Microbes and enzyme production

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Microbes and enzymes production
Enzymes
Enzymes are biocatalysts produced by living cells to bring about specific biochemical
reactions generally forming parts of the metabolic processes of the cells.
Enzymes are highly specific in their action on substrates and often many different enzymes are
required to bring about, by concerted action, the sequence of metabolic reactions performed by
the living cell. All enzymes which have been purified are protein in nature and may or may not
possess a non-protein prosthetic group.
Microbial enzymes
Microbial enzymes
Microbial enzymes are known to play a crucial role as metabolic catalysts, leading to
their use in various industries and applications. The end use market for industrial enzymes is
extremely wide-spread with numerous industrial commercial applications. Over 500 industrial
products are being made using enzymes. The demand for industrial enzymes is on a continuous
rise driven by a growing need for sustainable solutions. Microbes have served and continue to
serve as one of the largest and useful sources of many enzymes. Many industrial processes,
including chemical synthesis for production of chemicals and pharmaceuticals, have several
disadvantages:
 Low catalytic efficiency,
 Lack of enantiomeric specificity for chiral synthesis,
 Need for high temperature and
 Low ph and high pressure.
Also, the use of organic solvents leads to organic waste and pollutants. Enzymes are more useful
for these applications as they work under mild reaction conditions (e.g., temperature, ph,
atmospheric conditions), do not need protection of substrate functional groups, have a long half-
life, a high stereo-selectivity yielding stereo- and regio-chemically-defined reaction products at
an acceleration of 105
to 108
-fold, and, in addition, they work on unnatural substrates.
Furthermore, enzymes can be selected genetically and chemically-modified to enhance their key
properties: stability, substrate specificity and specific activity. There are drawbacks however, to
the use of enzymes, e.g., certain enzymes require co-factors. However, various approaches such
as cofactor recycling and use of whole cells can solve this problem. About 150 industrial
processes use enzymes or whole microbial cell catalysts.
Sources of enzymes
Initially, enzymes were extracted from the stomach of calves, lambs, and baby goats, but
now are produced by microorganisms like bacteria, fungi, yeast and actinomycetes. Enzymes
obtained from microorganisms are better than those of animal and plant origin.
Microorganisms can be genetically manipulated to improve the production of commercial
scale. A very wide range of sources are used for commercial enzyme production
from actinoplanes to zymomonas, from spinach to snake venom. Of the hundred or so enzymes
being used industrially, over a half are from fungi and yeast and over a third are from bacteria

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with the remainder divided between animals and plant. The different organisms and their
relative contribution for the production of commercial enzymes are given below:
● Fungi – 60%
● Bacteria – 24%
● Yeast – 4%
● Streptomyces – 2%
● Higher animals – 6%
● Higher plants – 4%
A real breakthrough for large scale industrial production of enzymes from microorganisms
occurred after 1950s.microbes are preferred to plants and animals as sources of enzymes
because:
1. They are generally cheaper to produce.
2. Their enzyme contents are more predictable and controllable,
3. Reliable supplies of raw material of constant composition are more easily arranged,
4. Plant and animal tissues contain more potentially harmful materials than microbes,
including phenolic compounds (from plants), endogenous enzyme inhibitors and
proteases.
Attempts are being made to overcome some of these difficulties by the use of animal and plant
cell culture.
Enzyme Source Intra/extra-
cellular
Industrial use
Bacterial enzyme
Amylase Bacillus E Starch
Asparaginase Escherichia coli I Health
Glucose isomerase Bacillus I Fructose syrup
Penicillin amidase Bacillus I Pharmaceutical
Protease Bacillus E Detergent
Fungal enzymes
Amylase Aspergillus E Baking
Aminoacylase Aspergillus I Pharmaceutical
Glucoamylase Aspergillus E Starch
Catalase Aspergillus I Food
Cellulase Trichoderma E Waste
Dextranase Penicillium E Food
Glucose oxidase Aspergillus I Food

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The technology of enzyme production—general considerations:
The first enzyme produced industrially was taka-diastase (a fungal amylase) in 1896, in
united states. It was used as a pharmaceutical agent to cure digestive disorders. A german
scientist (otto rohm) demonstrated in 1905 that extracts from animal organs (pancreases from pig
and cow) could be used as the source of enzymes-proteases, for leather softening. The utilization
of enzymes (chiefly proteases) for laundry purposes started in 1915. However, it was not
continued due to allergic reactions of impurities in enzymes. The techniques employed for
microbial production of enzymes are comparable to the methods used for manufacture of other
industrial products .the salient features are briefly described.
1. Selection of organism:
The most important criteria for selecting the microorganism are that the organism should
produce the maximum quantities of desired enzyme in a short time while the amounts of
other metabolite produced are minimal. Once the organism is selected, strain improvement
for optimising the enzyme production can be done by appropriate methods (mutagens, uv
rays). From the organism chosen, inoculum can be prepared in a liquid medium.
2. Formulation of medium:
The culture medium chosen should contain all the nutrients to support adequate growth of
microorganisms that will ultimately result in good quantities of enzyme production. The
ingredients of the medium should be readily available at low cost and are nutritionally safe.
Some of the commonly used substrates for the medium are starch hydrolysate, molasses, corn
steep liquor, yeast extract, whey, and soy bean meal. Some cereals (wheat) and pulses
(peanut) have also been used. The ph of the medium should be kept optimal for good
microbial growth and enzyme production.
3. Production process of enzyme:
Production of microbial enzymes by application of fermentation procedures involves
microbial propagation like bacteria, mold and yeast to get desired product. The process of
Lactase Aspergillus E Dairy
Lipase Rhizopus E Food
Rennet Mucor miehei E Cheese
Pectinase and Pectin lyase Aspergillus E Drinks
Protease Aspergillus E Baking
Yeast enzymes
Invertase Saccharomyces I/e Confectionery
Lactase Kluyveromyces I/e Dairy
Lipase Candida E Food
Raffinase Saccharomyces I Food

