SOURCES OF ENZYMES

Of the hundred or so enzymes being used industrially, over a half are from fungi and yeast and
over a third are from bacteria with the remainder divided between animal (8%) and plant (4%)
sources. Microbes are preferred to plants and animals as sources of enzymes because:

1. Faster growth rate of microbes.

2. They grow on relatively cheap media (compared to plants and animals)
3. Genetic manipulation of MO is easy.
4. Their enzyme contents are more predictable and controllable,
5. No seasonal fluctuations as in plants.
6. Plant and animal tissues contain more potentially harmful materials than microbes,
including phenolic compounds (from plants), endogenous enzyme inhibitors and
proteases.

Some important industrial enzymes and their sources.

Intra/extra
Enzymea EC numberb Source Industrial  use
-cellularc
Animal enzymes
Catalase 1.11.1.6 Liver I Food
Chymotrypsin 3.4.21.1 Pancreas E Leather
Lipasee 3.1.1.3 Pancreas E Food
Trypsin 3.4.21.4 Pancreas E Leather
Plant enzymes
-Amylase 3.2.1.1 Malted barley E Brewing
-Amylase 3.2.1.2 Malted barley E Brewing
Papain 3.4.22.2 Pawpaw latex E Meat
Bacterial enzymes
-Amylase 3.2.1.1 Bacillus E Starch
-Amylase 3.2.1.2 Bacillus E Starch
Asparaginase 3.5.1.1 Escherichia coli I Health
Glucose isomeraseh 5.3.1.5 Bacillus I Fructose syrup
Proteasei 3.4.21.14 Bacillus E Detergent
Fungal enzymes
-Amylase 3.2.1.1 Aspergillus E Baking
Cellulase 3.2.1.4 Trichoderma E Waste
Glucose oxidase 1.1.3.4 Aspergillus I Food
Lactasel 3.2.1.23 Aspergillus E Dairy
Lipasee 3.1.1.3 Rhizopus E Food
Pectinasen 3.2.1.15 Aspergillus E Drinks
 Proteasem 3.4.23.6 Aspergillus E Baking
Yeast enzymes
Invertasep 3.2.1.26 Saccharomyces I/E Confectionery
Lactasel 3.2.1.23 Kluyveromyces I/E Dairy
Lipasee 3.1.1.3 Candida E Food

In practice, the great majority of microbial enzymes come from a very limited number of genera,
of which Aspergillus species, Bacillus species and Saccharomyces species predominate.

Criteria for the selection of source -

 High yielding
 Produce only extracellular enzymes
 Able to grow on simple and cheap medium
 Not produce toxic by products
 Recovery and purification easy

SCREENING FOR NOVEL ENZYMES

1) The first stage of the screening procedure for commercial enzymes is to screen ideas, i.e. to
discuss the techno-economic viability of the process taking into account all the factors that
could affect the process.
2) The next stage involves the search for location of a source of the required enzyme. Cultures
may then be sought from any sources so revealed. If these first searches are unsuccessful, it is
probably necessary to screen for new microbial strains capable of producing the desired
enzyme.
3) The properties of the enzyme must be determined; i.e. temperature for optimum productivity,
temperature stability profile, pH optimum and stability, kinetic constants (Km, Vmax), whether
there is substrate or product inhibition etc.
4) Once an enzyme with suitable properties has been located, various decisions must be made
concerning the acceptability of the organism to the regulatory authorities, the productivity of
the organism, and the way in which the enzyme is to be isolated, utilised (free or
immobilised) and, if necessary, purified. (Development of the production process)
5) Once the biochemical engineers are satisfied that their initial criteria of productivity, activity
and stability can be met, the selected strain(s) of microbe will be grown in pilot plant
conditions. The enzyme pilot plant produces samples for safety and toxicological studies and
to be used by customers who may well also be at the pilot plant stage in the development of
the enzyme-utilizing process.
6) Screening for new enzymes is expensive so that the intellectual property generated must be
protected against copying by competitors. This is usually done by patenting the enzyme or its
production method or, most usefully, the process in which it is to be used.
MEDIUM FOR ENZYME PRODUCTION

 Defined media is not preferred(on cost grounds) unless when enzymes are to be produced
for high value uses, such as analysis or medical therapy where very pure preparations are
essential.
 Less-defined complex media are usually formulated based on cost and availability of
constituents. Waste materials and by-products from the food and agricultural industries
are often major ingredients.
 Thus molasses, corn steep liquor, distillers solubles and wheat bran are important
components of fermentation media providing carbohydrate, minerals, nitrogen and some
vitamins. Soybean meal and ammonium salts are frequently used sources of additional
nitrogen.

PREPARATION OF ENZYMES - Flow diagram for the preparation of enzymes

The first step in the preparation of enzymes is to separate the cells from the medium (liquid-
solid separation) which is achieved by Centrifugation or Filtration.
1) Centrifugation

Centrifugation separates on the basis of the particle size and density difference between the
liquid and solid phases. Sedimentation of material in a centrifugal field may be described by:

The efficiency of centrifugation can be improved if the particle diameter (d) is increased. This
can be done either by coagulating or flocculating particles. Coagulation is caused by the removal
of electrostatic charges (e.g. by pH change) and allowing particles to adhere to each other.
Flocculation is achieved by adding small amounts of high-molecular-weight charged materials
which bridge oppositely-charged particles to produce a loose aggregate which may be readily
removed by centrifugation or filtration.

Examples of commonly used centrifuges are :

 Tubular bowl centrifuge

 Continuous scroll centrifuge
 Continuous multichamber disc-stack centrifuge

2) Filtration

Filtration is one of the most common processes used at all scales of operation to separate
suspended particles from a liquid or gas, using a porous medium which retains the particles but
allows the liquid or gas to pass through. It separates simply on the basis of particle size.
Commonly used filter is the Rotary Vacuum Filter.

