Large-scale Enzyme Production

Tina C. Lung Syracuse University Dept. of Chemical Engineering and Material Science CEN 551-Biochemical Engineering January 22, 2004

Examples of Enzymes
Protease

(subtilisin, rennet) Hydrolases (pectinase, lipase, lactase) Isomerases (glucose isomerase) Oxidases (glucose oxidase) Produced using overproducing strains of certain organisms.

Separation and Purification

Disruption

of cells Removal of debris and nucleic acids Precipitation of proteins Ultrafiltration of the desired enzyme Chromatographic separation Crystallization Drying

Separation and Purification

The

process depends on whether or not the enzyme is intra or extracellular. Sometimes inactive (dead or resting) cells are used with desired enzyme activity in immobilized form.
No

separation and/or purification steps Reduces costs

Growing Enzymes
(1)

Cultivate the organisms producing the desired enzymes.

Production

can be regulated Fermentation conditions ca be optimized for overproduction.

Enzymes and Sources

Proteases

Overproducing strains of Bacillus, Aspergillus, Rhizopus, and Mucor.

Aspergillus niger. Yeast and Aspergillus. Certain strains of yeast and fungi. Flavobecterium arborescens or Bacillus coagulans

Pectinases

Lactases

Lipases

Glucose isomerase

Growing Enzymes (2)

(2) Cell separated from the media usually by filtration or something by centrfugation.

Depending on intra/extracellular nature of the enzyme, the cell or fermentation broth is further processed. Recovery of intracellular enzymes is more complicated and involves the disruption of cells and removal of debris and nucleic acids.

Increasing permeability of cell membrane (CaCl2 (salt) or dimethylsulfoxide (DMSO) or change in pH Last resort is cell disruption.

Figure 3.23

Medical and Industrial Utilization of Enzymes

Pharmaceutical
Wants

Uses

chirally pure compounds (IMPORTANT!). Often a particular enantiomer maybe useful while others cause side effects or no effect at all.

Medical and Industrial Utilization of Enzymes

Technological
Increasing

advances

in wider ranges of process

conditions. Enzymes grow in unusual environments (eg: deep ocean, salt lakes, and hot springs). New enzymes and better control allow the use of enzymes in more extreme environments.

Economics
Number

of enzymes made at high volume for industrial purposes evolves more slowly. 1996: sales of industrial enzymes--$372 million. 2006: expected to reach $686 million.

Table 3.5 Table 3.6

Industrial Protease
Hydrolyze

proteins into small peptide

groups. Obtained from:

Bacteria

(Bacillus) Molds (Aspergillus, Rhizopus, and Mucor) Animal pancreas Plants

Major Uses
Cheese Baking Meat

making (rennet)

tenderization (papain, trypsin) Brewing (trypsin, pepsin) Detergents (subtilisin Carlsberg) Tanning products Medical treatments of wounds

Hebei Shenzhou Animal Medicine Co., Ltd

Specialized

manufacturer of zinc bacitracin in China.

Genencor International

Second largest developer and manufacturer of industrial enzymes. Introduced the first industrial scale, recombinant enzyme in 1988. Leader in the areas of protein engineering, expression/secretion technology and enzyme-substrate interaction.

Novozymes

Novozymes.com Largest producers of enzymes. World Headquarter: Denmark (1941). More than 500 enzymes in over 130 countries.

Words of Wisdom
I

imagine a future where our biological solutions create the necessary balance between better business, cleaner environment, and better lives. ~Steen Riisgaard, President and CEO