Development of D-p-Hydroxyphenylglycine Bioprocessing

• Main Authors: Po Ting Chen, 陳柏庭
• Other Authors: Yun-Peng Chao
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/10020858401332489927

碩士 === 逢甲大學 === 化學工程學系 === 88 === Optically active D-amino acids are widely used in the pharmaceutical field as intermediates for the synthesis of b-Lactam antibiotics. Among the optically active D-amino acids, D-p-hydroxyphenylglycine (D-HPG) is the most importantprecursors used for the synthesis of semisynthetic cephalosporin and penicillin. D-HPG produced in a two-step reaction. First, D-hydantoinase converts DL-hydroxyphenylhydantoin (DL-HPH) to N-carbamoyl-D-p-hydroxyphenylglycine(CpHPG). Secondly,use either a chemical method or an enzymatic route enables to perform hydrolysis of CpHPG to D-HPG by N-carbamoyl-D-amino acid amidohydrolase (hereinafter carbamoylase). In this study, we cloned the gene of D-hydantoinase and carbamoylase from Agrobacterium radiobcter NRRL B11291 into Escherichia coli. By highly expressing both D-hydantoinase and carbamoylase, recombinant Escherichia coli strains were able to convert substrate DL-HPH to D-HPG with a conversion yield of 100%, accounting 20times productivity higher than that obtained by Agrobacterium radiobcter NRRL B11291. We also found that the ratio of D-carbamoylase to D-hydantoinase range between 1 and 2. In the second part of this study,we have developed a new T7 system which is under control by temperature. By using thermal shift,carbamoylase gene driven by T7 promoter was initiated to produce a large quantity of protein. It was found that the way by shifting temperature from 30℃ to 39℃ for 20 mins,followed by bringing down to 37℃ throughout the experiment appeared to the best method to optimize recombinant protein production. Besides experimenting, using the style of 30℃→39℃→37℃ to cultivate cell, we can obtain the optimal specific enzyme activity.