Industrial Production of Enzyme

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 of enzymes

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).

Enzyme EC Sources Application

-Amylase 3.2.1.1 Aspergillus E Baking

Catalase 1.11.1.6 Aspergillus I Food

Cellulase 3.2.1.4 Trichoderma E Waste

Dextranase 3.2.1.11 Penicillium E Food

Glucose oxidase 1.1.3.4 Aspergillus I Food

Lactase 3.2.1.23 Aspergillus E Dairy

Lipase 3.1.1.3 Rhizopus E Food

Rennet 3.4.23.6Mucor miehei

E Cheese

Pectinase 3.2.1.15 Aspergillus E Drinks

Protease 3.4.23.6 Aspergillus E Baking

E: extracellular enzyme; I: intracellular enzyme

Fungal enzymes

Enzyme Sources Application

-Amylase 3.2.1.1 Bacillus E Starch

-Amylase 3.2.1.2 Bacillus E Starch

Asparaginase 3.5.1.1Escherichia coli

I Health

Glucose isomerase

5.3.1.5 Bacillus IFructose syrup

Penicillin amidase

3.5.1.11 Bacillus IPharmaceutical

Protease 3.4.21.14 Bacillus E Detergent

Bacterial enzymes

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

-use all available databases to search for mention of the enzyme in the

academic and patents literature.

-screen for new microbial strains

ProceduresLocation of a sources 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

ProceduresDetermination of the Properties 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.

ProceduresDetermination of the acceptability of Enzyme

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.

ProceduresScale up of Production

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 purificationThe effect of number of steps on the yield and costsin a typical enzyme purification process.

StepSpecific activity

Total costCost 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

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.

ConstituentComposition

(%)

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%

Detergent

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 

-Lactamase 3.5.2.6 Penicillin allergy

Urokinase 3.4.21.31 Blood clots

Rhodanase 2.8.1.1 Cyanide poisoning

Uricase 1.7.3.3 Gout

Medical Application

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

V=K5

Estimation of inhibited enzyme kinetics

• Determine the type of inhibition.

• Determine the parameters for Michaelis-Menten equation without inhibition.

• Determine the parameter of KI for inhibited kinetics.

Substrate inhibition

2/1)'(max][ SIKmKS

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

2/1)'(max][ SIKmKS

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 50000 100000 150000

1/[s]

1/v

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

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.

In surface-bound enzymes on nonporous support materials.

Consider external diffusion rate (liquid film mass transfer rate)

EPk

ES 2E+S

Diffusion effects in surface-bound enzymes on nonporous support materials.

][

]['])[]([

sSmKsSmV

sSbSLksJ

At steady state, the reaction rate is equal to the external diffusion rate:

With the equation and known Sb, KL, Vm’ or Km, to determine numerically or graphically:- The substrate concentration at the surface.- The reaction rate.

Graphical solution for reaction rate per unit of surface area for enzyme immobilized on a non-porous support

])[]([ sSbSLksJ

Diffusion effects in surface-bound enzymes on nonporous support materials.

To increase the overall reaction rate with external diffusion limitation

-Increase the bulk concentration of substrate.

-Increase the liquid film mass transfer coefficient kL.

][

'

diffusion of rate maximum

reaction of rate maximum

bSLkmVDa

Summary of Diffusion Effects

At steady state, the reaction rate per unit volume is equal to the rate of net substrate gain in regard to the intraparticle diffusion.

In surface-bound enzymes on porous support materials.

Consider intraparticle diffusion rate.

is the effectiveness factor.

11

),( f

the rate is diffusion limited.

the rate is reaction limited.

][ sSmK

eDsSmVR"

.limitationdiffusion without ratereaction

limitationdiffusion cleintraparti with ratereaction

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-molecule-weight substrate may be reduced.

Steric hindrance