Lecture ii

Industrial fermentation is comprised of two main stages

· Upstream Processing (USP) · Downstream Processing (DSP)

• 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.

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

• 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

Downstream – ‘after the fermentation process’

Primary ‘unit operations’ of Downstream Processing Cell recovery/removal

Centrifugation Dewatering

Ultrafiltration Precipitation Spray drying

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

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

Adsorption chromatography Ion exchange 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(anionic) ion

exchange matrix

Net negatively charged (cationic)

protein at selected pHProtein binds to matrix

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

Matrix Spacer arm

Affinity ligand

+

Active-site-bound enzyme

1. Substrate analogue affinity chromatography

Matrix Spacer arm

Antibody ligand

+

Antibody-bound enzyme

2. Immunoaffinity chromatography

Protein epitope

Enzyme

• 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

Large protein Small protein

Short path length Longer path length

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