This document discusses the commercial production of enzymes. It begins by introducing enzymes and their industrial applications. The first enzyme produced commercially was taka-diastase in 1896 for pharmaceutical use. Most enzymes for industrial use come from fungi, bacteria, yeast and streptomyces. While plant and animal sources have limitations, microbial sources provide inexpensive production through fermentation. The document outlines the process of isolating and identifying microorganisms with desired enzyme activity, optimizing fermentation conditions, extracting intracellular and extracellular enzymes, and improving strains through mutagenesis or genetic engineering for higher yields.

1. COMMERCIAL
PRODUCTION OF ENZYMES

2. INTRODUCTION
Enzymes- Biocatalysts- Complex protein molecules that
bring about chemical reactions- Nontoxic and biodegradable.
Produced in large amounts by microorganisms- industrial
applications
Enzyme technology- broadly involves selection, production,
isolation, purification and use of enzymes
recombinant DNA technology and protein engineering-
efficient use of enzymes

3. The first enzyme produced industrially was taka-
diastase (a fungal amylase) in 1896, in United States. It
was used as a pharmaceutical agent to cure digestive
disorders.
Europe- softening of hides with faeces of dogs and
pigeons- before tanning- pancreases- German scientist-
Otto Rohm

4. Uses of enzymes from different
organisms
Fungi – 60%
Bacteria – 24%
Yeast – 4%
Streptomyces – 2%
Higher animals – 6%

5. ENZYMES FROM PLANT AND ANIMAL
SOURCES
Quantities are limited
Difficulties in isolating, purifying the enzymes,
and the cost factor
Wide variation in their distribution
Mammalian cell cultures- cost constraints-
Tissue plasminogen activator from cell

6. MICROBIAL SOURCES
Inexpensive media, short period, easy to manipulate,
isolation and purification are easy
Extracellular enzymes- safe, stable and active
Stable over a range of pH and temperature
Optimal fermentation conditions- cheap substrates, low
cost production
Co- production- amylase, lipase and protease- used in
detergent industry
rDNA- overproduce, substrate specific and stable
Bacillus and Aspergillus- most used

8. ISOLATION AND SCREENING OF
MICROORGANISMS
Sources: Plant bark, watery environment, skim milk , marine
sediment, municipal solid wastes and from grapes
Serial dilution and spread plate method- specific agar
Thermophilic microbes- thermostable enzymes
Enzyme inducers in media
Laccase- CuSO4 and MgSO4
Proteases- casein, skim milk, gelatin
Amylases, cellulases and lipases- starch, CMC, oil substances

10. IDENTIFICATION

11. SCREENING
Reaction Presence of enzyme
Starch hydrolysis amylase
Casein/ gelatin hydrolysis protease
Tributyrin/Tween 80
hydrolysis or change in
phenol red color to orange
Lipases (breakdown of oil to
FA)
Gram’s iodine solution amylases
0.1% Congo red solution
followed by 1 M sodium
chloride solution
cellulase
Phenol red to pink colour L-asparaginase
Sodium carbonate Alkalophilic organisms

12. IDENTIFICATION
Bergey’s manual of determinative
bacteriology- cultural, morphological,
microscopic and biochemical
characteristics
Fungi- 18S rRNA sequencing
Bacteria- 16S rRNA or 16S rDNA

13. PRODUC
TION
PROCES
S

15. PRODUCTION
Mostly used inocula- Acinetobacter, Pseudomonas,
Staphylococcus, Streptomyces, Fusarium, Mucor,
Penicillium, and Trichoderma species
Fermentation process- depends on plant equipment, yield,
convenience and application
•SmF- extracellular enzymes secreted in media, needs expensive
synthetic media
•SSF- inexpensive media, downstream processing is simple

17. Examples:
1. Invertase (β D fructofuranosidase)- produced by
Aspergillus sojae –SSF- orange peels moistened
with molasses -production of alcoholic beverages
2. Lignocellulosic biomass was used to produce
cellulase -with Aspergillus awamori
3. Detergent protease by the bacterium Alcaligenes
sp. -using fed batch fermentation

18. INDUCERS IN MEDIUM
Surfactants- Triton X-100/ Tween
Aromatic and phenolic
compounds
CHEAP SUBSTRATES
Chicken feathers- Bacillus megaterium-
keratinase
Orange peel- Aspergillus sojae- invertase
Wheat bran- Aspergillus sp.- keratinase, laccase and phytase

20. OPTIMIZATION
Optimization of various
nutritional parameters (C, N, and P sources)
physico-chemical aspects (inoculum age, incubation
time and temperature)
fermentation factors- inoculum level , agitation/
aeration

22. EXTRACTION OF EXTRA AND
INTRACELLULAR ENZYMES

25. STRAIN IMPROVEMENT
Cycles of random mutagenesis and screening
Target for-
1. secretion efficiency- EC enzymes
2. overcomes organism’s regulatory mechanism-
avoid catabolite repression
mRNA half life increased
Gene dosage increase- plasmid/ chromosomal
amplification
Use of GRAS listed organisms- Bacillus, Aspergillus and
Saccharomyces

26. PROTEIN ENGINEERING