The document discusses various industrially produced enzymes by microorganisms, focusing on amylases, pectinases, and proteases. It describes the production processes, fermentation conditions, and applications of these enzymes in industries such as food technology, brewing, and detergents. Key microorganisms, recovery methods, and purification techniques for each enzyme type are also detailed.

1. INDUSTRIALY PRODUCED
ENZYMES BY
MICROORGANISMS
M.S.SWETHA(BP231508)

2. ENZYMES:
• Enzymes are bio molecules that catalyse chemical reaction, Nearly all
enzymes re-proteins however certain RNA molecules can be effective
catalyst too. These are RNA molecule is known as Coenzyme.
• In Enzymatic reaction, the molecule at the Beginning of the process
are called Substrates, Enzymes converts then to different molecules
called Products.
• Industrial enzymes are these which are used for industrial production
and also these are produced in industry
Eg: Amylase, protease and pectinase

3. AMYLASE:
• Amylase is an enzyme that catalyses the hydrolysis of Starch to
Sugars.
• Amylases are the large group of enzymes. They are characterized by
their ability to hydrolyze 1,4 – glucosidic linkage in Polysaccharides
(E.g. Starch and Glycogen).
• Amylases play the most important part in Food technology (Bread
making, Beer making, etc.).
• Amylases are classified into two sub-groups. They are:
(i) α – Amylases
(ii) β - Amylases

4. MICROORGANISMS USED FOR AMYLAZE PRODUCTION
(BACTERIA AND FUNGI)
Bacterial strains:
Most are Bacillus species (B. subtilis, B.mesentericus, B. vulgaris, B.
megaterium, B. cereus).Amylases from Rhodothermus marinus,
Corynebacterium gigantea, Chromohalobacter sp., Lactobacillus fermentum
have also been isolated. Halophilic strains that produce amylases include
Haloarcula hispanica, Halobacillus sp., Chromohalobacter sp., Bacillus
dipsosauri, and Halomonas meridiana.
• Fungal strains:
Efficient amylase-producing species include those of genus Aspergillus (A.
oryzae, A. niger, A. fumigatus and A. flavus), as well as Penicillium species (P.
brunneum, P. expansum, P. chrysogenum and P. olsonii), Streptomyces
rimosus, Thermomyces lanuginosus, Cryptococcus flavus, Thermomonospora
curvata, and Mucor sp.

5. BACTERIAL AMYLASE
• Bacterial Amylase – Submerged fermentation.
• Fermentation Medium
Soya bean meal – 1.85 %
Brewer’s Yeast Fraction – 1.50 %
Distiller’s dried solubles – 0.76 %
N – Z Amine – 0.65 %
Lactose – 4.75 %
MgSO4.7H2O – 0.04 %
Antifoam - 0.05 %
Water - 90.40 L

6. FUNGAL AMYLAZE
• Fungal Amylase – Submerged fermentation, Wheat bran was used as
a substrate.
• Aeration and Agitation is problem due to high viscosity.
• Amylase synthesis is inhibited when medium contains Glucose.
• Fermentation Medium
Corn Starch – 24 g/L
Corn steep liquor – 36 g/L
KCl – 0.2 g/L
Na2HPO4 – 47 g/L
CaCl2 – 1 g/L
MgCl2.6H2O – 0.2 g/L

7. FERMENTATION CONDITIONS:
• Temperature – 30 °C to 40 °C.
• pH – 7.0; Neutral or close to Neutral; pH below 6 results in
denaturation of Amylase.
• Calcium carbonate is used to maintain Neutral pH.
• The Amylase production begins when the bacterial count reaches 109
to 1010 cells per ml after 10 – 20 hrs and continues for 100 – 150
hours.
• Duration of Fermentation – 4 to 6 Days.

8. RECOVERY OF AMYLASE:
• Separation of fungal mycelium is accomplished by Filtration of the
refrigerated broth.
• For Bacterial amylase, there is no need of Filtration.
• The suspended particles present in the broth are removed with
Flocculating agents like Calcium phosphate.
• The enzyme is precipitated, in order to get high degree of purity, by
using acetone or alcohol or even inorganic salts like ammonium
sulphate or sodium sulphate.
• Sometimes Fractional precipitation of the enzyme is done to obtain it
in purest form.

