Production of Enzymes Production of Enzymes

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Contents
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Production of enzymes
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LastBenchPharmacist.blogspot.com
Objectives
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• At the end of this lecture, student will be able to
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– Discuss on production of peroxidase, catalase and penicillinase
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Production of Enzymes_ Peroxidase
Peroxidase:
• Heme proteins and contain ferriprotoporphyrin IX as prosthetic group
• Higher plants, animals and microbes
• Catalyse oxidation of a substrate by hydrogen peroxide
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• Horseradish peroxidase catalyses the oxidation of a number of different reducing
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substances (dyes, amines, hydroquinones etc.) and the concomitant reduction of
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hydrogen peroxide
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• Oxidation of neutral ferrocene to ferricinium in the presence of hydrogen peroxide
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2[Fe(Cp)2] + H2O2 + 2H+= 2[Fe(Cp)2]+ + 2 H2O

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Peroxidase:
Classes:
• I – prokaryotic origin, intracellular
• II-Secretory fungal peroxidases
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• III- Extra cellular plant enzymes secreted into cell wall or surrounding medium
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Sources
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• Bacteria: Pseudomonas aeruginosa, P. stutzeri,
stutzeri P. qianpuensis, Bacillus megaterium,
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Serratia marcescens etc.,
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• Fungi: Phlebia radiata, A. niger, Chrysoporium spp, Coprinellus radians, Lentinus

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edodes, etc.,
Peroxidase:
Production: From A. niger by fermentation
Source: A. niger
Maintenance: Potato Dextrose Agar (PDA) media at 4oC and subcultured for every 30 days
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Inoculum preparation:
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• 5 days old sporulated PDA slant added with sterilized distilled water
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• Inoculum used for production
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Enzyme production:
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• Media: Glucose (10g), yeast extract (2

2g), Ammonium nitrate (0.2g), Magnesium
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sulphate (0.5g), Potassium hydrogen phosphate (1g), Sodium dihydrogen phospahte

(0.4g) pH 6.5, distilled water upto 1L
• Inoculum added to sterilized media and incubated at 25oC on rotary shaker at 160rpm
for 12 days
Peroxidase:
Production: From A. niger by fermentation
Purification:
• By ammonium sulphate precipitation at 70% concentration
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• Dialysis, ion exchange chromatography, gel filtration methods
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Applications:
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• In analysis and diagnostic kits: Immobilized enzyme based biostrip to estimate
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galactose
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• Detection of cholesterol level: Cholesterol esterase, HRP

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• Immunoassays: HRP
• Peroxide biosensors: Peroxidase based electrodes to determine H2O2 and organic
hydroperoxides
• Paper pulp industries, decolorization of synthetic dyes (HRP) etc.,
Production of Enzymes_ Catalase
Catalase:
• Catalyses hydrogen peroxide to water and molecular oxygen
• Used to remove residual hydrogen peroxide (Bleaching, Pasteurization)
Classes: Based on physical and biochemical properties
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Mono funtional haeme catalase (classical):
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• Animals, plants, microbes; 200-340 kDa, haeme in prosthetic group
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Catalase peroxidase (atypical):
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• Aerobic bacteria, 120-340 kDa, not found in plants and animals
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Non haeme catalase (Pseudocatalase):

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• Manganese in active center, 170-210 kDa

Minor catalases:
• Haeme containing proteins with low catalytic activity
• Includes bifunctional enzymes: Catalase phenol oxidase
Catalase:
Sources:
• Bacteria: Staphylococci, Pseudomonas, Bacillus, Micrococcus spp., etc.,
• Fungi: Aspergillus fumigatus, Candida albicans
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Production from Bacillus subtilis:
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Stock culture: Slant culture maintained in malt extract 1.5%, agar 2% at pH 7.5
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Seed inoculum: Liquid seed medium with malt extract incubated at 37oC for 48 hrs at 7.5
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pH to develop seed culture
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Production medium: Glucose (10g), Sodium nitrate (5g), A, Magnesium sulphate (0.5g),
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Potassium dihydrogen phosphate (0.6g), disodium hydrogen phospahte (9.5g), ferrous

sulphate (0.0025g), pH 7-7.5, distilled water upto 1L
Basic flask culture: Seed culture added to fermentation culture, incubated at 37oC for 36-
40 hrs at 200 rpm
Catalase:
Fermentation:
• Stirred fermentor
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• 5% 12 hr old basic flask culture added to fermentation media
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• Fermentation period of 48-53 hrs
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• 1 vvm aeration, 400 rpm
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• 37oC for first 10 hrs, 35oC for next 10-20 hrs and at 32oC later
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• Addition of proteinase inhibitors at 36 hrs increases catalase production

