ENZYME PRODUCTION

Surface and submerged fermentation

techniques

Surface = enzyme produced on the

surface of a solid medium
Submerged = the mould or bacterium
producing enzyme is grown throughout a
liquid medium

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 ENZYME PRODUCTION

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 ENZYME PRODUCTION

1. Removal of Whole Cells

2. Collect enzyme (extracellualar/intracellular
enzyme)
3. Concentration
4. Purification
5. Characterization

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 Enzymes and Sources
Proteases
Overproducing strains of Bacillus, Aspergillus,
Rhizopus, and Mucor.
From Animal pancreas, Plants
Pectinases
Aspergillus niger.
Lactases
Yeast and Aspergillus.
Lipases
Certain strains of yeast and fungi.
Glucose isomerase
Flavobacterium arborescens or Bacillus coagulans
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 Removal of Whole Cells

Centrifugation
5000 g for 15 min for cells
10 000 g for 45 min for cell debris
High capital and running costs

Filtration: membrane filters (0.1 -10 m)

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 Removal of Whole Cells

Removal of nucleic acids

Nucleic acids increases viscosity of cellular
homogenate
difficult to process
Methods: precipitation (by polyethylenimine) or
treatment with nucleases

Removal of lipids
Removal: Glass wool or a cloth of very fine mesh
size

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 Cell Disruption

For Intracellular enzyme

Animal cells (no Cell Wall):

Potter homogenizer
Osmotic shock
Freeze-thaw cycles

Plant cells (CW):

The Waring blender

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 Cell Disruption
Microbial cells (CW):

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 Concentration

In laboratory scale, Concentration by:

Ultrafiltration
Precipitation
Ion-exchange chromatography
Dialysis (using semi-permeable membrane)
Freeze drying

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 Concentration by precipitation

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 Concentration by precipitation

Advantages
One of the oldest methods
Uncomplicated equipment
High recovery of biological activity

Disadvantages
Many precipitants are highly corrosive
Inefficient if initial protein concentration is low
Some precipitants are highy inflammable, some are
expensive
Many precipitants must be disposed carefully
In many cases, precipitant must be removed totally
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 Concentration by Ion-Exchange
Isoelectronic point of proteins are different
(+)ly charged proteins cation exchanger (CM)
(-)ly charged proteins anion exchanger(DEAE)
Elution with a high ionic strength solution

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 Concentration by Ion-Exchange

Extracellular proteins from fermentation broths

or cell culture media
Cell debris from cell homogenates

Effective and relatively inexpensive

Easily regenerated
Considerable clarification of solution
Limited amount of protein purification

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 Concentration by ultrafiltration

Ultrafiltration membranes (pore diameters: 1 20 nm)

Molecular mass cut-off: 1 300 kDa (globular proteins)

Traditional materials: cellulose acetate and cellulose

nitrate

Modern materials: PVC and polycarbonate

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 Purification

Chromatography:
Separation of different protein types from each other
according to their differential partitioning between
two phases:
1. A solid stationary phase
2. A liquid mobile phase
Separation based on size and shape, overall charge,
presence of surface hydrophobic groups, and ability to
bind various ligands

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 Different Chromatographic Techniques

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 Gel Filtration Chromatography

Size Exclusion Chromatography

Separation based on size and shape
Porous gel matrix in bead form is used:
e.g. xlinked dextran, agarose, acrylamide

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 Gel Filtration Chromatography

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 Gel Filtration Chromatography

EXAMPLES
Sephadex: dextran based, G-25 to G-200: charged
groups attached to Sephadex G-25 or G-50

Sephacryl: allyl dextran based, more rigid and

physically stable suitable for large scale
Sepharose: agarose based, lack of physical stability

Bio-Gel P: acrylamide based

A: agarose based
Fractogel: A copolymer, very high degree of
mechanical stability
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 Ion-Exchange Chromatography

PRINCIPLE
Reversible electrostatic
attraction of a charged
molecule to a solid
matrix possessing
opposite charge

Elution is done by
increasing salt
concentration or
changing pH

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 Ion-Exchange Chromatography

FACTS
Proteins possess both (+) and (-) charges
At pH=7:
Aspartic and glutamic acid have negatively
charged side groups
Lysine, arginine, histidine have positively charged
side groups

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 Hydrophobic Interaction Chromatography

9 out of 20 commonly found a.acids in proteins are

classified as hydrophobic aa

In most proteins, the majority of hydrophobic

residues are buried inside the protein

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 Hydrophobic Interaction Chromatography

EXAMPLES
Ex: octyl- and phenyl-Sepharose gels

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 Affinity Chromatography

The most powerful & highly selective method

Most proteins to bind specifically and reversibly to
their ligands
Generally used in late purification steps
Support matrix: agarose, cellulose, silica and various
organic polymers

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 Affinity Chromatography

General ligand approach: Coenzyme (NAD+) or

lectins (group of proteins synthesized by plants,
vertebrates and some invertabrates )

Specific ligand approach: enzyme-substrate,

substrate analogues or inhibitors, antibodies

Immunoaffinity: using antibody for binding

Dye affinity chromatography: Triazine dyes

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 Affinity Chromatography

Metal chelate affinity: Ni, Cu, Zn, Fe

For basic groups: side chain of His
Mostly used in recombinant protein purification

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 Chromatofocussing

Separation based on isoelectic point of

proteins

A weak anion exchanger is used

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 High Pressure Liquid Chromatography (HPLC)

Silica gel, xlinked polystyrene are generally used

Superiour resolution due to small particle size

Fast
High degree of automation

Cost
Capacity
Generally used for high value proteins
intended for therapeutic use

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 Fast Protein Liquid Chromatography (FPLC)

