Protease

Microbial
production of
protease enzyme
Introduction
• Proteases are the enzymes which catalyse the
hydrolysis of peptide bonds of the proteins.
• Microbes have both intracellular and
extracellular proteases,
*the intracellular proteases are responsible
for the maintenance of amino acid pool inside
the cell by degrading the unwanted protein.
*the extracellular proteases hydrolyse
proteins outside the cells into peptides and
amino acid required by the cells for their
growth.
Classification of protease
 on the basis of pH optima of activity
1)alkaline protease
2)acid protease
3)neutral protease
 On the basis of the functional group present at the
catalytic site
1)serine proteases
2)cysteine proteases
3)aspartic proteases
4)threonine proteases
5)glutamic acid proteases
6)metalloproteases
The Importance of Proteases
• Proteolytic enzymes are very important in digestion as they breakdown the
peptide bonds in the protein foods to liberate the amino acids needed by the
body.
• Additionally, proteolytic enzymes have been used for a long time in various
forms of therapy. Their use in medicine is notable based on several clinical
studies indicating their benefits in oncology, inflammatory conditions, blood
rheology control, and immune regulation.
• Protease is able to hydrolyze almost all proteins as long as they are not
components of living cells. Normal living cells are protected against lysis by the
inhibitor mechanism.
• Parasites, fungal forms, and bacteria are protein.Viruses are cell parasites
consisting of nucleic acids covered by a protein film. Enzymes can break down
undigested protein, cellular debris, and toxins in the blood, sparing the immune
system this task. The immune system can then concentrate its full action on the
bacterial or parasitic invasion.
How Protease Deficiency Can Affect
Your Health:
• Acidity is created through the digestion of protein. Therefore a
protease deficiency results in an alkaline excess in the blood. This
alkaline environment can cause anxiety and insomnia.
• In addition, since protein is required to carry protein-bound

calcium in the blood, a protease deficiency lays the foundation for
arthritis, osteoporosis and other calcium-deficient diseases.
• Protease also has an ability to digest unwanted debris in the blood

including certain bacteria and viruses. Therefore, protease
deficient people are immune compromised, making them
susceptible to bacterial, viral and yeast infections and a general
decrease in immunity.
Industrial uses of proteases
• Baking industry:Neutral protease :Dough conditioner
• Dairy industry:Fungal acid proteases:Cheese
preparation
• Detergent industry:Alkaline protease from Bacillus
sp :Laundry detergent for protein stain removal
• Leather industry:Neutral proteases :Bating of leather
• Photographic industry:Alkaline protease :Digestion of
gelatin for recovery of silver from films
• Brewery industry:Neutral proteases :Hydrolyse cereal
mash to release peptides and amino acids for
utilization by yeast
Alkaline proteases
• Strains of Bacillus, Steptomyces, Aspergillus are the major
producers of alkaline proteases.
• Proteases from Bacillus (Bacillopeptidases) are mainly used in
detergents.
• Subtilisin carlsberg (protease from B. licheniformis) and Subtilisin
Novo (protease from B. amyloliquefaciens) are the best know
proteases used in detergents.
• These proteases have serine at the active site, and are not
inhibited by EDTA (ethylenediaminetetraacetic acid), but are
inhibited by DFP (diisopropyl flurophosphate).
• These proteases are stable at high temperature, active in alkaline
pH (9-11) and stable in presence of chelating and perborates,
which is an important characteristic of these enzyme for use in
detergents.
Production process
• The alkaline proteases are produced at large scale using 40-100 m 3
fermenters.
• Fermentation process is operated in fed-batch manner to maintain low
levels of ammonium and free amino acids, since at high concentrations
they repress the protease production.
• Continuous process for protease production has been developed but it is
not practiced at industrial scale.
• Oxygen is supplied to get the optimal protease titers. The fermentation
lasts for 48-72 h depending on the organism used.
• The proteases once produced are converted to particulate form by
encapsulation so that the persons involved in the production or use of
enzyme could avoid allergic reactions due to inhalation of dry enzyme
powder.
• The encapsulation is done by mixing the wet paste of enzyme at 50-70ºC
with hydrophobic substances (polyethylene glycol), and then making
tinny particles/globules.
Acid proteases
• Rennins from calf stomach, pepsin of humans are the well known
examples of acid proteases catalysing hydrolysis of protein around pH 2-
4.
PRODUCTION
PRODUCTION
• The rennin produced by Endothia parasitica was the first rennin of

microbial origin.
• Media containing 3 % soy meal, 1 % glucose, 1 % skim milk, 0.3 %
NaNO3, 0.005 % K2HPO4, 0.025 % MgSO4.7H2O; pH 6.8 is used for the
production of protease by the above strain.
• The fermentation is completed in 48 h at 28 ºC, after which the
mycelium is removed and enzyme is concentrated and precipitated by
evaporation process.
• This protease has a molecular weight of 34-37.5 kDa and is stable in the
pH range of 4.0-5.5 and temperature up to 50ºC.
• Rennin-protease from Mucor pusillus is produced by
solid surface process.
• The production of rennin-protease by Mucor miehei
is carried out in medium containing 4% potato starch,
3% soy meal, 10% ground barley, 0.5% CaCO3, at 40°C
for 5-6 days.
• The microbial rennins are stable at high temperature,
and cause harmful proteolysis.
• The calf rennin has been successfully cloned into E.
coli for production and use of rennin enzyme for
avoiding the problems encountered with microbial
rennin.
Neutral protease
• Neutral proteases are obtained from plants e.g. papain (from Carica papaya),
bromelain (from Ananas comorus) and ficin (from Ficus spp.), which are cysteine
proteases.
• Neutral proteases are produced by bacteria (Clostridium histolyticum,

Streptococcus spp., Bacillus subtilis, B. cereus, B. megaterium, B.
stearothermophilus, B. thuringiensis)and fungi (Aspergillus oryzae, A. sojae,
Penicillium spp., Pericularia oryzae).
• these microbial neutral proteases are either cysteine or

metalloproteases.Neutral metallo protease have specificity towards peptide
linkages that contain hydrophobic amino side.
• The neutral protease are unstable and require calcium,sodium and chloride ions
for their stability.not only the ph range of these protease is small but they also
get inactivated at elevated temperatures.

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Microbial

• In addition, since protein is required to carry protein-bound

• Protease also has an ability to digest unwanted debris in the blood

• The rennin produced by Endothia parasitica was the first rennin of

• Neutral proteases are produced by bacteria (Clostridium histolyticum,

• these microbial neutral proteases are either cysteine or

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