This document discusses industrial enzymes and their production through microbial sources. It describes that enzymes can be produced from plants, animals, and microorganisms, but microbes are preferred for large-scale production due to their ability to be genetically manipulated and grown at low costs. The key steps in microbial production include identifying a suitable source microbe, inoculum preparation through screening and isolation, cultivation through solid-state or submerged fermentation, enzyme extraction from cells or culture, and purification using techniques like chromatography, electrophoresis, or adsorbent gels.

1. INDUSTRIAL
ENZYME
BY M.Sc. STUDENTS, SESSION: 2017-18

2. Presented by
Md. Rakibul Islam
Dept. of Biotechnology & Genetic Engineering
Islamic University Kushtia, Bangladesh

3. Enzymes and Industrial enzymes
 Enzymes: Enzymes are biomolecule that catalyze chemical
reaction. Nearly all enzymes are proteins. However certain
RNA molecules can be effective biocatalysts too. These
RNA molecule is known as ribozymes.
 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.
 E.g. Protease, Lactase, Amylase etc.

4. Advantages of Industrial Enzymes
 Hazards free and environment friendly production is
possible
 Purity of the product can be ensured.
 Production cost is low.
 Simple nutritional value
 Cheap carbon and energy source
 Easy genetically manipulation
 Easy product recovery

5. Disadvantages of Industrial
Enzymes
 Sometimes product stability is not good.
 Risk of Pathogenicity.
 Chance of contamination.
 Low efficiency.
 Require additional measure such as sterilization.

6. Enzyme assay

7. Enzyme assay
Enzyme assays are laboratory methods for measuring
enzymatic activity. They are vital for the study of
enzyme kinetics and enzyme inhibition.
Two types measure
1. How much with react with substrate
2. How much production of product

8. Types of assay
1. Continuous assays
Continuous assays are most convenient, with one assay giving the
rate of reaction with no further work necessary. Normally, how
much with react with substrate
2. Discontinuous assays
Discontinuous assays are when samples are taken from an
enzyme reaction at intervals and the amount of product
production or substrate consumption is measured in this sample.
Normally, how much production of product.

9. Types of continuous assays
1. Spectrophotometric assays
In Spectrophotometric assays, the course of the reaction by
measuring a change in how much light the assay solution absorbs.
It is also called colorimetric assay . Such as the MMT assay,
redox assay etc.
2. Direct versus coupled assays
Here the product of one reaction is used as the substrate of
another, easily detectable reaction.

10. Types of continuous assays
3. Fluorometric assay
Fluorescence is when a molecule emits light of one wavelength
after absorbing light of different wavelength. Fluorometric assay
use a difference in the fluorescence of substrate from product to
measure the enzyme reaction.
4. Calorimetric
Calorimetric is the measurement of the heat released or absorbed
by chemical reaction. These assay can be used to measure
reactions that are impossible to assay in any other way.

11. Types of continuous assays
5. Chemiluminescent
Chemiluminescent is the emission of light by a chemical reaction.
Some enzyme reactions produce light and this can be measured to
detect product formation.

12. Discontinuous assays
1. Radiometric
Radiometric assays measure the incorporation of radioactivity into
substrate or it release from substrate. The radioactive isotopes most
frequently used in these assay are 14C, 32P, 35S and 125I.
2. Chromatographic
Chromatographic assays measure product formation by separating the
reaction mixture into its components by chromatography. This is
usually done by high performance liquid chromatography (HPLC) but
can also use the similar technique of thin layer chromatography.

13. Sources of enzymes

14. Sources of enzymes
 Industrial enzymes are produced from-
1. plant
2. Animal &
3. microorganisms
Manufacture from the plants and animals is limited for several
reasons.
Plant source limitation:
• Cultivation of plants has restricted to some areas
• Concentration of enzymes in plant tissues is generally low.
Animal sources limitation:
• Enzymes produced as by products in meat industry
• Concentration of enzyme is very low.

15. why we prefer microorganisms for
enzyme production?
 Produced sufficient amount according all demands of market
 Possible for genetic & environmental manipulation of bacteria
& fungi.
 The diversity of enzymes available from microorganisms is
very great.
 Microbial enzymes are utilizable for quit specific application.

16. Inducible enzymes
There are only few enzymes synthesized in substantial
concentration under all conditions of growth. But some enzymes
require the presence of substrate in the medium. They are
inducible enzymes. Such as starch acts as inducer for amylase.
Characteristics:
 Many of inducible enzymes use commercially
 There biosynthesis requires the presence of substrate in the
medium. Example is given below-
enzyme organism inducer
α-amylase Bacillus spp. Starch, dextrin
Catalase Aspergillus niger Oxygen, H2O2
Lipase Candida lipolytica Sorbitan monooleate
Lactase Escherichia coli lactose

17. Catabolic repression
 Characteristics
Catabolic repression is an important part of global control system of
various bacteria & other microorganisms.
Catabolic repression allows bacteria to adapt quickly to
preferred(rapidly metabolisable) carbon and energy source first.
Which can inhibit the synthesis of enzymes involved in catabolism
of carbon sources other than the preferred one.

