Large-scale Enzyme Production

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Large-scale Enzyme Production

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

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Examples of Enzymes

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

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Separation and Purification

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

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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

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Growing Enzymes

• (1) Cultivate the organisms producing the desired
  enzymes.
• Production can be regulated
• Fermentation conditions ca be optimized for
  overproduction.

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Enzymes and Sources

• Proteases
• Overproducing strains of Bacillus, Aspergillus,
  Rhizopus, and Mucor.
• Pectinases
• Aspergillus niger.
• Lactases
• Yeast and Aspergillus.
• Lipases
• Certain strains of yeast and fungi.
• Glucose isomerase
• Flavobecterium arborescens or Bacillus coagulans

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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.

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Figure 3.23
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Medical and Industrial Utilization of Enzymes

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

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Medical and Industrial Utilization of Enzymes

• Technological advances
• Increasing 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.

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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.

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Table 3.5
Table 3.6
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Industrial Protease

• Hydrolyze proteins into small peptide groups.
• Obtained from
• Bacteria (Bacillus)
• Molds (Aspergillus, Rhizopus, and Mucor)
• Animal pancreas
• Plants

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Major Uses

• Cheese making (rennet)
• Baking
• Meat tenderization (papain, trypsin)
• Brewing (trypsin, pepsin)
• Detergents (subtilisin Carlsberg)
• Tanning products
• Medical treatments of wounds

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Hebei Shenzhou Animal Medicine Co., Ltd

• Specialized manufacturer of zinc bacitracin in
  China.

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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.

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Novozymes

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

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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