INDUSTRIAL PRODUCTION OF

ENZYME PROTEASE

PRESENTED BY
SWATI . V . BALAPURE
MSc-II
( department of biotechnology)
CONTENTS

INTRODUCTION
SELECTION OF MICROORGANISM
SELECTION OF MEDIA
CULTURE CONDITIONS
FERMENTATION
PRODUCT RECOVERY
PACKING
APPLICATIONS OF PROTEASE
Introduction

Proteases: Proteases split protein

molecules. They are further classified by
their optimum
pH as acid, alkaline or neutral. They may
also be classified on the basis of their
active.
centers into the following
(i) Serine proteases: These have a residue
in their active center and are specifically
inhibited by diisopropyl phosphofluoridate
and other organophosphorus
derivates.
(ii) Thiol proteases: The activity of these
depends on the presence of an intact-SH
group
in their active center. They are specifically
inhibited by thiol reagents such as
heavy metal ions and their derivatives, as
well as alkylating and oxidizing agents.
(iii) Metal proteases: These depend on the
presence of more of less tightly bound
divalent
cations for their activity.
(iv) Acid proteases: Acid proteases contain
one or more side chain carboxyl groups in
their active center.
Selection of microorganisms

Isolation involves either the use of mix culture or a

pure culture.
For the production of protease the microorganisms
are used such as bacteria and fungi. e.g. Bacillus
subtilis Aspergillus oryzae,Penicillium roqueforti .
For the isolation of protease producing organisms
from soil the enrichment culture should be used .
For the first step the soil is incubated with the
substrate casein which is the protein and utilize by
the protease producing bacteria.
The microorganisms then characterize by various
methods such as microscopic detection and by
testing with comessi blue dye by replica plating
technique.
Skim milk medium (SM): Autoclave skim milk agar,
composed of skim milk
(10 g/L) and purified agar (15 g/L). Cool the
medium to about 60°C and add
Rose Bengal (2 μg/mL) or Fungizone (10 μg/mL)
to avoid development of fungi.
Final pH obtained is about 6.5–7.5 .
2. Minimal synthetic medium (ZM), with the
following composition: 1.0 g/L
(NH4)2SO4, 6.0 g/L K2HPO4, 3.0 g/L KH2PO4,
0.01 g/L MgSO4·7H2O, 0.05 g/L
CaCl2·2H2O, 0.01 g/L MnSO4·2H2O, 0.001 g/L
FeSO4·7H2O, 0.001 g/L
ZnSO4·7H2O, 1.0 g/L trisodium citrate, and 10.0
g/L casein (3).
3. Supplemented medium: ,
2.5 g/L yeast extract. This medium is utilized when
poor growth is observed
in the minimal medium. Vitamins and growth
factors are provided by yeast extract
the production of microbial rennet's soy bean
proteins are added into the medium to induce
protease production by most fungi
Culture conditions and fermentation

Enzyme protease can be produce by the two

methods by using
1. Semisolid medium
2. Submerged medium
Semisolid medium

The medium consists of moist sterile wheat or rice

bran
acidified with HCl; mineral salts including trace
minerals are added.

.
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 temperature of the bran is kept at
about 30°C by the circulating cool air.
The production period is usually 30-40 hours, but
could be as long as seven days. The
optimum production is determined by withdrawing
the growth from time to time and
assaying for enzyme. The material is dried with hot
air at about 37°C–40°C and ground
The enzyme is usually preserved in this manner. If
it is desired, the enzyme can be extracted. Growth
in a semi-solid medium seems sometimes to
encourage an enzyme
range different from that produced in submerged
growth. Thus, Aspergillus oryzae on
semi-solid medium will produce a large number of
enzymes, primarily amylase,
glucoamylose, and protease.
Submerged medium

Submerged production has replaced semi-solid

production wherever possible
because the latter is labour intensive and therefore
expensive where labour is scarce, and
because of the risk of infection and the generally
greater ease of controlling temperature,
pH and other environmental factors in a fermentor.
The medium must contain all the requirements for
growth.
sources of carbon, nitrogen, various metals, trace
elements, growth substances, etc.
However, a medium adequate for growth may not
be satisfactory for enzyme production
Product recovery

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.
enzymes precipitated using a variety of
chemicals such as methanol, acetone, ethyl
alcohol or ammonium sulfate. The precipitate
is 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 used.
Packing

The packing of enzymes has become extremely

important since the experience of the
allergic effect of enzyme dust inhalation by
detergent works. Nowadays, enzymes are
packaged preferably in liquid form but where solids
are used, the enzyme is mixed with
a filler and it is now common practice to coat the
particles with wax so that enzyme dusts are not
formed.
Application of protease

Manufacture of protein hydrolysates

Stabilization of evaporated milk
Curdling milk
Recovery of silver from spent film
Meat tenderization
Leather dehairing ,baiting
In medical field the protease used for treatment of
blood clots
To remove wound debrident such as dead or
damaged tissue .
As anti-inflammatory agent
References

Modern industrial microbiology and biotechnology

by Nauka okafor
Principles of fermentation technology by P.F .
STANBURY ,A.WHITAKER AND S.J.HALL