MICROBIAL ENZYMES AND ITS APPLICATIONS

DR. N. BANUASSOCIATE PROFESSOR

VELS UNIVERSITY

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.

Application of commercial enzymesIndustry Analytical Medicineamylases Glucose oxidase asparaginaseproteases Galactose oxidase proteasescatalases Alcohol

dehydrogenaselipases

isomerases hexokinasePenicillin acylases muramidase

Cholesterol oxidase

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.

Nutrient media

• Production media:• Natural raw materials• Synthetic• Medium must contains: carbon, nitrogen,

mineral substances, growth factors, energy. • Inducer.

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

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.

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.

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.

Amylases

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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)

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.

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.

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.

Flowchart for the hydrolysis of starch

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.

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.

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.

Features of detergent proteases

• Stability at high temp.• Stability in the alkaline range (pH 9 -11)• Stability in association with chelating agents

and perborates.

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.

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.

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.

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.

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.

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.