Chapter 3 enzyme production and purification 20141009

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 μm)

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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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