Contains information about protease productioin from different sources such as microbes, plants and animals.

1. PRODUCTION OF PROTEASE
ENZYME FROM DIFFERENT
SOURCES
L. THARRUN DANIEL PAUL,
DEPARTMENT OF BIOTECHNOLOGY,

2. INTRODUCTION
 WHAT ARE ENZYMES ?
 Enzymes are type of proteins that act as biocatalyst in our body.
 One of the most important roles of protease is the hydrolysis activity
(breaking down).
 WHAT ARE PROTEASES ?
 Proteases (also called as proteolytic enzymes, peptidases) are a group of
proteins that hydrolyze the long chain protein molecules into shorter
fragments and eventually into amino acids.
 Proteases denature proteins into their primary structure.
 Proteases have a wide range of industrial applications.

3. HISTORY
 1833- French chemist Anselme Payen- Diastase.
 1860s- Louis Pasteur- fermentation techniques with sugar.
 1897- Eduard Buchner- paper on ‘Study of yeast extracts’.
 1926- James B. Sumner- proved urease and catalase(1937) were proteins.
 1965- David Chilton Phillips- structure of lysozyme by x-ray crystallography.

4. TYPES OF PROTEASE
 Based on catalytic residue
 Serine proteases
 Cysteine proteases
 Threonine proteases
 Aspartic proteases
 Glutamic proteases
 Metalloproteases
 Asparagine peptide lyases
 Peptide lyases
 Classification based on optimal pH
 Acid proteases
 Neutral proteases
 Basic proteases (or alkaline proteases)

5. PROTEASE PRODUCTION IN LABORATORIES
 STAGES:
 Screening of enzyme activity- plate assay-well diffusion
 Substrate quantification using Folin’ s phenol
 Protein estimation- Bradford or Lowry’ s assay
 Optimization of substrate, temperature and pH.
 Precipitation using ammonium sulphate (salting out).
 Dialysis
 Qualitative test- SDS- PAGE


6. INDUSTRIAL PRODUCTION
 Commercially produced microbial proteases contribute to approximately 2/3 of all
enzyme sales.
 Bacteria- Bacillus, Pseudomonas, Clostridium, Proteus, and Serratia,
 Fungi- Aspergillus niger, Aspergillus oryzae, Aspergillus flavus, and Penicillium
roquefortii.
 Bacillus species are mostly used in the commercial production of proteases.
 The fungal proteases present a wider pH activity range- wider range of uses.
 There are two types of proteases: (a) alkaline serine proteases and (b) acid proteases.
 Alkaline serine proteases- Bacillus licheniformis by submerged culture method.
 Acid proteases- fungi by either semisolid culture or submerged culture method.

7. MICROBIAL PROTEASES
 Microbial proteases are one of the important groups of industrially and
commercially produced enzymes.
 Emphasis is on the microorganisms producing proteases with desired
characters.
 Demand for novel proteases.
 Microorganisms represent an excellent source- broad biochemical diversity,
susceptibility to genetic manipulation.
 Optimization of culture media is important to yield an economically viable
amount of proteases.
SOURCES FOR PROTEASE PRODUCTION

8. FUNGAL PROTEASE
 Commercial production of fungal protease- Aspergillus flavus, Aspergillus wentii,
Aspergillus oryzae, Mucor delemar, Mucor miehei and Amylomyces rouxii.
 The fungus is usually grown on wheat bran, although other media are sometimes
employed, under fermentation conditions similar to those for amylase production.
 At sporulation, the various fungal proteolytic enzymes are present in the medium,
and the proteases are recovered by procedures similar to those for mold amylases.
amylases.
 The optimum temperature of the fermentation is 30°C & requires 3 days for
completion.
 Mucor miehei- Acid proteases by submerged culture method (The optimum
temperature of the fermentation is 30°C but requires 7 days for completion).

9. BACTERIAL PROTEASE
 Bacterial protease production- strains of Bacillus subtilis, and the fermentation
conditions are similar to those for amylase production by this organism.
 However, the Bacillus subtilis strains are specially selected for high protease activity
and not for amylase activity.
 As stated previously, a high carbohydrate content medium is utilized to stimulate
protease activity and depress amylase production, although the final product does
contain some amylase activity.
 The fermentation is incubated 3 to 5 days at 37°C in pans containing a shallow layer
of fermentation medium, and the harvest procedure is similar to that for bacterial
amylase.
 Bacillus licheniformis - alkaline serine proteases by submerged culture method.

10. PLANT PROTEASES
 EXAMPLES:
 Papain, bromelain and ficin represent some of the well-known proteases of
plant origin.
 Papain- latex of Carica papaya fruits- active between pH 5-9 and is stable up
to 90°C.
 Bromelain- stem and juice of pineapples- also called as cysteine protease.
 A neutral protease- purified from Raphanus sativus leaves.
 An aspartic protease- potato leaves.
 Thiol Protease- pineapple Crown Leaf.
 Serine protease- artificially senescing parsley leaves.
 Endo proteases- alfalfa, oat and barley senesced leaves.

