Chapter 3 enzyme production and purification 20141009

Chromatography: Separation of different protein types from each other according to their differential partitioning between two phases: 1.. A liquid mobile phase Separation based on siz

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

ENZYME PRODUCTION

ENZYME PRODUCTION

1 Removal of Whole Cells

2 Collect enzyme (extracellualar/intracellular

enzyme)

3 Concentration

4 Purification

5 Characterization

Enzymes and Sources

• Proteases

– Overproducing strains of Bacillus, Aspergillus,

Rhizopus, and Mucor.

– From Animal pancreas, Plants

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 μm)

Removal of Whole Cells

• Removal of nucleic acids

– Nucleic acids increases viscosity of cellular

Cell Disruption For Intracellular enzyme

Animal cells (no Cell Wall):

– Potter homogenizer– Osmotic shock

– Freeze-thaw cycles

Plant cells (CW):

– The Waring blender

Cell Disruption

Microbial cells (CW):

Concentration by precipitation

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

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

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

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

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

Different Chromatographic Techniques

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

Gel Filtration Chromatography

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

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

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

Hydrophobic Interaction Chromatography

EXAMPLES

– Ex: octyl- and phenyl-Sepharose gels

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

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:

Affinity Chromatography

• Metal chelate affinity: Ni, Cu, Zn, Fe

− For basic groups: side chain of His

− Mostly used in recombinant protein purification

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

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

• Pharmacia’s 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

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

Expanded bed chromatography

• The use of beads with an appropriate diameter range is

important for the generation of a stable expanded bed

(100-300 μm)

Purification of recombinant proteins

• Specific peptide or protein tags can be incorporated

for rapid purification

– Polyarginine or polylysine tag: cation exchange

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

Protein deactivation

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

• 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

• 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

Characterization

• Functional Studies

– Determination of specific activity

– Determination of substrate range and specifity

– Kinetic characteristics

– Effect of various factors on activity

• 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

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

• 2-D Electrophoresis: combines SDS-PAGE with isoelectric

focussing

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

• 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

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

Enzyme Assay Methods

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

Spectrophotometric methods

Spectrophotometric methods

Fluorescence Method: (Fluorimetric method)

Fluorescence Method: (Fluorimetric method)

Manometric Method

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

Polarimetric method

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,

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

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