Isolation of an Enzyme

Deva Krisna Kadarani MSi

Chemistry Department of FST
UIN MAULANA MALIK IBRAHIM MALANG
 Scopes

• Enzymes source characteristics (bacteria, yeast,

plant, etc)
• Buffer solution
• Isolation of enzymes
Basic step of isolation an enzyme

1) Cell disruption
2) Centrifugation
3) Decantation
 Important things on isolation of enzyme

• Objective
• Lab scale : high purity, high cost production
• Industrial scale : medium purity, low cost production
and high yields
• Source: plant, animal, microbes
• Location: intracellular and extracellular
• Stability of enzymes
 Source of enzymes

Microorganism
Plants (4%) Animal (8%) (>80%)
(Papain, bromelain) (Rennet) (Yeast, Fungi and
Bacteria)
 Plants as sources of enzymes
Industrial
Enzymes No (EC) Sources Extra/Intra
use
Actinidin 3.4.22.14 Kiwi fruit E Food

-Amylase 3.2.1.1 Malted barley E Brewing

-Amylase 3.2.1.2 Pancreas E Food

Bromelain 3.4.22.4 Pineapple latex E Brewing

-Glucanase 3.2.1.6 Malted barley E Brewing

Ficin 3.4.22.3 Fig latex E Food

Lipoxygenase 1.13.11.12 Soybeans I Food

Papain 3.4.22.2 Pawpaw latex E Meat

 Animal as sources of enzymes

Industrial
Enzymes No (EC) Sources Extra/Intra
use
Catalase 1.11.1.6 Liver I Food

Chymotrypsin 3.4.21.1 Pancreas E Leather

Lipase 3.1.1.3 Pancreas E Food

Rennet 3.4.23.4 Abomasum E Cheese

Trypsin 3.4.21.4 Pancreas E Leather

 Yeast as sources of enzymes

Enzymes No (EC) Sources Extra/Intra Industrial use

Invertase 3.2.1.26 Saccharomyces I/E Confectionery

Lactase 3.2.1.23 Kluyveromyces I/E Dairy

Lipase 3.1.1.3 Candida E Food

Raffinase 3.2.1.22 Saccharomyces I Food

 Fungi as sources of enzymes

Enzymes No (EC) Sources Extra/Intra Industrial use

Aminoacylase 3.5.1.14 Aspergillus I Pharmeceutical

-Amylase 3.2.1.1 Aspergillus E Starch

Catalase 1.11.1.6 Aspergillus I Food

Cellulase 3.2.1.4 Trichoderma E waste

Rennet 3.4.23.6 Mucor miehei E Cheese

Protease 3.4.23.6 Aspergillus E Baking

Pectinase 3.2.1.15 Aspergillus E Drinks

 Bacteria as sources of enzymes

Enzymes No (EC) Sources Extra/Intra Industrial use

Asparaginase 3.5.1.1 E. coli I Health

-Amylase 3.2.1.1 Bacillus E Starch

-Amylase 3.2.1.2 Bacillus E Starch

Glcose
3.5.1.5 Bacillus I Fructose syrup
isomerase
Penicillin
3.5.1.11 Bacillus I Pharmeceutical
amidase

Protease 3.4.21.14 Bacillus E Detergent

Pullulanase 3.2.1.41 Klebsiella E Starch

 Why microbe as best source of enzyme ?
• Low-cost production
• Easy and quick to extracted and purified
• Predictable and controlable products
• Able to used genetical manipulation
• Less toxic
General flowchart of Isolation and purification
Animal Plant Microorganisms

Grinding Fermentation
Extracellular Intracellular
Extraction enzyme enzyme

Filtration Disruption

Concentration Purification Pure Enzyme

 Type of microbial enzyme

• Intracellular enzyme • Extracellular enzyme

• Inside cell, precisely on membrane
cell
• Unstable in outer cell environment
• Isolation depend on buffer and
temperature
• Endogen enzymes
• Outside cell, diffused into
environment
• No cell wall degradation
• Using centrifugation
(supernatant), filtration and
concentration
• Exogen enzymes
 Production of microbial enzyme

• Submerged Fermentation • Surface Fermentation

• Produced enzyme by microbes on • Produced enzyme on surface of
liquid medium solid medium
• High yields • Medium yields
• Aeration is essential
Industrial Fermentors
(submerged fermentation – liquid medium)
 Growth Curve

discontinous Semi continous

 Disruption cell method
• Physical • Chemical
• Freeze thawing : N2 liquid H2O • Organic solvent : ethyl
• Osmotic pressure acetate, toluene
• Mechanic • Detergent : SDS, Triton X-100
• Solid shear : plant, bacteria with sand tween
• Liquid shear : plant and animal tissue
by using blender
• Enzymatic
• High pressure disruption : animal cell • Cellulase : plant cell
(1000 psi) and bacteria (1000-2000 • Lysozyme : bacteria
psi) • Citinase : fungi
• Ultrasonication
 Isolation method
Isolation method
Cell lysis (osmotic shock)
Enzymatic digestion
Grinding with abrasive (sand)
Ultrasonication
French press
Fractional precipitation
Protein or enzyme source
Intracellular protein (low yields but
reduced protease release)
Intracellular protein (for lab scale using
lysozyme, 37oC, 15 min, combined with
mechanical disruption
Plant tissue, bacteria
Intracellular protein
Plant tissue, bacteria
Extracellular protein
Buffer Advantage Disadvantage
Phosphate Low cost Weak at pH 8-11
Compatible into : Inhibit enzymes : dehydrogenase, kinase
• Gel permeation and DO NOT SUITABLE in anion-exchange
cation-exchange chromatography
chromatography
• Cross-linking reagents
TRIS Low cost Poor buffer below pH 7
Suitable for gel permeation Passes through biological membrane
and anion exchange Sensitive to temperature
chromatography
Borate Low cost Form complexes with oligosaccharides,
mono-
Citrate Low cost Binds to some protein and metal complex
Carbonate Low cost Limited solubility
 Effective buffer criteria
• Water-soluble
• Does not permeate biological membranes
• pKa between 6 and 8
• Minimal effect on biochemical reaction
• Chemically stable
• Does not absorb UV light
• Easy to purify
 Assignment
• Create a group (about 3-4 people) then make a presentation about
isolation and characterization of an enzyme.
• Please send me via google classroom assignment by 3 may 2021,
23:59 pm
• If you have any que about this, do not hesitate to ask me personally
or in our group chat
Thank You