11. ANIMAL PROTEASES
 EXAMPLES:
 The most common proteases of animal origin are pancreatic trypsin,
chymotrypsin, pepsin, and rennins.
 Trypsin- intestinal digestive enzyme- type of serine protease-
hydrolyzes peptide bonds.
 Chymotrypsin- animal pancreatic extract- expensive enzyme used
only for diagnostic and analytical applications- used extensively in
the de- allergenizing of milk protein hydrolysates.
 Pepsin- acidic protease- present in the stomachs of vertebrates.
 Rennet- pepsin-like protease (rennin, chymosin)- used exclusively in
the dairy industry to produce good flavored curd.

12. BIOREACTOR
 A bioreactor may refer to any manufactured or engineered device,
(large fermentation chamber), for growing organisms such as bacteria or
yeast under controlled conditions that which supports a biologically
active environment or a chemical process is carried out which
organisms or biochemically active substances derived from such
organisms.
 This process can either be aerobic or anaerobic.
 These bioreactors are commonly cylindrical, ranging in size from liters to
cubic meters, and are often made of stainless steel.
 Bioreactors are used in the biotechnological production of substances
such as pharmaceuticals, antibodies, or vaccines, or for the
bioconversion of organic waste.

13. TYPES OF BIOREACTORS
Types of Bioreactor
Continuous Stirred Tank Bioreactor
Airlift Bioreactor
Fluidized Bed Bioreactor
Packed Bed Bioreactor
Photobioreactor
Membrane Bioreactor

14. CSTR Air lift bioreactor

15. Packed Bed Bioreactor Photobioreactor

16. Fluidized bed bioreactor Membrane Bioreactor

17. FERMENTATION
 Fermentation is a metabolic process that produces chemical changes in
organic substrates (sugars) through the action of microbes and
 It is also defined as the extraction of energy from carbohydrates in the
absence of oxygen or refer to any process in which the activity
of microorganisms brings about a desirable change to a foodstuff or
beverage.
 The science of fermentation is known as zymology.
 It is carried out in a fermenter, which is type of bioreactor.
 Humans have used fermentation to produce foodstuffs and beverages
since the Neolithic age .

18. SOLID STATE FERMENTATION
 Solid state fermentation (SSF) is an industrial process, in which
mostly metabolites generated by microorganisms grown on a solid
support selected for this purpose. This technology for the culture of
microorganisms is an alternative to liquid or submerged fermentation,
used predominantly for industrial purposes (bread, maturing of
cheese).
SUBMERGED STATE FERMENTATION
 Submerged fermentation is an industrial process in which enzymes and
other reactive compounds are submerged in a liquid such as alcohol,
or a nutrient broth. The process is used for a variety of purposes,
in industrial manufacturing (enzymes, antibiotics..).

19. SOLID STATE FERMENTOR

20. SUBMERGED FERMENTOR

21. INDUSTRIAL APPLICATION
 Detergent industry
 Leather industry
 Food industry
 Dairy industry
 Baking and brewing industry
 Soy sauce production
 Meat tenderization
 Synthesis of aspartame
 Pharmaceutical industry
 Therapeutics
 Photography industry
 Management of industrial wastes
 Degumming of silk

22. RECENT ADVANCES AND FUTURE PROSPECTS
 Extension of biotechnological approach in terms of both quality and
quantity.
 Qualitative improvements: low-temperature screening methods,
protein engineering, r-DNA technology, novel cold-active proteases.
 Quantitative enhancement needs: Strain improvement, especially
through site-directed mutagenesis, and standardizing the nutrient
media for the overproduction.
 Strain development- achieved by changes at the gene level.
 Extensive attempts to engineer cold-adapted proteases from subtilisin
BPN have previously been made.

23.  Genetic and protein engineering can bring about a great yield of the
enzymes by the microbes.
 Offer a new strategy for site specific drug targeting and tumor imaging
imaging (cross linking).
 Proteases from extremophiles have a great market value due to their
desirable unique features, eg., microbes in hot springs.

24. CONCLUSION
 Microbes are a reliable source for protease production in order to attain a
standard output.
 Plant and animal sources are not reliable for industrial production as they
are limited.
 Advancement in biotechnology leads to a constructive position in
protease production.
 The need for the protease enzyme is constantly increasing till now and
there will be a great need for industries producing proteases.
 Future of enzyme technology also depends on the research and
development sector in modifying a microbial strain to tolerate various
physical conditions and produce a good yield.

25. THANK YOU