| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 90 === The subtilisin YaB produced by alkalophilic Bacillus YaB is a serine protease with special substrate specificity. Subtilisin YaB has potential to be developed into a favorable meat tenderizer due to its better hydrolytic activity toward elastin and collagen than actin and myosin. In the past years, we have improved the substrate specificity by substitution the Gly124, Gly151 at the side and the Gly159 at the bottom of the S1 binding pocket for Ala. We obtained a mutant enzyme YaB-G124A which only cuts at the Ala and Gly residues on the peptides, and expected it is a better tenderizer than wild type subtilisin YaB.
The structural gene for subtilisin YaB named ale and its promoter region was cloned in pEG124A, but it was not expressed in E. coli, but was weakly expressed in B. subtilis. To increase the production of YaB-G124A, we used different expression vector systems to express ale(G124A) in B. subtilis. Meanwhile, we modified the medium composition with expectation of improving the production of subtilisin YaB-G124A. When we use the expression vector pULI7 with spac-I promoter and increase the nutrient broth concentration from 1.6 to 4.8 %, the subtilisin YaB-G124A yield 4.2 times more pEG124A.
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