1. MICROBIAL ENZYMES AND ITS
APPLICATIONS
DR. N. BANU
ASSOCIATE PROFESSOR
VELS UNIVERSITY

2. PRODUCTION OF MICROBIAL
ENZYMES AND ITS APPLICATIONS
• Enzymes are the catalysts of biological systems.
• They classifies into
• Oxidoreductases, transferases, hydrolases, lyases,
isomerases and ligases.
• Sources of enzymes:
• Animal, plant and microbial.
• Amylase takadiastase, first fungal enzyme used for digestive
disorders.
• proteases – detergent industry.
• Amylases and amyloglucosidases – glucose from starch.
• Glucose isomerase – production of fructose.
• Microbial renin – cheese production.

3. Application of commercial enzymes
Industry Analytical Medicine
amylases Glucose oxidase asparaginase
proteases Galactose oxidase proteases
catalases Alcohol
dehydrogenase
lipases
isomerases hexokinase
Penicillin acylases muramidase
Cholesterol oxidase

4. Selection of suitable strains
• High yield in the shortest time.
• Extracellular enzymes are preferred
• A production strains must not produce toxic
substances and antibiotics.
• Grow on cheap nutrients.

5. Nutrient media
• Production media:
• Natural raw materials
• Synthetic
• Medium must contains: carbon, nitrogen,
mineral substances, growth factors, energy.
• Inducer.

6. Fermentation
• Surface cultures (solid-substrate cultures)
• Submerged cultures (liquid cultures)
• Surface cultures:
• Basic substrate with a high content of nutrients and a
large surface . E.g. wheat bran, rice bran, cereal
meal,with minerals and salts.
• Low water content.
• Types of processess:
• Tray process
• Drum process
• High heap process

7. Submerged cultures
• Stirred tank of 10000 to 100,000lts capacity.
• Batch operation
• Continuous – glucose isomerase.
• Drawback:
• Instability of highly mutated production
strains and
• Difficultly in sterilization of technical media.

8. Isolation of enzymes
• Mechanical: intracellular enzymes
• High-pressure homogenizers
• Agitator bead mills
• Freeze dispersion
• Ultrasonic treatment
• Enzymatic method of disintegration:
• Precipitation of proteins:
• Organic solvents
• Alcohols (ethanol, methanol, isopropanol, acetone)
• Polymers(PEG and PEI)
• Sodium sulfate and ammonium sulfate.
• Precipitation of nucleic acids:
• Manganese ii salts, streptomycin sulfate, protamine sulfate,
cetyltriethylammonium bromide and other salts.

9. Separation of solids
• Separation of cells from the culture broth
• Clarification of the crude extract after the
disintegration of the cells
• Elimination of cell fragements.
• Centrifugation, filtration, extraction, purification,
adsorption and ion-exchange chromatography,
gel filtration, affinity, column chromatography,
electrophoretic processes, concentratin,
ultrafiltration, desalting, diafiltration, dialysis,
drying, vacuum drying, freeze-drying and spray
drying.

10. Amylases
Glucose
Disaccharide maltose
Short chain polymer
Starch

11. Uses of amylases
• Productin of sweeteners for food industry
• Maltose syrup (low viscous, not crystalline and
slightly sweet and heat stable).
• Fructose production.( glu fructose)by glucose
isomerase.

12. Starch saccharification enzymes
• α – amylase, β-amylase, glucoamylases, glucose
isomerases, pullulanases and isoamylases.
• α – amylase:(1,4 glucan glucanohydrolases)
• Extracellular, hydrolyse 1,4 glycosidic bonds.
• Classification:
• Liquefaction
• Saccharogenic or gelatinization.
• pH, temp and stability plays a key role.

13. α – amylase
• Starch liquefying amylases breakdown starch
polymers but do not produce free sugars.
• Saccharogenic amylases produce free sugars.
• α – amylase producing bacteria:
• Bacillus subtilis, B. cereus, B. amyloliquefaciens,
B. coagulans, B. polymyxa, B. licheniformis,
Lactobacillus, Micrococus, Pseudomonas,
Arthrobacter, Eschericha, Proteus,
Thermomonospora and serratia.
• Fungi:
• Aspergillus oryzae, Penicillium Cephalosporium,
Mucor, Candida, Neurospora and Rhizopus.

