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1.  Lab scale production of
bacterial enzyme:
M.Phool Badshah

2. Enzymes:
 Enzyme are macromolecular biological catalysts.
 Enzymes accelerate or catalyze chemical reaction.
 The molecules at the beginning of the process are called
substrates and enzymes converts these into different
molecules called products.
 Microbial enzymes are biological catalysts for the
biochemical reactions leading to microbial growth and
respiration, as well as to the formation of fermentation
products.

3. Methods of enzyme production:
 There are two methods of enzyme production
which are generally used at industry level :
 Solid-state Fermentation..
 Submerged Fermentation.
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4. a
Solid-State Fermentation
o There are many biotechnological processes that involve
the growth of organisms on solid substrates in the
absence or near absence of free water.
o Solid state method is another method used for the
production of enzymes, which involves the cultivation of
microorganisms on solid substrate, such as grain , rice
and wheat.
o Solid state fermentation employs natural raw materials as
carbon source such as cassava , barley, wheat bran etc.

5. a
o Solid State Fermentation (SSF) is a fermentation
method used by several industries like
the pharmaceuticals, food, textile etc., to produce
metabolites of microorganisms using solid support in
place of the liquid medium.
o Solid state fermentation (SSF) deals with substrates
that are solid and contain low moisture levels.
o The most regularly used solid substrates are cereal
grains (rice, wheat, barley, and corn), legume seeds,
wheat bran, ligno-cellulose materials such as straws,
sawdust or wood shavings, and a wide range of
plant and animal materials.

7. a
 Selection of Microorganisms:
o This is one of key factor to improve yields of the
product. Bacteria, Yeast and Filamentous fungi can
be used.
o Filamentous fungi has shown better results growing
in the solid substrate fermentation.
 Substrate:
o Substrate also plays important role in determining
the growth of microorganisms, there by increasing
the product yield.
o Substrate is chosen in such a way that it should
provide as well as nutrients to the growing culture.

8. a
Applications
o Solid-state fermentation has emerged as a potential
technology for the production of microbial products
such as feed, fuel, food, industrial chemicals, and
pharmaceutical products.
o It is widely applied to producing several enzymes,
organic acids, flavoring compounds etc., which must
be extracted and purified and then used in different
products.
o Its application in bioprocesses such as
 bioleaching,
 bio-beneficiation,
 bioremediation,
 bio-pulping, etc.

10. a
Submerged Fermentation
o Submerged liquid fermentations are traditionally used
for the production of microbial derived enzymes.
o In the submerged process, the substrate used for
fermentation is always in liquid state which contains
the nutrients needed for growth.
o The fermentor which contains substrate is operated
continuously and the product biomass is continuously
harvested from the fermenter by using different
techniques then the product is filtered or centrifuged
and then dried.
o Submerged fermentation is the method of
manufacturing bimolecular in which enzymes and
other reactive compounds are submerged in a liquid
such as alcohol, oil and nutrient broth.

11. a
o The process is used for a variety of purposes, mostly
in industrial manufacturing.
o The process can be used to make products such as
citric acid, glycerol or lactic acid.
o Submerged culture fermentation has been widely
used for the production of enzyme because in
submerged fermentation unwanted metabolites are
not produced and purification of enzymes takes place
in an easy way.
o Parameters like temperature, pH, oxygen
consumption and carbon dioxide formation are
measured and controlled to optimize the fermentation
process.
 Fermentation takes place in large vessels (fermenter)
with volumes of up to 1,000 cubic meters.
 The fermentation media sterilizes nutrients based on
renewable raw materials like maize, sugars, and

12. a
Applications
o Submerged Fermentation/Liquid Fermentation
utilizes free-flowing liquid substrates, such as
molasses and broths.
o The bioactive compounds are secreted into the
fermentation broth.
o The substrates are utilized quite rapidly; hence need
to be constantly replaced/supplemented with
nutrients.
o This fermentation technique is best suited for
microorganisms such as bacteria that require high
moisture.
o An additional advantage of this technique is that the
purification of products is easier.

