7-2.5 Production of recombinant microbial enzymes:

The production of enzymes by fermentation process was an established commercial process before modern microbial biotechnology. The microorganisms used for enzyme production are grown in fermenters using optimized growth medium. The enzymes produced by the microorganism may be intracellular or secreted into the extracellular medium.

Recombinant DNA technology was generally used for the improvement of enzyme production. Commercially important enzymes are usually of the following types:

1) Enzymes of industrial importance: A mylases, Proteases, Chymosin, Catalases, Isomerases recombinant Lipases.

2) Enzymes used for analytical purposes, such as glucose oxidase (GOs), alcohol dehydrogenase (ADH), hexokinase, cholesterol oxidases, horseradish peroxidase (HRP), alkaline phosphatase etc.

3) Enzymes of medicinal importance: Trypsin, Asparaginase, Proteases, Lipases etc.

Industrial enzymes have now reached an annual market of US$1.6 billion. Recombinant therapeutic enzymes already have a market value of over US$2 billion, being used for thromboses, gastrointestinal and rheumatic disorders, metabolic diseases and cancer. They include tissue plasminogen activator (tPA), human DNase and Cerezyme.

•  The most common strategy employed to date has been the use of recombinant DNA techniques to engineer microorganisms to over-express the desired enzymes, including heterologous enzymes derived from other species which are not naturally found in the host species.

•  Genetic engineering could be used to give ethanol fermenting microbes the metabolic pathways needed to utilize sugar sources such as the 5-carbon xyloses and other pentoses that are released from hydrolysis of woody biomass.

•  The alternative approach would be to splice genes encoding the enzymes making up the ethanol fermentation pathway and into an organism lacking that trait but having the ability to digest the complex cellulosic components. 

•  A variation of this strategy is to create and use engineered microbes to manufacture novel or improved industrial enzymes, which can then be used as a catalyst in fuel fermentations to enhance or accelerate biofuel production processes. Enzymes like cellulases, amylases, and other degradative enzymes can be used to pre-treat cellulosic feedstocks, or can be added to ethanol production process at any other suitable time. Calf chymosin (prochymosin) was cloned and expressed in E. coli (first genetically engineered protein approved for human consumption, 1990)

•  Current focus is to use synthetic biology and metabolic engineering to create novel or synthetic microorganisms possessing enzymatic capabilities not found in the original host organism.

•  This strategy might involve designing an “optimal” organism using combinations of enzymes from other sources, or even completely new enzymes designed and created using protein engineering to have maximal catalytic activity.