14. α – amylase
• Batch or fed-batch.
• Formed very low during exponential growth.
• Just before the growth rate decreases and spore
formation begins, amylase production increases.
• Medium for α – amylase by B. subtilis:
• Starch-5%, NH4NO3-0.56%, sodium citrate-
0.28%, KH2PO4 – 0.13%, MgSO4. 7H2O-0.05%,
CaCl2.2H2O-0.01%, Peptone – 0.5%, yeast extract
– 0.2%, pH – 6.8.
• Max. production 45C after 18 hrs.
• Low temp. 27-30C
• 53C for Thermomonospora.

15. α – amylase from fungi
• A. oryzae, A. niger.
• They need lower deactivation temp., high
saccharifying action and low opt. pH value (4 -
5).
• Less suitable for liquefaction
• Used for manufacture of baked products.
• Manufacture of maltose rich syrups.

16. α – amylase from fungi
• Production
• Solid substrate or submerged fermentation
• Glucose acts as a repressor
• Medium
• Starch-8%,NaNo3-1.2%,K2HPo4-0.1%,MgSo4-
0.1%, Kcl-.05%, FeSo4-.003%,Mg(No3)2-.08%,
Mg(H2PO4)2-.05%, Malt – 2%, temp.-28-30C,
duration-3-4 days.

17. β-amylases
• α-1,4, glucan-maltohydrolases
• Plant origin
• Bacillus polymyxa, B. cereus, B. megaterium,
Streptomyces sp., Pseudomonas sp. and Rhizopus
japanicus.
• Wild strain-low yield
• Mutant strain-200times more enzymes.
• Bacterial amylases
• greater heat resistant
• pH opt. higher (7)
• Calcium ions not necessary for stabilization and
activation.
• Uses
• Production of maltose syrup.

18. Glucoamylase
• α- 1,4-glucan glucohydrolases
• Glucose, maltose and limited dextrins- end
products of glucoamylase action.
• Microbes:
• A. niger, A. oryzae, A. awamori, A. phoenices
or A. usamii, Rhizopus niveus, R. delemar, R.
formosaenesis,and R. Javanicus.
• Glucoamylase isoenzymes: A. awamori var.
kawachi produces 3 glucoamylases.

19. Glucoamylase
• Production
• Aspergillus sp. cultivated under submerged
fermentation.
• Temp. 30 – 35Cand 4-5 days.
• Rich medium with high starch content (20%)
• Liquefied with bacterial amylases before
sterilization.
• Not repressed by glucose.
• During fermentation, pH drops to 3 to 4.
• Culture supernatant contains proteases, cellulase,
lactase, amylase and transglucosidases together
with sugar alcohol and organic acids.

20. pullulanase
• Bacillus amylopullulyticus or Klebsiellla
pneumoniae.
• Intracellular or extracellular.
• Hydrolyse α-1,6 branches in amylopectins.
• It shorten the saccharification times with
glucoamylase and decrease the accumulation of
isomaltose and panose.
• Uses:
• Maltose rich syrup.

21. Glucose isomerase
• D-glucose ketoisomerase.
• Isomerization of glucose to fructose.
• Reversible reaction and a mixture of glucose and
fructose is produced.
• Liquid sugar: 42% fructose, 50% glucose and 8%
of other mono and disaccharides.
• Use: fructose syrup.
• Glucose isomerase is a D-xylose isomerase and
occurs in microbe that uses xylose.
• This enzyme uses bivalent cations(CO2+, Mg2+
and Mn2+ but no cofactors.
• First intracellular enzyme used at large scale
(immobilization technique)

22. Glucose isomerase
• Producers:
• Bacillus coagulants – sweetzyme Novo
Industries.
• Streptomyces phaeochromogenes
• S. rubiginosus – Optisweet,
• S. olivochromogenes
• Arthrobacter sp.Actinoplanes missouriensis
(Ketozyme, Maxazyme)
• Microbispora rosea, Nocardia asteroides,
Flavobacterium arborescens – Taka-sweet.

23. Glucose isomerase
• Production: B. coagulans.
• It is a xylose isomerase, xylose must be added
for the induction.
• Xylose is replaced by xylan or wheat bran.
• Mg2+ needed to stabilize and activate at a
conc. of .5 to 5 mmol/l.
• Calcium – competitive inhibitor.
• Glucose – repressor.
• Max. enzyme activity – 24 hrs.