18. Catabolic repression
.
Enzyme Organism Repressor
α- amylase Bacillus stearothermophilus Fructose
Catalase Endomycopsis bispora Glucose, maltose
Cellulase Trichoderma viride Glucose, starch
Protease Bacillus subtilis glucose

19. Selection criteria of
microorganism for large scale
enzyme production

20. Selection criteria
Some selection criteria should be followed in the large
scale production of industrial enzyme:
 Extracellular enzyme production
 High production rate
 Productivity stabile
 Cheap substrate
 Genetically stable
 Easy product recovery
Extracellular
Intracellular
 Profitable byproduct

21. Microbial Production of
Industrial Enzyme

22. Microbial Production of Industrial
Enzyme
 Industrial enzymes are produced from plants, animals and
microorganisms, but manufacture from plant and animals is
difficult and yield is low.
 On the other hand, microbial enzymes can be produced in
meeting all demands.
 The enzyme production through microorganisms have
following steps
 Identify the source
 Inoculums preparation
 Cultivation
 Enzyme recovery
 Purification

23. 1.Identification of Source
The microorganism which will be used as source has to meet
some criteria such as:
The microorganism must be stable with respect to
productivity, requirement for culture conditions and
sporulations.
The organism must be grown in cheap substrate.
The organism should have genetic stability.
The organism should be allow genetic manipulation.
The organism should not produce toxic products.
Extracellular enzymes are preferred.

24. 2.Inoculums Preparation
Inoculums preparation consists of following
steps:
a. Screening of Microorganism:
 Screening can defined as the procedure of isolation ,detection
and separation of micro-organism from mixed population of our
interest by using highly selective procedure is called Screening.
 The main aim of screening is selection of valuable and
important micro-organism and removal of valueless micro-
organism from microbial population.

25. Inoculums Preparation
b. Isolation of Microorganisms:
Some common techniques employed for the isolation of
microorganisms are given below:
 Direct sponge of the soil
 Soil dilution
 Gradient plate method (Pour plate and streak plate
technique)
 Aerosol dilution
 Flotation
 Centrifugation.

26. Inoculums Preparation
General criteria of Isolation of Microorganisms:
 The sample (soil or water) is diluted with sterile water to which an
emulsifying agent (Tween) is added.
 Sample is thoroughly mixed and allowed to stand at room
temperature.
 Supernatant is diluted, 10-1 to 10-10
 Various culture media are inoculated with diluted samples and
incubated.
 Colonies from the plates are isolated and identified.
 The required pure strains are maintained and preserved.

27. Inoculums Preparation
c. Preservation of Pure Culture
Once a microorganism has been isolated and grown in pure culture,
it becomes necessary to maintain the viability and purity of the
microorganism by keeping the pure culture free from
contamination.
The methods involved in preservation of pure culture are:-
Periodic Transfer to Fresh Media
Refrigeration
Preservation by overlaying cultures with mineral oil
Cryopreservation
Lyophilization (Freeze-Drying)

28. Inoculums Preparation
d. Culture
 Microbiological culture, or microbial culture, is a
method of multiplying microbial organisms by letting them
reproduce in predetermined culture medium under
controlled laboratory conditions.
Isolated microorganism which is preserved is taken and
allowed to grow in small scale in culture medium.

29. 3. Cultivation
Solid Substrate Cultivation
Solid Substrate Cultivation is generally used for the
microorganism who produce extracellular enzyme.
Solid Substrate Cultivation is a biomolecule manufacturing
process used in the food, pharmaceutical, cosmetic, fuel and
textile industries.
These biomolecules are mostly metabolites generated by
microorganisms grown on a solid support selected for this
purpose.

30. Cultivation
Advantages of Solid Substrate Cultivation :
 Enzyme yield per unit volume of incubator is high.
 Power requirement is low.
 Extraction yields highly concentrated enzyme solutions.
Disadvantages of Solid Substrate Cultivation :
 Continuous operation is not possible
 Feeding substrates during cultivation is not possible

31. Cultivation
Submerged State Cultivation:
Submerged State Cultivation involves the growth of the
microorganism as a suspension in a liquid medium in which
various nutrients are either dissolved or suspended as particulate
solids in many commercial media.
Advantages Submerged State Cultivation:
 Submerged fermentation technology has the advantages of short
period, low cost and high yield.
 Purification of products is easier.
 Continuous culture is possible.

