MICROBIAL PRODUCTION OF PROTEASE ENZYME

INTRODUCTION

Proteases are the enzymes which catalyze the hydrolysis of peptide bonds of the proteins. The
amino acid composition of proteins is very diverse so the proteases responsible for their
hydrolysis are also diverse. Microbes have both intracellular and extra cellular proteases, the
intracellular proteases are responsible for the maintenance of amino acid pool inside the cell by
degrading the unwanted proteins and the extra cellular proteases hydrolyze proteins outside the
cells into peptides and amino acid required by the cells for their growth.

Proteases are classified into two major groups: the exopeptidases (peptidases) and the
endopeptidases (proteinases). The peptidases hydrolyze the protein from C- or N-terminus
releasing single amino acid and the endopeptidases as the name suggests hydrolyses the peptide
bond in the middle of the amino acid chain.

Further the proteases are also classified into alkaline, acid and neutral proteases based on their
pH optima of activity. On the basis of the functional group present at the catalytic site these
proteases are classified as serine proteases, cysteine proteases, aspartic proteases, threonine
proteases, glutamic acid proteases and metalloproteases.

The proteases represent one of the three largest groups of industrial enzymes others being the
amylases and lipases. The proteases find their application in detergents, leather, food,
pharmaceutical industries and bioremediation processes.
Both bacterial and fungal proteases are produced commercially and their production conditions
are very different from each other. Some organisms are thermophilic especially grown to obtain
thermostable proteases for use in detergents.

The Importance of Proteases

Proteases refer to a group of enzymes whose catalytic function is to hydrolyze (breakdown)

proteins. They are also called proteolytic enzymes or proteinases.

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.

 Because protein is converted to glucose upon demand, inadequate protein digestion leads
to hypoglycemia, resulting in moodiness, mood swings and irritability.

 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

Industry Protease Application

Baking Neutral protease Dough conditioner

Dairy Fungal acid proteases Cheese preparation

Detergent Alkaline protease from Laundry detergent for protein stain removal
Bacillus sp

Food processing Several proteases Modification of protein rich material (soy

protein or gluten), meat tenderization

Leather Neutral proteases Bating of leather, dehairing of hides

Brewery Neutral proteases Hydrolyse cereal mash to release peptides

 and amino acids for utilization by yeast

Photographic Alkaline protease Digestion of gelatin for recovery of silver

from films

Biopharmaceutical Several proteases Digestive syrup, contact lens cleaner,

necrotic tissue removal, blood clot digestion

Peptide synthesis Thermolysin, α-chymosin Reverse reaction to synthesise aspartame and

and several other protease other peptides of pharmaceutical importance

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.

 Screening of organism producing alkaline proteases is done using strongly basic media
and the colonies are tested on protein agar plates (pH 10). The wild strains have been
improved by altering the amino acid sequences of the proteases by genetic engineering
tools, changing their substrate specificity, pH optimum, and stability to the bleaching
agents. Extensive genetic manipulations have also been carried out to improve the yields
of the proteases during the fermentation.

 Proteases to be used as detergent additive should be stable and active in the presence of
surfactants, bleaching agents, bleach activators, fillers, fabric softeners and various other
formulation of a typical detergent. In textile industry, proteases may also be used to
remove the stiff and dull gum layer of sericine from the raw silk fibre to achieve
improved luster and softness. Protease treatment also modifies the surface of wool and
silk fibers to provide unique finishes. The alkaline proteases also have potential
application in removal of gelatin from the used photographic films vis-à-vis recovery of
silver from them.

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 at the rate of 1vvm 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. Some of the fungi also produce
acid proteases which are rennin-like and used mainly in cheese production. Acid
proteases are also used in the preparation of digestive syrup, soy protein digestion during
sauce preparation, hydrolyzing the gluten from wheat dough used for preparing biscuits
in bakery making them crispy. Silver from the film roll is recovered by digesting the
gelatin by acid proteases. Alcaligens, Bacillus, Corynebacterium, Lactobacillus,
Pseudomonas, Serratia, Streptococcus, and Streptomyces are the bacteria, and
Aspergillus, Candida, Coriolus, Endothia, Endomophthora, Irpex, Mucor,
Penicillium¸Rhizopus, Sclerotium, and Torulopsis are the some of the fungi producing
rennin like proteases which find applications in cheese processing. There are three strains
being used for acid protease production and divided into two groups on the basis of
culture conditions
The rennin produced by Endothia parasitica was the first rennin of microbial origin
marketed in the year 1967. 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, B.
pumilus, B. polymyxa, B. licheniformis, B. amyloliquefaciens. B. stearothermophilus,
Pseudomonas aeruginosa, Streptomyces griseus) 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. commercial
fungal neutral protease are used in banking ,food processing, protein modification and in
leather, animal feeds and pharmaceutical industries.