions as each competes for the binding sites on
the resin.
The ion exchange chromatography is commonly used in the first place due to its high capacity for
loaded protein. This technique is normally characterized by the great degree of lipase purification
due to the establishment of strong electrostatic interactions between the enzyme and the gel.

GEL FILTRATION CHROMATOGRAPHY AND AFFINITY CHROMATOGRAPHY

In gel filtration chromatography, the proteins are separated based solely on the molecular sizes.
The filtration is achieved by use of a porous matrix which for steric reasons, the molecules have
differing access. I.e. the smaller molecules are retained by the porous matrix while the bigger
molecules are not. Thus molecules are eluted from the gel chromatography column in decreasing
order of size.
In affinity chromatography, the separation of biochemical mixtures is based on a highly specific
interaction between the antigen and the antibody, the enzyme and the substrate, or ligand and
the receptor. It makes use of the specific binding interactions between molecules. The ligand is
chemically attached to a solid support, such that when the biochemical mixture is passed through
the column, the molecules with a high affinity for the ligand become bonded to that ligand. When
the sample molecules have been washed away, the bonded molecules are stripped away from
their support resulting into it’s purification. This method offers a high selectivity, high resolution
and generally high capacity for the desired protein. The degree of purification can be thousands
of times and the yield can be usually very good
The gel and the affinity chromatography are also important processes used in more than 60%
and 27% of the purification apparatus respectively
The Affinity chromatography can be applied in an early stage of the purification process, however
the materials are expensive and thus gel filtration or ion exchange chromatography are preferred
due to the lower costs associated with the consumables. Although gel filtration represents lower
purification capacities for loaded proteins, it can be applied in both cases, as an initial step of
concentration or in the final contributing to the product polishing.
The number of works dealing with these methods is significant, with the chromatographic
techniques being used principally as purification platforms in processes where a high level of
purity is required and for which the level of knowledge generated allows the manipulation of the
process even at large scales.
However, for lipase a different scenario is found, the usual procedures are at a deficient
performance mainly because they promote a decrease in the lipase activity, they are of difficult
manipulation principally due to the high number of chromatographic units connected in the
same process which results in time consuming processes with low final yields of usually <20%.
Therefore, novel purification techniques are needed to increase the overall enzyme yields as well
as to reduce the number of steps in the downstream processing.
LIQUID-LIQUID EXTRACTION
An approach to the goal is to greatly increase the efficiency of the initial stages of product
purification by the continuous processing of fermentation broths, where the enzyme is produced,
yielding a stream of concentrated partially purified enzyme. This can be achieved by liquid-liquid
extraction, as this unit has the potential to work efficiently for the continuous recovery and it is
readily scalable and has relatively high capacity and selectivity for the desired product. Liquid-
liquid extraction also allows the processing of solid containing streams, reaching simultaneous
biomass removal and product isolation and partial purification and in certain cases allow the
integration of fermentation and the first steps of product isolation in a single step.
Liquid-liquid extraction can be achieved by two techniques;
- Reversed micelles
- Aqueous two phase systems
Aqueous two-phase systems

The aqueous two-phase systems used in bio separation are composed of two incompatible
polymers (e.g. dextran vs PEG) in water solution or in a high salt concentration (e.g. Phosphate).
The partitioning of proteins in aqueous two phase systems depends on the physio-chemical
properties, e.g. protein hydrophobicity, charge and size. The partitioning is influenced by
changing polymers, polymer molecular mass, or pH, or by the addition of salts or detergent to
the system. The advantages of aqueous two phase extraction lie in volume reduction, high
capacity, rapid separations and mildness. The technique can be used early in the purification on
process streams containing whole cells or cell debris. Compared with other separation
techniques, two-phase extraction is relatively straightforward to scale-up. The aqueous two-
phase system is an interesting technique with properties suitable for the separation and
purification of macromolecules and particles that are difficult to purify with other existing
techniques

For lipases, the hydrophobic nature of the enzyme is exploited in aqueous two-phase systems by
employing detergents or surfactants during the purification.

DRYING AND PACKAGING

The concentrated form of lipase enzyme can be obtained by drying. This can be done by film
evaporators or freeze dryers (lyophilizers). The dried lipase enzyme can be packed and marketed,
it’s stability achieved by keeping them in ammonium sulfate suspensions

PROPERTIES OF MICROBIAL LIPASES

1) PH and Temperature Kinetics.