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fermentation is classified based on specific parameters. There are two methods of
fermentation used to produce enzymes. These are
 Submerged fermentation
 Solid-substrate fermentation
Submerged fermentation
Submerged fermentation involves the production of enzymes by microorganisms in a
liquid nutrient media. In this technique, the yields are more and the chances of infection are
less. Hence, this is a preferred method.
Solid-substrate fermentation
Solid-state fermentation is the cultivation of microorganisms, and hence enzymes on a
solid substrate. However, solid substrate fermentation is historically important and still in use
for the production of fungal enzymes e.g. Amylases, cellulases, proteases and pectinases. The
medium can be sterilized by employing batch or continuous sterilization techniques.
Surface fermentation Sub-merged fermentation
 Requires much space for tray
 Requires much hand labor
 Uses low pressure air blower
 Little power requirement
 Minimum control necessary
 Little contamination problem
 Recovery involves extraction with
aqueous solution, filtration or
centrifugation, and perhaps
evaporation and/or precipitation
 Uses compact closed fermenters
 Requires minimum of labor
 Requires high pressure air
 Needs considerable power for air
compressors and agitators
 Requires careful control
 Contamination frequently a serious
problem
 Recovery involve filtration or
centrifugation, and perhaps evaporation
and/or precipitation
Procedure
The fermentation is started by inoculating the medium. The growth conditions (ph,
temperature, o2 supply, nutrient addition) are maintained at optimal levels. The froth formation
can be minimized by adding antifoam agents. The production of enzymes is mostly carried out
by batch fermentation and to a lesser extent by continuous process. The bioreactor system
must be maintained sterile throughout the fermentation process. The duration of fermentation is
variable around 2-7 days, in most production processes. Besides the desired enzyme(s), several
other metabolites are also produced. The enzyme(s) have to be recovered and purified.
4. Recovery and purification of enzymes:
The desired enzyme produced may be excreted into the culture medium (extracellular
enzymes) or may be present within the cells (intracellular enzymes). Depending on the
requirement, the commercial enzyme may be crude or highly purified. Further, it may be in
the solid or liquid form. The steps involved in downstream processing i.e. Recovery and
purification steps employed will depend on the nature of the enzyme and the degree of
purity desired.

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In general, recovery of an extracellular enzyme which is present in the broth is relatively
simpler compared to an intracellular enzyme. For the release of intracellular enzymes, special
techniques are needed for cell disruption. Microbial cells can be broken down by physical
means (sonication, high pressure, glass beads). Or by enzymatic method which are
expensive. The most important consideration is to minimise the loss of desired enzyme
activity.
5. Removal of cell debris and nucleic acids:
Filtration or centrifugation can be used to remove cell debris. Nucleic acids interfere with
the recovery and purification of enzymes. They can be precipitated and removed by adding
poly-cations such as polyamines, streptomycin and polyethyleneimine.
6. Enzyme separation
 Enzyme precipitation: enzymes can be precipitated by using salts (ammonium sulfate)
organic solvents (isopropanol, ethanol, and acetone). Precipitation is advantageous since
the precipitated enzyme can be dissolved in a minimal volume to concentrate the enzyme.
 Liquid-liquid partition: further concentration of desired enzymes can be achieved by
liquid-liquid extraction using polyethylene glycol or polyamines.
 Separation by chromatography: there are several chromatographic techniques for
separation and purification of enzymes. These include ion-exchange, size exclusion,
affinity, hydrophobic interaction and dye ligand chromatography .among these, ion-
exchange chromatography is the most commonly used for enzyme purification.
7. Drying and packing:
The concentrated form of the enzyme can be obtained by drying. This can be done by
film evaporators or freeze dryers (lyophilizers). The dried enzyme can be packed and
marketed. For certain enzymes, stability can be achieved by keeping them in ammonium
sulfate suspensions. All the enzymes used in foods or medical treatments must be of high
grade purity, and must meet the required specifications by the regulatory bodies. These
enzymes should be totally free from toxic materials, harmful microorganisms and should not
cause allergic reactions.