Operating pressure is significantly lower

Glass or inert plastic columns in stead of stainless steel
Economically more attractive than HPLC
Pharmacias BioPilot and BioProcess systems are
commercial FPLC systems designed for pilot and
industrial scale use
Flowrates up to 400 L/h are achievable in BioProcess
system

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 Expanded bed chromatography

Particulate matter in protein sample should be

removed before conventional purification procedures
Expanded bed chromatography aims to overcome
this requirement

Duration and cost decrease

Design considerations:
Bead density
Flow rate of mobile phase
Bead size distribution

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 Expanded bed chromatography

The use of beads with an appropriate diameter range is

important for the generation of a stable expanded bed (100-
300 m)

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 Purification of recombinant proteins

Specific peptide or protein tags can be incorporated

for rapid purification
Polyarginine or polylysine tag: cation exchange
chromatography
Polyhistidine tag: metal chelate chromatography
Flag (a synthetic peptide) tag: immunoaffinity
chromatography
Removal of the tag is generally desirable afterwards

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 Purification of recombinant proteins

Recombinant DNA technology is a very useful tool for

protein purification

for 'overproduction' of proteins using expression

vectors

for application of 'tags' to proteins

for excretion of proteins into the culture medium

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 Protein deactivation

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 Protein stabilization

Buffered solution
Temperature control
Minimization of processing time
Avoid vigorous agitation or addition of denaturing
chemicals
Add substances inactivating known inactivators
Include stabilizing agents
Glycerol, sugars and PEG: they decrease free
water activity
BSA: as bulking protein

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 Storage

Optimum T and pH for maximum stability

In liquid format: add stabilizing agents, filter-
sterilization is advised
In frozen format: quickly freeze the solution,
preferably in liquid nitrogen, then store in -70OC
In dry format: protein may be more stable

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 Lyophilization

Lyophilization involves the drying of protein directly

from frozen state
Freeze the sample
Apply vacuum
Increase the temperature sublimation

Many commercial proteins (e.g. vaccines, hormones,

antibodies) are marketed in freeze-dried form

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 Characterization

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 Characterization

Functional Studies

Determination of specific activity

Determination of substrate range and specifity
Kinetic characteristics
Effect of various factors on activity

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 Characterization

Evidence of purity

1-D SDS-PAGE: The most common method used is 1-D

polyacrylamide gel electrophoresis in the presence of
sodium dodecyl sulphate (SDS)

Purpose:
Determination of purity
Determination of molecular mass

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 Characterization

Isoelectric focussing: in stead of SDS, a mixture

of low molecular mass organic acids and bases
are used
A pH gradient forms in the gel
Protein will stop moving when it comes to the
pH equals its pI value

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 Characterization

2-D Electrophoresis: combines SDS-PAGE with isoelectric

focussing

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 Characterization

Capillary Electrophoresis: not in polyacrylamide gel but

along a narrow capillary tube packed with a fused
silica matrix, generally for low Mw substances.

HPLC: superior peak resolution and fast

At least 2 different HPLC column types are used

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 Characterization

Molecular Mass Determination

Mass Spectroscopy
Gel filtration analysis
Non-denaturing electrophoresis (Ferguson plot)
Analytical ultracentrifuge:
Specially designed sample cells are used
Svedberg equation is used to find molecular
mass from sedimentation coefficient

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 Enzyme Assay Methods

An assay requires to determine the concentration

of a product or substrate at a given time after
starting the reaction.

Different enzymes require different estimation

methods depending on the type of reaction
catalyzed, the nature of S and P or coenzyme.

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 Enzyme Assay Methods

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 Spectrophotometric methods

Many substrates and products of enzyme reactions absorb

light either in the visible region or in the U.V. region.

Mostly the spectra of S and P are not the same.

The conversion of one into another is followed by a
considerable change of absorption and by measuring this
change, the progress of the reaction can be followed
quantitatively.

The enzyme is allowed to react with substrate and the

decrease in the conc. of substrate or the increase of
product produced will be followed spectrophotometrically
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 Spectrophotometric methods

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 Spectrophotometric methods

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 Fluorescence Method: (Fluorimetric method)

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 Fluorescence Method: (Fluorimetric method)

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 Manometric Method

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 Electrode Method

To follow reactions which involve the production

of acids.

Use glass or platinum electrode.

In this method, pH meter is used to measure

change in H+ conc.during enzyme reactions.

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 Radiometer pH meter

Keeps pH constant by addition of acid or alkali

Automatic apparatus

Convenient

Plots a curve of amount added against time.

With this apparatus, progress cures of many

enzyme reactions can be obtained automatically
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 Polarimetric method

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 Sampling method

Many enzyme reactions are followed by withdrawing

samples at intervals and estimating the substrate or
product by chemical methods

Fiske and SabbaRow method: for inorganic

hosphate.

It can be used for phosphatase, phosphorylase,

nucleotides and all enzymes involving ATP or ADP
including some kinase and synthetase.

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 Characterization of Enzyme

1. Determine its amino acid sequence and 3-D structure.

Compare these basic structural properties of the enzyme to
other known amino acid sequences using the computer
databases very helpful in identifying invariant amino acid
residues important in the enzyme's structure and
functionality

2. Study the kinetics and substrate specificity of the enzyme

and identify inhibitors

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 Characterization of Enzyme

3. Identify key functional amino acid side chains and do 'site-

directed mutagenesis: Are they essential for catalytic activity?
Are they important for substrate binding? Are they important for
stability of the folded native state of the enzyme?

4. Make hypothesis of the chemical events and bond

rearrangements occurring during catalysis. Test this hypothesis
by 'site-directed mutagenesis' and methods to identify
'intermediates' in catalysis

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