Proteases are the enzymes formed by mixture of proteinases and peptidases which provides platform for hydrolysis of proteins. About 500 tons of proteases are produced per year making protease 2nd largest producing enzyme. Alkaline serine proteases are specifically manufactured on larger scale for production of detergents, as it have maximum stability in highly basic conditions and they are stable even at high temperature. Also they have stability towards the complexation, they do net directly get inhibited by the EDTA (Ethylene Diamino Tetra Acetic acid). proteases can be inhibited by the DFP (Di Isopropyl Flurophosphate)

1. Production of
Proteases
Presented by:- Lokesh Patil
B. Pharm 5th Sem, 3rd Year
Smt B.N.B Swaminarayan Pharmacy college Salvav,
Vapi

2. Contents of presentation
Introduction to proteases General information related to proteases
Classification 1 Classification based upon the residues in catalytic site
Classification 2 Classification based upon the pH in which the proteases are active
Alkaline Serine Proteases Information and production bacterias
Neutral Protease Information and production bacterias
Acid Protease Information and production bacterias
Production process of
proteases
Briefly explained 4 step process
Applications of Proteases Unveiling the Enzymatic Elegance: Exploring the Fascinating Applications of Proteases
Production of enzyme, Proteases

3. Proteases
● Proteases are the mixture of peptidase and
proteinases that platform the hydrolysis of peptide
bond.
● Proteinases are the extracellular and peptidases are
endocellular
● Proteases are the 2nd most important enzyme
produces on larger scale after amylase
● About 500 tons of the enzymes are produced per
year.
● This can be produced commercially from bacteria and
fungi.

4. Classification based upon the
residues in the catalytic site
❖ Serine protease
❖ Threonine protease
❖ Aspartate protease
❖ Metalloprotease
❖ Cysteine protease
❖ Glutamic acid protease

5. Classification based upon the pH
in which the protease are active
❖ Alkaline Protease
❖ Acid protease
❖ Neutral protease

6. Alkaline Serine Protease
⮚ Alkaline proteases have some features which makes its applications in industrial scales like stability at
high temperature, stability in alkaline range (pH 9-11), stability in association with chelating agents.
⮚ Many bacteria and fungi excrete alkaline protease and the most important producer are Bacillus strains
like Bacillus licheniformis, Bacillus amylotiquefaciens, B. megaterium, B.
purilis and Streptomyces strains like Streptomyces fradiae, S. griseus, S. rectus and fungi
like Aspergillus niger, A. sojae, A. oryzae, A. flavus
⮚ Alkaline proteases are commonly used in detergents.
⮚ pH of the production medium is kept at 7.0 for satisfactory results.
⮚ Optimum temperature to be maintained during the production is 30-400 C.
⮚ Enzymes used in detergents are mainly proteases from bacillus strains (Bacillopeptidases).
⮚ These enzymes are not inhibited by EDTA but are inhibited by DFP (Di isopropyl fluorophosphate).

7. Neutral Protease
⮚ Neutral proteases are relatively unstable and calcium, sodium and chloride must be added for maximal
stability.
⮚ Its pH range for activity is narrow and sensitive to increased temperature.
⮚ Neutral proteases are produced by bacteria and fungi. E.g. Bacillus subtilis, B. coreus, B. megaterium,
Pseudomonas aeruginosa, Streptomyces griseus, Aspergillus oryzae, Aspergillus sojae.
⮚ These are quickly inactivated by alkaline proteases.
⮚ Because of these limitations, they have restricted industrial applications but can be used in leather
industries and in food industries (For manufacture of breads and rolls).

8. Acid Protease
• Acidic proteases are found in animal cells, yeast and molds but never in bacteria.
• Pepsin like acid proteases are derived from Aspergillus species and Rhizopus spp.
• These microbial renin-like enzymes are derived from Mucor michei, Mucor pusillus,
Mucor racemaeus, Mucor bacilliformis.
• Similar to mammalian pepsin, it consists of Renin like proteases are commonly used in
cheese production in optimum pH 2-4.
• They are used in medicine, in the digestion of soy protein for soya sauce production and
to break down wheat gluten in the baking industry.

9. Production process of Protease
1. Isolation of proteolytic microbes
2. Media formulation
3. Fermentation
4. Purification

10. Step I: Isolation of proteolytic
microbes:
⮚ Proteolytic microbes can be isolated by observing hydrolysis in casein agar.
⮚ After isolating the suitable strain, it necessarily increases enzyme production
by optimizing process parameter like media composition, pH, volume,
moisture content (in-case of solid-state fermentation), concentration of
mineral salts, age and size of inoculum, fermentation time and temperature,
organic and inorganic supplements.
⮚ Among the various proteases, bacterial proteases are the most significant as
compared with animal and fungal proteases.
⮚ Among bacteria, Bacillus spp are specific producer of extracellular
proteases.
⮚ Corresponding changes in enzymatic properties such as substrate
specificity, pH optimum and stability to bleaching agent.

11. Step II: Media formulation
⮚ Media rich in nitrogen sources such as soyabean
milk, casein, gelatin and carbohydrate sources
such as starch, or lactose are generally used for
protease production.

12. Step III: Fermentation
Cultures are stored in lyophilized state or under liquid
nitrogen.
Initial cultures are carried out in shaken flasks &
small fermenters at 30-370C.
Fed batch culture is generally used to keep down the
concentration of ammonium ions & amino acids as
they suppress the protease production.

13. Step III: Fermentation contd….
High oxygen partial pressure is generally necessary
for optimal protease titers.
Time span for fermentation is 48-72 hours depending
upon the organism.
Particulate must be converted in a particulate form of
feasible size before they are added to detergents.

14. Figure showing typical fermenter

15. Step IV: Purification
⮚ Different methods can be applied for purification of enzyme like ultrafiltration.
⮚ Chromatographic technique (ion exchange) purification by treatment with
activated charcoal and H2O2.

17. Summary
1. Isolation
3.Fermentation
2. Media
formulation
4. Purification

18. Applications
⮚ Used in cheese production
⮚ Used in medicine (similar to mammalian pepsin)
⮚ Used in digestion of soya-protein for soy-sauce production.
⮚ Break down wheat gluten in baking industries.
⮚ In textile industry to remove proteinaceous ring
⮚ Silk industry to liberate silk from naturally occurring proteinaceous material in
which they are embedded.
⮚ Tenderizing meat ie; for breaking down of collagens in meat to make it
palatable.
⮚ Used in detergent and food industries.

19. Proteases for use in detergent
industries
⮚ Stability at high temperature.
⮚ Stability in alkaline range. (pH 9-11)
⮚ Stability in association with chelating agent
⮚ But shelf life is affected in presence of
surface active agents

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