32. Cultivation
Disadvantages of Submerged State Cultivation :
Low volumetric productivity
Relatively lower concentration of the products
More effluent generation
Complex fermentation equipments

33. 4.Enzyme Recovery
Concentration and Extraction
In enzyme production, there is a very unfavorable ratio between
input of raw material and output of product.
This requires the installation of concentration procedures. For
economic purpose, a concentration up to 10-fold is usually
satisfactory for industrial enzyme preparations.
For example, enzyme products employed in detergents contain
about 5-10 percent protease while amylase preparations for use in
flour treatment contain only about 0.1 percent pure α–amylase.

34. Enzyme Recovery
Extraction of Culture:
Enzymes produced by solid substrate cultivation used to be of the
extracellular type.
Extraction of Cells:
When enzymes are produced as intracellular enzyme then
extraction of cells is necessary.
There are a number of methods for cell disruption such as chemical
and biochemical methods such as autolysis, treatment with solvents,
detergents, or lytic enzymes etc.

35. 5. Purification of Enzymes
 After extraction the second step is to purify the
enzymes. Enzyme purification has several technique
such as
 Adsorbent gel
 Electrophoresis
 Chromatography: Chromatography has several types:
 Column chromatography
 Thin layer chromatography
 HPLC
 Ion exchange chromatography
 Gel filtration

36. Extraction and purification
methods of enzyme

37. Extraction methods of enzyme
 The first step of enzyme isolation is their extraction. The
extraction technique has two types
 Extraction from solid substrate culture
 Extraction of cells
Extraction from solid substrate culture:
 Enzyme produce by solid substrate cultivation used to
be of the extracellular type.
 Microorganism produces enzymes and the enzymes
secreted in extracellular substrate.
 Enzymes are collected by extracting the extracellular
media.

38. Extraction methods of enzyme
 Extraction of cells:
 Many microorganism produce enzyme as intracellular
enzyme.
 To isolate enzyme it is must to break down the cell
wall.
 There are number of methods for cell disruption. Chemical
and biochemical methods such as autolysis, treatment with
solvent , detergents or lytic enzyme .

39. Purification of enzyme
 After extraction the second step is to purify the
enzymes. Enzyme purification has two steps:
 Preliminary purification
 Further purification

40. Preliminary purification
 After extraction, the extracting solution contains large
number of cell debris, others protein, amino acids, salt,
polysaccharide, media content etc.
 All precipitates and cell debris are then removed by
centrifugation and discarded. Polysaccharide may also be
removed by high speed centrifugation.
 The next stage of purification is to precipitate the enzyme
of interest from solution which contains nucleotides, free
amino acids, many others protein molecule.

41. Preliminary purification
 This may be achieved by altering the pH and organic
salt concentrations of the medium.
 For example, pH may be adjusted to iso-electric point
of the enzyme.

42. Further purification
 The crude extract are partially purified. The purification
may be increased by treating a variety of ways. Such as
a) Adsorbent gels
b) Electrophoresis
c) Chromatography

43. a. Adsorbent gels
 Adsorbents gels such as zinc hydroxide have been used
to remove pigments from enzymes preparations.
 A mixture of enzyme may also be absorbed by a
suitable gel like aluminium hydroxide and then
fractionated by elution with buffers of increasing ionic
solution.

44. b. Electrophoresis
 Electrophoresis is mainly an analytical procedure, it is
ideally suited to separation of small amounts of material
,but it has also been used for purification of protein.
 It is the movement of charged particle under the influence
of an electrical field.
 The ions migrate based on the electric charge of the field.
The velocity of migration of ions given by : Charge of the
particle, electrical potential , viscosity of the liquid.

45. c. Chromatography
 Chromatography is a laboratory technique for the
separation of a mixture.
 The mixture is dissolved in a fluid called the mobile phase,
which carries it through a structure holding another
material called the stationary phase.
 The various constituents of the mixture travel at different
speeds, causing them to separate.

46. Chromatography
 Chromatography has several types:
 Column chromatography
 Thin layer chromatography
 HPLC
 Ion exchange chromatography
 Gel filtration

47. Column chromatography
 Column Chromatography consists of two phases: one
mobile phase and one contiguous stationery phase.
 The stationery phase is solid and the mobile phase is
liquid.
 The compound mixture moves along with the mobile
phase through stationery phase and separates
depending on the different degree of adhesion (to the
silica) of each component in the sample or the
compound mixture.

48. Column chromatography

49. Thin layer chromatography
 Thin layer chromatography is done exactly as it says - using
a thin, uniform layer of silica gel or alumina coated onto a
piece of glass, metal or rigid plastic.
 The silica gel (or the alumina) is the stationary phase. The
stationary phase for thin layer chromatography also often
contains a substance which fluoresces in UV light.
 The mobile phase is a suitable liquid solvent or mixture of
solvents.

