Industrial Production of Enzyme
Sources of enzymes
Biologically active enzymes may be extracted from
any living organism:
Of the hundred enzymes being used industrially,
- over a half are from fungi
- over a third are from bacteria with the
remainder divided between animal (8%) and
plant (4%) sources .
Sources f Enzyme
Microbes are preferred to plants and animals as
sources of enzymes because:
- they are generally cheaper to produce.
- their enzyme contents are more predictable and
controllable.
- plant and animal tissues contain more potentially
harmful materials than microbes, including
phenolic compounds (from plants).
Fungal enzymes
Enzyme
EC
Sources
a-Amylase
3.2.1.1
Aspergillus
E
Baking
Catalase
1.11.1.6 Aspergillus
I
Food
Cellulase
3.2.1.4
E
Waste
Dextranase
3.2.1.11 Penicillium
E
Food
Aspergillus
I
Food
Lactase
3.2.1.23 Aspergillus
E
Dairy
Lipase
3.1.1.3
Rhizopus
E
Food
Rennet
3.4.23.6
Mucor
miehei
E
Cheese
Pectinase
3.2.1.15 Aspergillus
E
Drinks
Protease
3.4.23.6 Aspergillus
E
Baking
Glucose oxidase 1.1.3.4
Trichoderma
Application
E: extracellular enzyme; I: intracellular enzyme
Bacterial enzymes
Enzyme
Sources
a-Amylase
b-Amylase
3.2.1.1
3.2.1.2
Asparaginase
3.5.1.1
Glucose
isomerase
Penicillin
amidase
Protease
Application
Bacillus
Bacillus
Escherichia
coli
E
E
Starch
Starch
I
Health
5.3.1.5
Bacillus
I
3.5.1.11
Bacillus
I
3.4.21.14
Bacillus
E
Fructose
syrup
Pharmace
utical
Detergent
PROCEDURES
The screening procedure for commercial enzymes
is to screen ideas:
- to determine the potential commercial need
for a new enzyme.
- to estimate the size of the market and to
decide how much potential users of the
enzyme will be able to afford to pay for it.
E.g. entirely novel substance, or to improve a
process
agreement, discussions with potential users
Procedures
Location of a sources of enzyme
-use all available databases to search for
mention of the enzyme in the
academic and patents literature.
-screen for new microbial strains
Procedures
Determination of the Properties of Enzyme
- temperature for optimum productivity and stability
- pH optimum and stability
- kinetic constants (Km, Vmax)
- whether there is substrate or product inhibition
- the ability to withstand components of
the expected feedstock other than substrate.
- select a reactor
Procedures
Determination of the acceptability of Enzyme
Various decisions must be made concerning the acceptability
of the organism to the regulatory authorities:
-the productivity of the organism.
-the way in which the enzyme is to be isolated,
utilised (free or immobilised) and, if necessary, purified.
If the organism is unacceptable from a regulatory viewpoint
two options exist;
- to eliminate that organism & continue the screening
operation.
- to use recombinant DNA technology.
Procedures
Scale up of Production
The selected strain(s) of microbe will be grown
in pilot plant conditions.
- achieve accurate costing of processes.
-reveal imperfections, or at least areas of ignorance
which must be corrected at the laboratory scale.
-produce samples of the enzyme preparation to be used
by customers.
-produces samples for safety and toxicological studies.
Protect intellectual property generated by patenting the
enzyme or its production method or the process.
Determination of Enzyme Activity
Specific activity: the number of units of
enzyme activity per amount of total
protein.
Unit: the amount of enzyme that gives a
predetermined amount of catalytic activity
under specific conditions.
Determination of Enzyme Activity
To measure the amount of glucoamylase in a crude
enzyme preparation, 1 ml of the crude enzyme
preparation containing 8 mg protein is added to 9 ml of a
4.44% Lintner starch solution.
One unit of activity of glucoamylase is defined as the
amount of enzyme which produces a µmol of glucose
per min in a 4% solution of starch at pH 4.5 and at 60oC.
Initial rate experiments show that the reaction produces 0.6
µmol of glucose/ml-min. What is the specific activity of
the crude enzyme preparation?
Determination of Enzyme Activity
To determine the total amount of glucose produced:
10 ml X 0.6 µmol of glucose/ml-min = 6 µmol of glucose/min
= 6 units of activity
The specific activity is:
6 units of activity / total protein added
= 6 units of activity / (1ml protein solution X8 mg protein/ml)
= 0.75 units/mg protein
Cost of purification
The effect of number of steps on the yield and costs
in a typical enzyme purification process.
Specific
activity
Total cost
Cost per
weight
Cost per
activity
1
1.00
1
1.00
1
3
1.10
4
1.47
2
9
1.20
19
2.13
3
27
1.30
83
3.08
4
81
1.40
358
4.92
5
243
1.50
1536
6.32
Step
Enzyme Production at a Large
Scale
• Hydrolase: proteases, pectinase, lipase,
lactase
• Isomerases: glucose isomerase
• Oxidases: glucose oxidase
• Transferases: Rhodanase
Application of Industrial Enzyme
• Food industrial:
Starch saccharification: amylase
cheese: rennase cleaves the principal protein of milk and