13. a
Solid-state fermentation
o Organism require less water
for growth and preferred such
as filamentous fungi.
o Less chances of
contamination because of less
availability of water.
o Less consumption of energy
for aeration and gas transfer.
o Liquid waste is not produced.
o Downstream processing is
easy, cheaper and less time
consuming.
o Limiting factor for growth is
diffusion for nutrients.
Submerged Fermentation
o Media concentration is very
much lower as compared to
water content.
o Higher water activity
becomes the major cause of
contamination in SLF.
o Higher air pressure
consumes more power and
there is poor transfer of gas
in SLF.
o High quantity of liquid waste
is produced, causing
difficulties in dumping.
o Water makes downstream
process difficult and very
expensive.
o Vigorous mixing makes
diffusion easy.

14. Protease Enzyme
o Protease (mixture of peptidases and proteinases )
are enzymes that perform the hydrolysis of peptide
bonds.
o Peptide bonds links the amino acids to give the final
structure of a protein.
o Proteinases are extracellular and peptidases are
intracellular.
o Second most important enzyme produced on a large
scale after amylase.

15. a
 Classification based upon the residues in the
catalytic sites:
o Serine protease.
o Threonine protease.
o Aspartate protease.
o Cysteine protease.
o Glutamic acid protease.
o Metalloprotease.
 Classification based upon the PH in which the
proteases are active:
o Alkaline serine proteases
o Acid proteases.
o Neutral proteases.

16. a
Alkaline serine Proteases
o PH of the production medium is kept at 7.0 for
satisfactory results.
o Optimal temperature maintained is 30’ to 40’C.
o Important producers are B, lechinoformis ,
B.amyloliqueifaciens, B.firmus , A.flavus .
o Enzymes used in detergents are chiefly proteases
from bacillus strains (Bacillopeptidases).
o Best Known proteases are Subtilisin Carlsberg
from B,licheniformis and Subtilisin BPN and
Subtilisin Novo from B. Amyloliquefaciens.

17. a
 Substrate for Protease:
o Molasses.
o Nutrient broth.
 SCREENING:
o Because enzyme should be stable in alkaline
conditions, screening for better producers is done by
using highly alkaline media.
o It was found that B.Lichenoformis and B.subtilis
showed growth in the range of PH 6,7 by new strains
were found to grow even in PH 10-11.
o Genetic manipulation can also be carried out.

18. a
Fermentation Process
o Culture was stored in the lyophilized state or under liquid
nitrogen.
o Initial culture are carried out in shaken flasks and small
fermenters (40-100m3) at 30-37’C.
o Fed-Batch culture is generally used to keep down the
concentration of ammonium ions and amino acids as
they may repress protease production.
o High oxygen partial pressure is generally necessary for
optimal protease titers, time span for fermentation is 48-
72 hours depending upon the organism.
o Protease must be converted in a particulate form before
they are added to detergents.
o To produce a suitable encapsulated product, a wet paste
of enzyme is melted at 50’ to 70’C with a hydrophobic
substance such as poly-ethy lene-glycol and then
converted into tiny particles.

19. a
Neutral protease:
o They are relatively unstable and calcium, sodium
and chloride must be added for maximum stability.
o Not stable at higher temperature.
o Producing organisms are B.subtilis and
B.megaterium etc.
o They are quickly inactivated by alkaline protease.
Acid protease
o Similar to mammalian pepsin.
o It consist of Rennin like proteases from fungi which
are chiefly used in cheese production.
o They are used in medicine, in the digestion of soy
protein for soy sauce production and to break down
wheat gluten in the baking industry

20. a
Application
o Textile industry to remove proteinaceous sizing.
o Silk industry to liberate silk fibers from naturally
occuring proteinaceous material in which they are
embedded.
o Tanderizing of meat.
o Used in detergent and food industries.