Diagram of a simple filtration apparatus :

CELL BREAKAGE
A variety of cell disruption techniques have been developed for the release of intracellular
enzymes from their respective cells. These techniques can be classified into :
 Physical Methods
 Chemical Methods

1) Use of Abrasives

 Use of pestle and mortar

 Grinding with glass beads or carborundum that act as mechanical agitators
 Use of Dyno mills and Bead mills

2) Use of Liquid and Solid shear

 The process of passing suspension of cells at high pressure through a small orifice
into a chamber at atmospheric pressure is termed Liquid shear. The cells are literally
blown apart by the sudden drop in pressure.
 When a cell paste is frozen to -20degree celsius and forced through a small orifice at
high pressure, the process is termed Solid shear.

3) Osmotic Shock

The cells are suspended in distilled water, endo-osmosis takes place and the cells burst
releasing the enzymes into the environment.

4) Ultrasonic Disruption

The treatment of microbial cells in suspension with inaudible ultrasound (greater than about
18 kHz) results in their inactivation and disruption. The sonic energy is converted into
mechanical energy in the form of shock waves equivalent to several thousand atmospheres
(300 MPa) pressure. This energy imparts motions to parts of cells which disintegrate when
their kinetic energy content exceeds the wall strength.

5) Use of Lyzozymes

Lyzozymes digests the polymeric compounds that impart the cell wall its rigidity.

6) Use of EDTA

EDTA removes the Mg ions that are essential for preserving the overall structure of cell
membrane.
7) Use of Detergents
 Detergents disrupt membranes and extract the lipoprotein fragments into the medium as
components of micelles.
 Natural bile salts – cholate
 Anionic detergents – SDS
 Cationic detergents – C-TAB
 Non-ionic synthetic detergents – Triton X-100
 Ionic synthetic detergents – CHAPS

8) Use of Alkali

Cell lysis occurs at very high pH – 11-12. But the enzymes should be stable at that pH for
atleast half an hour.

Once the cell is lysed, the components should be extracted into a medium, The nature of
EXTRACTION MEDIUM –

 EM should be cold (below 4 degrees)

 EM should be set at high pH (far from pI of the enzyme)
 Should contain reducing agent/stabilizers– mercaptoethanol
 Should contain anti-oxidant – ascorbic acid
 Should contain inhibitors of proteolytic enzymes that attack the enzyme of interest –
PMSF (PhenylMethylSulphonylFluoride)
 Chelating agents – EDTA reduce deleterious effect of phenol oxidases

Once the components are extracted, next step is Purification. The preliminary purification
procedures are:

NUCLEIC ACID REMOVAL

Extracted nucleic acids cause problems by increasing the viscosity but they can be digested by

 Addition of nuclease enzymes

 Precipitated by the addition of streptomycin, CTAB
 Divalent metal ions like Mn, Mg
 Long fragments of nucleic acids are sheared by sonication

CONCENTRATION by PRECIPITATION
Proteins show minimal solubility at their pI, but at this pH the enzymes usually loose their
biological activity and hence this concentration technique is not widely used.

The most common method of protein concentration is Ammonium Sulphate Precipitaion

(Salting-out). The salt concentration is increased in stages, the aim being to ppt other
proteins(which are then discarded) befor precipitationg the fraction containing the relevant
enzyme. This is then re-dissolved in buffer, and residual ammonium sulphate removed by
dialysis or SEC.

Further Purification Techniques -

CHROMATOGRAPHY

It is based on the partitioning of components of a mixture between two immiscible phases –

stationary and mobile phases and represents the most popular collective technique for the
purification of enzymes. The most Common amongst these are :

1) Ion-Excange Chromatography –

It relies on the attraction of oppositely charged components.

 At 1 pH above pI of a protein, its surface will have an overall negative charge and it
will bind to positively charged resin like DEAE. The binding can be reversed by
lowering the pH.
 At 1pH below pI of a protein, its surface will have an overall positive charge and it
will bind to negatively charged reisn like CarboxyMethyl group. The binding can be
reversed by increasing the pH.

2) Affinity Chromatography –

It exploits the bio-specificity of a protein like the substrate-enzyme and antigen-antibody

(bio-affinity ELISA) interactions.
 The column is packed with an inert matrix like agarose to which ligands for the
required enzyme have been attached. If the ligand is small, a spcer-arm is provided for
attachment with matrix.
 The protein mixture is applied to the column.
 The relevant enzyme binds to the immobilized ligand while the other proteins pass
through and are discarded.
 The bound is liberated by eluting with a deforming buffer which changes the
characteristics of the enzyme and no longer allows it to bund ti the ligand or using a
competitive counter ligand which displaces the immobilized ligand.
3) Size-Exclusion Chromatography
 Also called, Gel Filtration, Gel Permeation or Molecular-Sieve Chromatography
 It consists of column packed with swollen porous beads which separate the
components of a sample based on its molecular Weight/size. Cross-linked dextrans
(Sephadex), cross-linked agarose (Sepharose) and cross-linked polyacrylamide
(Biogel) are commonly used in SEC.
 Large molecules because of their size pass only through the buffer surrounding the
beads and pass quickly through the column.
 Smaller molecules can travel through the buffer surrounding the beads but in addition
they only travel through the pores present in the beads moving a larger volume of the
buffer.

ELECTROPHORESIS

ISO ELECTRIC FOCUSSING

The purified enzyme is likely to be dilute, so it can be concentrated by Ammonium-Sulphate

pptn , Ultra-filtration etc. The enzyme can be packaged in the form of powder (Lyophilization)
or crystals (crystallization).

SAFETY AND REGULATORY ASPECTS OF ENZYME USE