50. Thin layer chromatography

51. HPLC
 High performance liquid chromatography is basically a highly
improved form of column chromatography. Instead of a solvent
being allowed to drip through a column under gravity, it is forced
through under high pressures of up to 400 atmospheres. That
makes it much faster.
 It also allows to use a very much smaller particle size for the
column packing material which gives a much greater surface area
for interactions between the stationary phase and the molecules
flowing past it. This allows a much better separation of the
components of the mixture.

52. HPLC

53. Ion exchange chromatography
 Ion exchange chromatography is a technique used to separate
molecules according to their charge, for example, it can be used to
purify charged molecules such as proteins, amino acids and
nucleotides.
 An ion exchange column is composed of a gel matrix made from
beads with charged functional groups. The functional group that is
used will depend on the molecule being targeted for separation.
 For example, if the molecules to be extracted from the sample have
a positive charge, the functional groups in the column will have a
negative charge.

54. Ion exchange chromatography

55. Immobilization of Enzyme

56. Immobilization of Enzyme
 Immobilization of enzyme may be defined as confining the
enzyme molecules to a distinct phase.
 This may be achieved by fixing the enzyme molecules to
some suitable material.
 Substrates and products can move freely in and out of the
phase in which enzymes are confined.
 The material used for immobilization called carrier
matrices are usually inert polymer or inorganic material.

57. Methods of immobilization
 The methods used for immobilization of enzyme may
be classified into four groups
 Adsorption
 Covalent bonding
 Entrapment and
 Membrane confinement.

58. Adsorption
 ADSORPTION: Involves the physical binding of the
enzyme on the surface of carrier matrix.
 Carrier may be organic or inorganic.
 The process of adsorption involves the weak
interactions like Vander Waal or hydrogen bonds.
 Carriers: - silica, bentonite, cellulose, etc.
 e.g. catalase & invertase

59. Covalent bonding
 Covalent bonding based on the binding of enzymes
and water-insoluble carriers by covalent bonds.
 The strength of the binding is very strong and no
enzymes loss during use.
 The functional groups that may take part in this
binding are Amino group, Carboxyl group, Sulfhydryl
group, Hydroxyl group, Imidazole group, Phenolic
group, Thiol group, etc

60. Entrapment
 In entrapment, the enzymes or cells are not directly attached
to the support surface, but simply trapped inside the
polymer matrix.
 Enzymes are held or entrapped within the suitable gels or
fibres.
 It is done in such a way as to retain protein while allowing
penetration of substrate.
 Inclusion in gels: Poly acrylamide gel, Poly vinyl alcohol
gels.
 Inclusion in fibers: Cellulose.

61. Membrane confinement
 Enzyme molecules usually are in an aqueous solution and
they are confined within a semi-permeable membrane.
 Which generally allows the movement to the either
direction of substrate and products but does not allow the
enzyme molecule to escape.
 Here, the reaction vessel may be patitioned into two
chamber by a semi permeable membrane. One side for
enzyme and other side for substrate and product.

62. Manipulation of enzyme
biosynthesis

63. Manipulation of enzyme biosynthesis
A number of methods are available to overcome anyone
of the control mechanisms which may exert an
inhibiting effect on the production of large amounts of
a given enzyme. These techniques can be divided into
two main categories.
 Manipulation during production
 Manipulation during end product

64. Manipulation during production
 Manipulation during production can be performed by two ways.
1. Genetic manipulation
Mutation: there are two ways in which mutation can cause
overproduction of enzyme.
The first involves in the alteration in the regulation mechanism.
This mutational effect can be done in
 removal of inducer requirement,
 resistant to end product repression, and
 resistant to catabolic repression

65. Manipulation during production
 The second is the increase on copies of the gene responsible
for the production of enzyme.
 Genetic Engineering: Genetic engineering means transfer
of genes from one strain to another. Where desired gene is
isolated from one strain the transfer to another strain or
organism for maximum production.

66. Manipulation during production
2. Environmental Factor:
Manipulation in environmental factors enables the
biochemical engineer to overcome inhibition of enzyme
biosynthesis caused by regulatory mechanism.
Such environmental factors are:
 Select suitable media
 Light
 pH
 Heat

67. Manipulation during end product
End product repression of enzyme biosynthesis can be
overcome by:
 Avoiding presence of end product as medium constituent.
 Selection of regulatory mutants which are not repressed by end
products.
 Avoiding the repressing carbon source in the medium. For
example replacing of fructose by glycerol increase α-amylase
production of Bacillus stearothermophilus more than 25 fold.