Commercial pro

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