Generally, bacterial lipases have neutral or alkaline pH optima with the exception of P.
fluorescens SIK W1 lipase, which has an acidic optimum at pH 4.8. Bacterial lipases possess
stability over a wide range, from pH 4 to pH 11.
Bacterial lipases generally have temperature optima in the range 30–60°C. However, reports exist
on bacterial lipases with optima in both lower and higher ranges. Thermal stability data are
available only for species of Bacillus, Chromobacterium, Pseudomonas and Staphylococcus. The
thermostability of the enzyme from Bacillus sp. is enhanced by the addition of stabilizers such as
ethylene glycol, sorbitol, glycerol, with the enzyme retaining activity at 70°C even after 150 min.

2) Stability in Organic Solvents

Stability in organic solvents is desirable in synthesis reactions. From the available literature, it can
be inferred that lipases are generally stable in organic solvents, with few exceptions of
stimulation or inhibition.

3) Effect of Metal Ions

Cofactors are generally not required for lipase activity, but divalent cations such as calcium often
stimulate enzyme activity. This has been suggested to be due to the formation of the calcium
salts of long-chain fatty acids.
 4) Lipase Inhibitors

Lipase inhibitors have been used in the study of structural and mechanistic properties of lipases.
Further, the search for lipase inhibitors is also of pharmacological interest. Lipase inhibitors are
used for designing drugs for the treatment of obesity and the problem of acne. Following is an
account of general inhibitors. Broadly, inhibitors of enzymes are classified as reversible or
irreversible. The reversible inhibitors can be further classified as nonspecific and specific
reversible inhibitors.

Non-specific inhibitors - Compounds that do not act directly at the active site, but inhibit lipase
activity by changing the conformation of lipase or interfacial properties. Examples are proteins,
bile salts. Specific inhibitors - Specific inhibitors are those compounds, which directly interact
with the active site of the enzyme. Such inhibitors can be either reversible or irreversible.
Examples are Boronic acid derivatives

5) Substrate Specificity

Microbial lipases may be divided into three categories: namely nonspecific, regio-specific and
fatty acid-specific, based on the substrate specificity. Nonspecific lipases act at random on the
triacyl glyceride molecule and result in the complete breakdown of triacyl glyceride to fatty acid
and glycerol. In contrast, regio-specific lipases are 1,3-specific lipases which hydrolyze only
primary ester bonds. The third group comprises fatty acid-specific lipases, which exhibit a
pronounced fatty acid preference.

Thus, bacterial lipases are highly robust enzymes, since they are active over a wide range of pH
and temperature. They belong to the group of serine hydrolases and are not sulfhydryl proteins.
They may be regio-specific or nonspecific towards triacyclglycerols. Some lipases also possess
fatty acid-specificity with reference to the carbon- chain length. Besides these features, the
enantioselective nature of lipases provides them with an edge over other hydrolases, particularly
in the field of organic chemistry and pharmaceuticals.

CONCLUSION

Lipases are the biocatalysts of choice for the present and future, owing to their properties such
as activity over a wide temperature and pH range, substrate specificity, diverse substrate range
and enantioselectivity. Their importance is increasing by the day in several industries, such as
food, detergents, chemicals, pharmaceuticals, etc. However, the commercial exploitation of
lipases is still in its infancy, due to the economics of the lipase industry.
Thus, there is a need today to develop production and downstream-processing systems which
are cost-effective, simple and not time-consuming. The growing demand for lipases has shifted
the trend towards prospecting for novel lipases, improving the properties of existing lipases for
established technical applications and producing new enzymes tailor-made for entirely new areas
of application. This has largely been possible due to outstanding events in the field of molecular
enzymology. The number of novel microbial lipases being cloned and biochemically characterized
is on the rise. Rational protein engineering, by way of mutagenesis and directed evolution, has
provided a new and valuable tool for improving or adapting enzyme properties to the desired
requirements. The upcoming trend to access novel natural sequenced space, via the direct
cloning of metagenomic DNA, is significantly contributing to the screening and identification of
hitherto unexplored microbial consortia for valuable biocatalysts.
However, the success of these techniques demands the development of faster high-throughput
screening systems. Thus, the modern methods of genetic engineering combined with an
increasing knowledge of structure and function are allowing further adaptation to industrial
needs and the exploration of novel applications.