9. APPLICATIONS OF AMYLASE
• Alpha and beta amylases are important in brewing beer and liquor
made from sugars derived from starch.
• Amylases are used in bread making and to break down complex
sugars, such as starch (found in flour), into simple sugars.
• Amylase also has medical applications in the use of Pancreatic
Enzyme Replacement Therapy.
• Bacilliary amylase is also used in clothing and dishwasher detergents
to dissolve starches from fabrics and dishes.

10. PECTINASE:
• Pectinase is the collective term for row of enzyme that are able to breakdown or
transform pectins.
• Pectinase is a general form for enzymes such as pectolyase, pectozyme,
polygalacturonase etc. commonly referred as Pectic enzymes.
• Pectinase breakdown Pectin, a polysaccharide that is found in the cell wall of plants.
Pectinase is involved in the metabolism of the cell wall as well as in the growth of
the cell, senescence (biological ageing), ripening of fruits, pathogenesis and
abscission process (shedding of various parts).
• The basic structure of Pectin is 1,4 – linked galuctronic acid with up to 90 % of its
Carboxyl group, esterified with Methanol.
• According to the Enzyme Commission and the International Union of Biochemistry,
Pectinase enzymes are classified under Hydrolase group.
• The majority of commercial enzymes are obtained by employing fungal cultures.
• Pectinases have been commercially prepared from the microorganisms especially
from the fungi since the 1970s.

11. MICROBES USED FOR PECTINASE PRODUCTION:
• Pectinase Produced by various bacteria and fungi although
commercial microbial pectinase production utilizes species of
Penicillium and Aspergillus.
Microbial strains:
Aspergillus niger, Aspergillus japonicum, Bacillus subtilis and
Penicillium italicum are common pectinase producers.

12. FERMENTATION CONDITIONS:
• Solid or submerged fermentation process are used.
• Substrates - Wheat bran, Rice bran, Potato starch, Rice husk, 0.1%
NH4NO3, NH4H2PO4 and MgSO4.
• Enhancer - 2% sucrose and 2% pectin
• Inoculum - Spore of Aspergillus niger (107 spores per gram of dry
substrate)
• Aeration is necessary.
• Temperature – 37 °C.
• pH – 3 to 4
• Duration of Fermentation – 60 to 80 hours.

13. RECOVERY AND PURIFICATION:
• Pectinase can be extracted from the cell and the medium so can be
recovered from both sources.
• In the process of harvesting, the mycelium is filtered or centrifuged,
dried, grind and pectinase extracted with water.
• Recover aqueous portion is concentrated by evaporation to yield
pectinase concentrate.
• As an alternate recovery procedure, pectinase can be precipitated
from aqueous solution by the addition of cold acetone, ethanol,
isopropanol or ammonium sulphate.

14. APPLICATIONS OF PECTINASE:
• Pectinases are widely used in the food industry -To improve the cloud stability of fruit and
vegetable nectars and also For production and clarification of fruit juices For haze removal
from wines.
• Pectinases are also involved in Clarification of wine,Oil extraction,Removal of citric fruit
peels,Degumming fibres
• Pectinase usage accelerates Tea fermentation and also destroys the Foam forming property of
Instant tea powders by destroying pectins.
• Pectinase used in Coffee fermentation to remove mucilaginous coat from coffee beans.
• Pectinase used in the extraction of pigments, food coloring from plant based raw materials.
• Pectinase used in the Industrial manufacture of Feeds.
• Pectinase used in Retting (separation of Fiber from the plant parts).