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Discovery of new enzymes
Nature provides a vast amount of microbial enzyme resources. Our ability to tap into
such immense biodiversity depends on the tools available to expand the search for new enzymes
by
 Metagenome screening
 Genome mining in more than 2,000 sequenced microbial genomes and
 Exploring the diversity of extremophiles.
Metagenomic Screening
Although numerous microbes inhabit the biosphere, less than 1% can be cultivated
through standard laboratory techniques. Metagenomics has appeared as an alternative strategy to
conventional microbe screening by preparing a genomic library from environmental DNA and
systematically screening such a library for the open reading frames potentially encoding putative
novel enzymes. Metagenomic screening is mostly based on either function or sequence
approaches.
 Function-based screening is a straightforward way to isolate genes that show the desired
function by direct phenotypical detection, heterologous complementation, and induced
gene expression. Function-based screening of metagenomic libraries is somehow
problematic mainly due to insufficient, biased expression of foreign genes in Escherichia
coli as this bacterium is usually used as surogate host. To overcome such hurdles,
alternative bacterial host and expression systems are currently being examined
 On the other hand, sequence-based screening is performed using either the polymerase
chain reaction (PCR) or hybridization procedures. Usually, the common procedure is to
use a set of degenerated primers that have been designed based on consensus amino acid
sequences.
Microbial Genomes
Recent success of genome sequencing programs has resulted in an explosion of
information available from sequence databases(NCBI database), thus creating an opportunity to
explore the possibility of finding new natural products (including enzymes) by database mining.
Two approaches are being followed to discover new enzymes.
 On one hand, genome hunting is based on searching for open reading frames in the
genome of a certain microorganism. Sequences that are annotated as putative enzymes
are subjected to subsequent cloning, over-expression and activity screening.
 Another approach, called data mining, is based on homology alignment among all
sequences deposited in databases. Using different bioinformatics tools (e.g., BLAST), a
search for conserved regions between sequences yields homologous protein sequences
that are identified as possible candidates for further characterization.
Extremophiles
Due to their capability to survive under environments of extreme conditions, both
physical as temperature (−2 to 12 °C, 60–110 °C), pressure or radiation, and geochemical such as

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salinity (2–5 nacl) and ph (<2, >9), extremophiles are a very interesting source of enzymes with
extreme stability under conditions regarded as incompatible with biological materials.
Strategies to Improve Properties of Microbial Enzymes
The continuously expanding application of enzymes is creating a growing demand for
biocatalysts that exhibit improved or new properties. Although enzymes have favorable turnover
numbers, they do not necessary fulfill all process requirements and need further fine tuning to
achieve industrial scale production. Genetic modification is very important and recombinant
DNA techniques have increased production by 100-fold. Development of new/improved
biocatalysts is a challenging and complex task. There are two major ways in which enzymes can
be modified to adapt their functions to applied ends:
 Rational redesign of existing biocatalysts and
 Combinatorial methods which search for the desired functionality in libraries generated at
random.
Rational Design
This approach includes site-directed mutagenesis to target amino acid substitutions, thus
requiring knowledge of detailed information about the 3-D structure and chemical mechanism of
the enzymatic reaction, some of which may not be available.
Comparison of the sequence of a new biocatalyst identified in a screening program with the
thousands deposited in the databases can identify related proteins whose functions or/and
structures are already known. Because new enzymes have evolved in nature by relatively minor
modification of active-site structures, the goals of homology-driven experiments include
engineering binding sites to fit different substrates as well as construction of new catalytic
residues to modify functions and mechanisms
Computational protein design starts with the coordinates of a protein main chain and uses a force
field to identify sequences and geometries of amino acids that are optimal for stabilizing the
backbone geometry.
Directed Evolution
Combinatorial methods such as directed evolution create a large number of variants for
screening for enantioselectivity, catalytic efficiency, catalytic rate, solubility, specificity and
enzyme stability, but do not require extensive knowledge about the enzyme.
 Directed evolution is a fast and inexpensive way of finding variants of existing enzymes
that work better than naturally occurring enzymes under specific conditions.
 Directed evolution includes an entire range of molecular biological techniques that allow
the achievement of genetic diversity mimicking mechanisms of evolution occurring in
nature.
 It involves random mutagenesis of the protein-encoding gene by different techniques
including
 The error-prone polymerase chain reaction (PCR) repeated oligonucleotide
directed mutagenesis. Error prone PCR accomplishes introduction of random
point mutations in a population of enzymes. Such molecular breeding techniques

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(DNA shuffling, Molecular breedingtm
) allow in vitro random homologous
recombination, typically between parent genes with homology higher than 70%.
After cloning and expression, a large collection of enzyme variants (104
–106
) is
typically generated and is subjected to screening or selection.
All the approaches mentioned above are not mutually exclusive as the fields of rational, semi-
rational and random redesign of enzymes are moving closer. Thus, directed evolution techniques
make use, where possible, of smaller enzyme variant libraries designed by rational or semi-
rational methods to reduce the screening effort but without compromising the likelihood of
finding better variants.