Improvement of the thermostability of expandase from Streptomyces clavuligerus

• Main Authors: Yu-Ching Lin, 林育靖
• Other Authors: Ying-Chieh Tsai
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/78257844643820553929

碩士 === 國立陽明大學 === 生物化學研究所 === 90 === The deacetoxycephalosporin C (DAOC) synthase from Streptomyces clavuligerus catalysed the oxidative ring expansion that converts penicillin N into DAOC and so called expandase. 7-aminodeacetoxy cephalosporanic acid (7-ADCA) is a key precursor for cephalosporin synthesis. However, the process of oxidative ring expansion is very complicated, and it’s necessary to use very dangerous chemical reagents which cause environmental pollution. Therefore, in order to reduce the environmental pollution from the expansion, scientists expect to use the characters of expandase to replace the complex chemical expansion. Streptomyces clavuligerus expandase was encoded from cefE gene and the molecular weight of expandase is 34.6 kDa. It’s not a thermostable enzyme because expndase loses 50% of activity after heated under 40 ℃ for 10 min.. So it is necessary to enhance the thermostability of expandase to make immobilized enzyme technique possible to be applied in the product of 7-ADCA in the industry. In this study, cefE gene in the recombinant plasmid pYB4 was modified by random mutagenesis. After screening 11,352 transformants, we selected 6 thermostable mutants by using the screening system established from our laboratory. The 6 mutants were named as YH 2, YH 3, YH 4, YH 5, YH 6 and YH 7 and were purifed by anion-exchange and gel filtration chromatographies. In thermostability study, t1/2 of YH 2 mutant was 60 minutes at 45 ℃, and it was increased 20 minutes than wild type enzyme. The optimal temperature of YH 2 mutant was 30 ℃ as well as wild type enzyme. The Km and kcat value of YH 2 for substrate penicillin G were 2.45 mM and 0.0450 S-1 and were almost the same as the wild type enzyme (Km= 2.58 mM, kcat= 0.0302 S-1). These results have demonstrated that the mutation of YH 2 mutant enhances the thermostability of expandase and can’t affect catalysis of expandase.