15. PROTEASES:
• Proteases represent a broad group of enzymes which break down or
hydrolyze proteins or peptides. Complex mixtures of Proteinases and
Peptidases are called Proteases.
• Proteases act on the peptide bonds joining the adjacent amino acid
residues in a protein molecule and cleave them leading to formation of
shorter peptides and amino acids.Proteases occupy a central position
in commerce, accounting for nearly 65% of the global enzyme market.
• Proteases are hydrolytic enzymes are ubiquitous in nature and have
been found in eukaryotes (plants, animals, fungi) and prokaryotic
domains of bacteria and archaea. Even several Viruses are also known
to encode their own proteases. Cysteine proteases, identified in
Adenovirus, Alphavirus; Serine protease reported in Hepatitis C
Virus; Aspartic protease found in HIV.

16. TYPES OF PROTEASES:
• Proteases are classified into three sub-groups. They are:
(i) Alkaline protease (pH range 9 to 11) – Serine protease, Cysteine
protease and Subtilisin Carlsberg (most widely used Detergent
protease)
(ii) Acid protease (pH range 3.8 to 5.6) – Aspartic protease, Pepsin and
Retropepsin.
(iii) Neutral protease (pH range 5 to 8) – Neutrase (Thermolysin)

17. Alkaline protease
Bacteria - Bacillus sp.,Bacillus megaterium Enterococcus hirae, Pseudomonas
aeruginosa,Streptomyces griseus and Streptomyces rectus.
(Proteases are mainly produced from Bacillus licheniformis)
Fungi – Aspergillus niger, Aspergillus oryzae, Aspergillus flavus, Aspergillus
sojae & Chetomium globosum
Acid protease
Acid proteases are found in animal cells, yeast and molds but never in bacteria.
Microbial renin-like enzymes are derived from Mucor sp..Aspergillus sp. And
Rhizopus spp.
Neutral protease
Bacteria - Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Pseudomonas
aeruginosa and Streptomyces griseus.
Fungi – Aspergillus oryzae and Aspergillus sojae

18. ALKALINE PROTEASE(FERMENTATION
MEDIUM)
• Subtilisin Carlsberg from Bacillus licheniformis is the most widely
used Detergent protease obtained by Submerged culture method.
• In 1971, Japanese Scientist Koki Horiloshi first reported the
production of Alkali Protease from Bacteria.
• Fermentation Medium
Starch hydrolysate – 50 g/L
Soya bean meal – 20 g/L
Casein – 20 g/L
Disodium phosphate – 3.3 g/L

19. FERMENTATION CONDITIONS
 Temperature – 30 °C to 40 °C.
 pH – 7.0
 Calcium carbonate is used to maintain Neutral pH.
 The Acid Protease production begins after 10 – 20 hrs of microbial
cultivation.
 Duration of Fermentation – 2 to 3 Days.
 Yield – 10 % of the initial Protein content of the medium.
 Enzyme stability – Stable in Liquid preparation and the Liquid
preparation contains 2 % of enzyme

20. ACID PROTEASE(FERMENTATION MEDIUM)
Mostly produced by Fungi through Semi-solid culture or Submerged
culture.
Normally, the Fermentation medium contains 60 % of Wheat bran with
water.
Fermentation Medium
Starch – 4 g/L
Soya bean meal – 3 g/L
Ground Barley – 10 g/L
Calcium carbonate – 0.5 g/L

21. FERMENTATION CONDITIONS
• Temperature – 30 °C.
• pH – 7.0
• Duration of Fermentation – 3 Days.
• Yield – 3200 Soxhlet units per gram of Wheat bran in Mucor pusillus
fermentation and 10000 to 150000 Soxhlet units per ml of
Fermentation medium in Mucor miehei fermentation

22. RECOVERY AND PURIFICATION
(ALKALINE AND ACID PROTEASE)
Different methods can be applied for purification of enzyme like
Ultrafiltration.
Chromatographic technique (ion exchange) purification by treatment
with activated charcoal and H2O2.

23. APPLICATIONS OF PROTEASE:
• Alkaline protease – Detergent industry and Leather industry.
• Acidic proteases - Soy sauce, Protein hydrolysate, Digestive aids and
in production of Seasoning material, Clearing beer and fruit juice,
Improving texture of flour paste and Tendering the fibril muscle.
• Neutral protease - Food industry and Brewing industry.

24. THANK YOU…