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Acquisition of Heat Stable Enzymes from Thermophilic Microorganisms: Peroxidases, Ureases, and Glucose Oxidases.
Final rept. Aug 89-Aug 91,
J K RESEARCH CORP BOZEMAN MT
Pagination or Media Count:
Thermophilic microoganisms producing heat stable peroxidases, ureases and glucose oxidase have been collected and isolated. Optimization methodology for the production and purification of these enzymes has been investigated in detail. The 16S rRNA and fatty acid analyses suggest the three organisms submitted to US Army Chemical. Research, Development and Engineering Center CRDEC have not been previously isolated and identified. The most noteworthy observation was a heat stable urease with a molecular weight MW between 120, 000 and 130,000. Considering the poor heat stability of Jack Bean urease and MW of 550,000, this heat stable urease is a particularly valuable enzyme for conjugation reactions used for a host of diagnostic assays. The bacterium producing a peroxidase with slightly better stability than a recent Japanese isolate, was observed to have a distinctive microscopic appearance and may later be classified in a new genius. A total of 4 thermophiles were submitted to CRDEC 197 for peroxidase. 408 and 429 for urease and 370 for glucose oxidase. Detailed methodology for culturing, fermenting, maintaining and assaying the enzymes are described. Fermentation evaluation in reactors to 1 5 L was accomplished and detailed instructions for these organisms for enzyme production and purification were developed. Fermentations will require close monitoring of several parameters to achieve optimum production. Further investigation on the purification of all three enzymes is essential to achieve efficient removal of protease and other non specific proteins without losing major portions of the enzymes of interest. Bacteria, Ureases, Biochemistry, Oxidase, Fermentation, Heat Stable Enzymes, Biotechnology, Thermophilic Microorganisms.
APPROVED FOR PUBLIC RELEASE