causes milk to curdle and aids digestion.
Bread : amylase, protease, hemicellulases.
Fruit juice: pectinases to degrade pectins in cell walls of
fruits and vegetables
Beer: amylase, acetolactate decarboxylase.
Detergent
Constituent
Composition
(%)
Sodium tripolyphosphate (water softener, loosens
dirt)a
38.0
Sodium alkane sulphonate (surfactant)
25.0
Sodium perborate tetrahydrate (oxidising agent)
25.0
Soap (sodium alkane carboxylates)
3.0
Sodium sulphate (filler, water softener)
2.5
Sodium carboxymethyl cellulose (dirt-suspending
agent)
1.6
Sodium metasilicate (binder, loosens dirt)
1.0
Bacillus protease (3% active)
0.8
Fluorescent brighteners
0.3
Foam-controlling agents
Trace
Perfume
Trace
Water
to 100%
Medical Application
Enzyme
EC number
Use
Asparaginase
3.5.1.1
Leukemia
Collagenase
3.4.24.3
Skin ulcers
Glutaminase
3.5.1.2
Leukemia
Lysozyme
3.2.1.17
Antibiotic
Ribonuclease
3.1.26.4
Antiviral
b-Lactamase
3.5.2.6
Penicillin allergy
3.4.21.31
Blood clots
Rhodanase
2.8.1.1
Cyanide poisoning
Uricase
1.7.3.3
Gout
Urokinase
Summary of Enzyme
• Enzyme classification
• Enzyme have common catalytical features
- decrease the reaction activation energy
- does not affect equilibrium
• Enzyme special catalytic features
- Efficient
- Specific
- regulated
- versatile
Summary of Simple Saturation
Kinetics
• Michaelis-Menten Approach
• Briggs-Haldane Approach
• Use these two approaches to derive
enzyme catalytic reaction.
• Use experimental data to obtain
parameters of Michaelis-Menten kinetics.
V= K5[ES]2
K5
V=
Estimation of inhibited enzyme
kinetics
• Determine the type of inhibition.
• Determine the parameters for MichaelisMenten equation without inhibition.
• Determine the parameter of KI for inhibited
kinetics.
Substrate inhibition
' K )1/ 2
[S ]max  (K m
SI
Summary of Inhibited Kinetics
• For reversible enzyme inhibition, there are
- competitive
- noncompetitive
- uncompetitive
- substrate inhibition
• Determine parameters for all these types
of inhibition kinetics.
Estimation of inhibited enzyme
kinetics
Substrate inhibition
' K )1/ 2
[S ]max  (K m
SI
0.14
0.12
1/v
0.1
0.08
0.06
0.04
0.02
0
0
50000
100000
1/[s]
150000
Summary of Immobilization
Methods
Methods of Enzyme immobilization:
- Entrapment
- matrix
- membrane (microencapsulation)
- Surface immobilization
- physical adsorption
- ionic binding
- covalent binding
- Cross-linking
Summary of Diffusion Effects in
Immobilized Enzyme System
- Determine the support to be non-porous or porous.
- Identify the substrate determining the reaction rate.
- Conduct mass balance of the substrate of interest.
Accumulation of substrate of interest =
rate of substrate gain - substrate consumption rate
(production formation rate, or reaction rate)
At steady state,
Rate of substrate gain = substrate consumption rate
Summary of Diffusion Effects
In surface-bound enzymes on nonporous support
materials.
Consider external diffusion rate (liquid film mass transfer rate)
At steady state, the reaction rate per unit surface area is
equal to the rate of net substrate gain in regard to the
external diffusion.
E+S
k2
ES  P  E
Diffusion effects in surface-bound enzymes
on nonporous support materials.
At steady state, the reaction rate is equal to
the external diffusion rate:
Vm '[ S s ]
J s  k L ([ Sb ]  [ S s ]) 
K m  [S s ]
With the equation and known Sb, KL, Vm’ or Km,
to determine numerically or graphically:
- The substrate concentration at the surface.
- The reaction rate.
J s  kL ([Sb ]  [Ss ])
Graphical solution for reaction rate per unit of surface area
for enzyme immobilized on a non-porous support
Diffusion effects in surface-bound enzymes
on nonporous support materials.
To increase the overall reaction rate
with external diffusion limitation
Vm '
maximum rate of reaction
Da 

maximum rate of diffusion k L [ Sb ]
-Increase the bulk concentration of substrate.
-Increase the liquid film mass transfer coefficient kL.
Summary of Diffusion Effects
In surface-bound enzymes on porous support
materials.
Consider intraparticle diffusion rate.
At steady state, the reaction rate per unit volume
is equal to the rate of net substrate gain in regard
to the intraparticle diffusion.

is the effectiveness factor.
reactionrate with intraparticlediffusion limitation

reactionrate without diffusion limitation.
  f (, b )
 R
Km
b
[S s ]
"
Vm
S s De
 1
 1
the rate is diffusion limited.
the rate is reaction limited.
β
η
Ф
η
At specific conditions (T, P) for a fixed system,
To increase the intra-particle mass transfer rate:
- Decrease the size of immobilized enzyme particle
- Increase the substrate concentration
- Increase the porosity or specific surface area of
the particle
Electrostatic and Steric Effects in Immobilized
Enzyme Systems
- The optimum pH for immobilized enzyme
system will shift from that of soluble free enzyme
Electrostatic effect
- The activity of enzyme toward a high-moleculeweight substrate may be reduced.
Steric hindrance