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Catalase:
Purification:
• After fermentation cells were recovered by centrifugation and suspended in 50mM
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phosphate buffer at pH 7.0
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• Then mixed with 300 ppm lysozyme and incubated at 30oC for 1 hr
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• Filtered through diatomaceous earth followed by membrane filtration and further
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concentrated by ultrafiltration
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• Purified by ammonium sulphate precipitation to obtain 30-35% ammonium sulphate

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saturation
• Finally purified by eluting with 50 mM phosphate buffer through DEAE A-500 column
Catalase:
Applications:
• Detection of calcium in milk
• Removal of H2O2 after pasteurization
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• As preservatives: catalase with glucose oxidase
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• As anticorrosive agent
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• Removal of H2O2 from beverages
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• As functional food
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• For its immunomodulatory potential, removal of H2O2 from blood, artificial blood
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substitute with other enzymes
Production of Enzymes_ Penicillinase
Penicillinase
• Bacterial enzyme
• Inactivates penicillin and certain cephalosporins
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• Several other hydrolytic enzymes also hydrolyse penicillin
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• Produced by many pathogenic bacteria, Staphylococcus spp, some gram negative
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bacteria of intestinal origin
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• Some species had resistance towards penicillin, especially patients infected with
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S. aureus
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• Penicllin amidase is produced by many common fungi, like Penicillium, Aspergillus and
Mucor
Penicillinase
• Divided in to two major classes based on activity
 Penicillin amidase or penicillin acylase
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 Penicillinase or β lactamase
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Penicillinase-Production
• Organism: Alkalophilic Bacillus spp.,
• Isolation medium: Glucose, 10 g; polypepton,
polypepton 5 g; yeast extract, 5 g; K2HP04, 1 g;
MgSO4 7H2O, 0.2 g; agar, 20 g and Andrade's indicator
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• Culture medium:
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 Glucose or glycerol, 2 g; polypepton,
polypepton 5 g; yeast extract, 5 g; K2HPO4., 1 g and
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MgSO4 7H2O, 0.2 g
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 pH of the medium was adjusted to 9.0
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Production:
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• In 300 ml-flask containing 100 ml of medium, Isolated strain cultivated at 30oC for 23
hr on a rotary shaker
• 19 hr after inoculation 10,000 units of benzyl penicillin was added as an inducer of
penicillinase, and cultivation was further continued for 4 hr
Summary
 Peroxidase have heme proteins and contain ferriprotoporphyrin IX as prosthetic
group that catalyse oxidation of a substrate by hydrogen peroxide
 Catalase catalyses hydrogen peroxide to water and molecular oxygen Used to
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remove residual hydrogen peroxide
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 Penicillinase is a bacterial enzyme that inactivates penicillin and certain
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cephalosporins
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 Penicillinase is produced by many pathogenic bacteria, Staphylococcus spp, and
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some gram negative bacteria of intestinal origin
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Production of Enzymes Production of Enzymes

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LastBenchPharmacist.blogspot.

2[Fe(Cp)2] + H2O2 + 2H+= 2[Fe(Cp)2]+ + 2 H2O

• Fungi: Phlebia radiata, A. niger, Chrysoporium spp, Coprinellus radians, Lentinus

• Media: Glucose (10g), yeast extract (2

sulphate (0.5g), Potassium hydrogen phosphate (1g), Sodium dihydrogen phospahte

• Detection of cholesterol level: Cholesterol esterase, HRP

Non haeme catalase (Pseudocatalase):

• Manganese in active center, 170-210 kDa

Potassium dihydrogen phosphate (0.6g), disodium hydrogen phospahte (9.5g), ferrous

• Addition of proteinase inhibitors at 36 hrs increases catalase production

• Purified by ammonium sulphate precipitation to obtain 30-35% ammonium sulphate

substitute with other enzymes

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