24. Glucose isomerase from streptomyces
• Batch process, mutant requires xylose or
cobalt.
• Temp. – 30C
• 24-48hrs, shaken culture.
• Medium: soymeal, yeast extract, glucose,
starch, phosphate and deionised water.
• 5% inoculum, fermentor- 80 – 150 m3 vol.
• S. olivaceus NRRL 3588.

25. Flowchart for the hydrolysis of starch
42% fructose syrup (71% dry matter)
Purification, filtration and concentration
Isomerization, glucose isomerase, pH 7 – 8.5, 60 – 65C, 0.5 – 4 hrs.
Purification, filtration, ion exchange, concentration.
Saccharification Glucoamylase
pH 4.0 – 4.5 55-60C
Liquefaction by α –amylase
pH 6.7 80-105C, 2-3hrs
Starch suspension
27 – 40% pH adjustment with lime

26. L – Asparaginases (L- Asparagine
amidohydrolases)
• Antitumor agents against leukemias and
lymphomas.
• Principle: tumor cells require L-asparagine.
Asparagenase, splits the asparagine into aspartic
acid and ammonium ions.
• Production: 80 m3 fermenters. Intracellular
enzyme.
• Strains: mutants of E. coli ATCC 9637
• Serratia marcescens ATCC 60 and Erwinia
carotovora.
• Medium: 3% csl, .6% sodium acetate, .2%
ammonium sulfate , pH – 7.

27. Proteases
• Uses: detergents, pharmaceutical, leather,
protein hydrolysates production, food, film,
waste processing industry.
• Production organisms – bacteria and fungi.
• Based on the pH optimum – alkaline proteases
• Acid proteases and neutral protease.

28. Alkaline proteases
• Bacillus strains – B. licheniformis, B.
amyloliquefaciens, B . Firmus, B. megaterium and
B. pumilis.
• Streptomyces – S. fradiae, .S. griseus and S.
rectus.
• A. niger, A. sojae, A. oryzae and A. flavus.
• Best known proteases: serine proteases
• Subtilisin carlsberg- B. licheniformis
• Subtilisin BPN and subtilisin Novo – B.
amyloliquefaciens.

29. Features of detergent proteases
• Stability at high temp.
• Stability in the alkaline range (pH 9 -11)
• Stability in association with chelating agents
and perborates.

30. Fermentation
• Lyophilized cultures are used.
• Shaken flasks-initial growth, small fermenter at
30 – 37C
• Extracellular protease production regulated by
the medium composition.
• Fed-batch method, aeration and high oxygen
potential is necessary.
• Duration – 48 – 72 hrs.
• Enzyme concentrates are marketed in a
microencapsulated form.

31. Neutral proteases
• Organisms: B. subtilis, B. cereus, B. megaterium,
Pseudomonas aeruginosa, Streptomyces griseus,
A. oryzae, A. soja and Pericularia oryzae.
• Unstable. To stablilize calcium, sodium and
chloride must be added.
• Neutral pH.
• Increase in temp. – not stable.
• Inactivated by alkaline proteases.
• Uses: restricted industrial applications.
• Used in leather and food industry.

32. Acid proteases
• Acid pH range.
• Mucor and Aspergillus.
• Mucor proteases – Mucor pusillus and M.
miehei.
• Aspergillus proteases from A. oryzae and A.
niger.
• Used for the hydrolysis of soybean proteins in
the production of soy sauce.

33. Pectinases
• Hydrolysis of pectin.
• Mixture of endo and exoenzymes and also
esterases.
• A. niger and A. wentii.
• Cultivation: submerged or solid substrate.
• Use: depectinizing fruit and vegetable juices.
• Α1,4 galacturonic acid with upto 95% of the
carboxyl groups esterified with methanol.

34. Classification
• Based on point of attack on pectin.
• The methyl ester is split by pectinesterase.
• The glucoside bonds are split by hydrolysis
with endo – polygalacturonase or
exopolygalacturonases.
• Bacterial endopectate lyases
• Fungal exopectate lyase
• Endopectate lyases.

35. Fermentation with Aspergillus niger
• 60 – 80 hrs in fed-batch cultures.
• pH 3-4, 37C.
• Medium: 2% sucrose, and 2% pectin.
• Purification: biomass removal by filtration or
centrifugation
• Stabilized with chemicals
• Enzyme precipitated with organic solvents.
• Crude protein is dried.
• Uses: clarify fruit juices and grape must
• Maceration of vegetable and fruits.
• Extraction of olive oil.