| Budget Amount *help |
¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1994: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1993: ¥500,000 (Direct Cost: ¥500,000)
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| Research Abstract |
For the future biotechnological application, it is important to develop microbial enzymes which are highly active at low temperatures such as below 15ﾟC.Two approaches were taken for this purpose : (1) screening of natural bacterial strains possessing cold-adapted enzyme activities of interest, (2) genetic adaptation of a pre-exsisted enzyme to lower temperatures by means of experimental evolution. 1. In the first year of research term, "Approach 1" was tried. We screened candidates of cold-adapted protease- and lactase-producing bacteria in water and soil samples from various points of Antarctica, Lake Baikal and Japan. By an improved screening method, several strains with high activities at 10ﾟC were obtained for both model enzymes out of each 50-80 candidate strains. However, these high activities were not specific for low temperature. 2. In the second year, we constructed a novel experimental evolution system for "Approach 2", using a typical Bacillus protease "subtilisin" as a model enzyme. This system is characterized by localized and random mutagenesis for primary (activity decrease) and secondary (activity increase) mutations and selection using E.coli host-vector system. Ca.30 evolvants were obtained, one of which possessed 1.5 times k_<cat>/Km value of that of the wild-type enzyme at various temperatures including 10ﾟC.3. In the third year, the experimental evolution system was improved for obtaining subtilisin evolvants which are more cold-adapted than, or of different types from those obtained in the second year. As the result, many such evolvants were successfully obtained ; e.g., one evolvant showed ca.70% increase in activity at 10ﾟC compared with the wild-type enzyme, and another evolvant acquired a novel type of cold-adaptation (the activity increase was specific for low temperature).
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