Microbial enzymes
Dr. Umbreen Rashid
PRODUCTION OF ENZYMES
Fermentation for Enzyme Production
Most enzyme production is carried out in deep
submerged fermentation; a few are best produced in semi-
solid media.
Semi solid medium
Submerged production
This system, also known as the ‘Koji’ or ‘moldy bran’ method of ‘solid
state’ fermentation is still widely used in Japan.
The medium consists of moist sterile wheat or rice bran acidified
with HCl; mineral salts including trace minerals are added.
An inducer is also usually added: 10% starch is used for amylase;
gelatin and pectin for protein and pectinase production,
respectively.
Semi solid The organisms used are fungi, which appear amenable to high
enzyme production because of the low moisture condition and
medium high degree of aeration of the semi-soluble medium.
The moist bran inoculated with spores of the appropriate fungi is
distributed either in flat trays or placed in a revolving drum.
Moisture (about 8%) is maintained by occasionally spraying water
on the trays and by circulating moist air over the preparation.
The optimum production is determined
The temperature of the bran is kept at The production period is usually 30–40
by withdrawing the growth from time to
about 30°C by the circulating cool air. hours.
time and assaying for enzyme.
Growth in a semi-solid medium Thus, Aspergillus oryzae on semi-solid
The material is dried with hot air at sometimes seems to encourage an medium will produce a large number of
about 37°C–40°C and ground. enzyme range different from that enzymes, primarily amylase,
produced in submerged growth. glucoamylase, and protease.
Similarly, if Aspergillus oryzae producing
takadiastase (a commercial powder
containing amylase and some
protease) is grown in submerged
In submerged culture, amylase
culture, four protease components are
production rises at the expense of the
formed whereas on semi-solid medium,
other enzymes.
not only are merely two proteases
formed, but these are less heat resistant
than those produced in submerged
fermentation.
Submerged production
• Enzyme production by submerged cultivation in a deep fermenter has
replaced semi-solid production wherever possible because the latter is
labor intensive and therefore expensive, and due to the risk of infection
and the generally greater ease of controlling temperature, pH, and
other environmental factors in a fermenter.
• The medium must contain all the requirements for growth, including
adequate sources of carbon, nitrogen, various metals, trace elements,
growth substances, etc. However, a medium adequate for growth may
not be satisfactory for enzyme production.
• For the production of inducible enzymes, the inducers must be present.
• Thus, pectic substances need to be in the medium when pectinolytic
enzymes are the desired product.
• Similarly, in the production of microbial rennet, soy bean proteins are
added into the medium to induce protease production by most fungi.
• The inducer may not always be the substrate; sometimes, a breakdown
or end-product may serve such as cellobiose which stimulates cellulose
production.
Thus, α-amylase synthesis is
Some easily metabolizable repressed by glucose in It is therefore common to
components of the medium Bacillus licheniformis and B. replace glucose by more
Strong repression is often seen
may repress enzyme subtilis. In many organisms, slowly metabolized
in media containing glucose.
production by catabolite protease synthesis is carbohydrates such as partly
repression. repressed by amino acids as hydrolyzed starch.
well as by glucose.
Thus, isoleucine and proline Temperature and pH
Some specific amino acids are involved in the case of B. requirements have to be
End-product inhibition has
inhibit protease production in megaterium, while sulfur worked out for each
also been widely observed.
some organisms. amino acids inhibit protease organism and each desired
formation in Aspergillus niger. product.
The temperature and pH
requirements for optimum The temperature adopted for
growth, enzyme production, the fermentation is usually a
and stability of the enzyme compromise taking all three
are not necessarily the same requirements into account.
for all enzymes.
The oxygen requirement is Vigorous aeration and Batch fermentation is
usually high, as most of the agitation are therefore usually employed in
organisms employed in done in the submerged commercial enzyme
enzyme production are fermentations for enzyme fermentation and lasts
aerobic. production. from one to seven days.
In a few cases, the enzyme
production is highest Furthermore, different Thus, A. niger produces
during the exponential enzymes are produced at mostly α-amylases in the
phase of growth, but in different stages of the first 72 hours but mainly
most others, it occurs post- growth cycle. maltase thereafter.
exponentially.
Enzyme Extraction
In order to limit contamination and degradation of the enzyme, the broth is cooled to about
20°C as soon as the fermentation is over.
Stabilizers such as calcium salts, proteins, sugar, and starch hydrolysates may be added and
destabilizing metals may be removed with EDTA.
Antimicrobials if used at all are those that are normally allowed in food such as benzoates and
sorbate.
Most industrial enzymes are extracellular in nature. In the case of cell bound enzymes, the cells
are disrupted before centrifugation and/or vacuum filtration.
The extent of the purification after the clarification depends on the purpose for which the
enzyme is to be used.
Sometimes, enzymes may be precipitated using a variety of chemicals such as methanol,
acetone, ethyl alcohol, or ammonium sulfate.
The precipitate may be further purified by dialysis, chromatography, etc. before being dried in
a drum drier or a low temperature vacuum drier depending on the stability of the enzymes to
high temperature.
Ultra-filtration separation technique based on molecular size may be used.
Packaging and
Finishing
Enzymes are packaged preferably in
liquid form but where solids are used, the
enzyme is mixed with a filler.
It is now common practice to coat the
particles with wax so that enzyme dusts
are not formed.
