1. Lecture ii

2.  Industrial fermentation is comprised of two
main stages
 · Upstream Processing (USP)
 · Downstream Processing (DSP)

3. • Involves all factors and processes leading to and
including
• the fermentation.
• · It consists of three main areas:
• PRODUCER MICROORGANISM.
• This include processes for
• a. obtaining a suitable microorganism
• b. strain improvement to increase the productivity and
• yield
• c. maintenance of strain purity
• d. preparation of suitable inoculum
• FERMENTATION MEDIA.
• FERMENTATION PROCESS.

4.  It is the collective term for the processes
that follows fermentation .
 a. cell harvesting
 b. cell disruption
 c. product purification from cell extracts or
the growth medium

5. • Recovery of cells and/or medium
(clarification)
– For intracellular enzyme, the cell fraction is
required
– For extracellular enzymes, the culture medium is
required
• On an industrial scale, cell/medium
separation is almost always performed by
centrifugation
– Industrial scale centrifuges may be batch,
continuous, or continuous with desludging

6.  Downstream – ‘after the fermentation process’
 Primary ‘unit operations’ of Downstream
Processing
 Cell recovery/removal
 Centrifugation
 Dewatering
 Ultrafiltration
 Precipitation
 Spray drying

7.  Dewatering of whole cell fraction (use
centrifugation)
 Dewatering of culture medium or a lysed cell
fraction (for recovery of a soluble protein fraction)
 S Precipitation
 Salting out – addition of a high concentration of a soluble
salt (typically ammonium sulphate) causes proteins to
aggregate and precipitate
 Ultrafiltration
 The solution is forced under pressure through a membrane
with micropores, which allows water, salts and small
molecules to pass but retains large molecules (e.g.,
proteins)
 Spray drying
 Requires use of heat to evaporate water – unsuitable for
most proteins

8.  Sonication
 Use of high frequency sound waves to disrupt cell walls and
membranes
 Can be used as continuous lysis method
 Better suited to small (lab-scale) operations
 Can damage sensitive proteins
 Pressure cells
 Apply apply high pressure to cells; cells fracture as pressure
is abruptly released
 Readily adapted to large-scale and continuous operations
 Industry standard (Manton-Gaulin cell disruptor)
 Enzymic lysis
 Certain enzymes lyse cell walls
 Lysozyme for bacteria; chitinase for fungi
 Only useful on small laboratory scale

9.  Adsorption chromatography
 Ionexchange chromatography – binding and
separation of proteins based on charge-charge
interactions
 Proteins bind at low ionic strength, and are eluted
at high ionic strength
+ + - - -
+ +
+ + + +
+ + +
+ + + +
+ - - + -
+ -
+ +
+
Positively charged Net negatively
(anionic) ion charged (cationic) Protein binds to matrix
exchange matrix protein at selected pH

10.  Binding of a protein to a matrix via a protein-
specific ligand
 Substrate or product analogue
 Antibody
 Inhibitor analogue
 Cofactor/coenzyme
 Specific protein is eluted by adding reagent
which competes with binding

11. 1. Substrate analogue affinity chromatography
Affinity Enzyme
ligand
+
Matrix Spacer arm Active-site-bound enzyme
2. Immunoaffinity chromatography
Antibody Protein epitope
ligand
+
Matrix Spacer arm Antibody-bound enzyme

12. • Also known as ‘size exclusion
chromatography’ and ‘gel filtration
chromatography’
• Separates molecules on the basis of
molecular size
• Separation is based on the use of a porous
matrix. Small molecules penetrate into the
matrix more, and their path length of elution
is longer.
• Large molecules appear first, smaller
molecules later

13. Large protein Small protein
Short path length Longer path length

14. 1. Enzyme preparations for animal feed
supplementation (e.g., phytase) are not
purified
2. Enzymes for industrial use may be partially
purified (e.g., amylase for starch industry)
3. Enzymes for analytical use (e.g., glucose
oxidase) and pharmaceutical proteins (e.g.,
TPA) are very highly purified