Process Development for the Production of Amidohydrolase in Escherichia coli

• Main Authors: Tzong-Yuan Juang, 莊宗原
• Other Authors: Yun-Peng Chao
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/92416520184252794114

碩士 === 逢甲大學 === 化學工程學系 === 87 === Abstract Optically active D-amino acids are widely used in the pharmaceutical field as intermediates for the synthesis of b-Lactam antibiotics. The optically active D-p-hydroxyphenylglycine (D-HPG) can be produced in a hydantoin-transforming reaction, starting from DL-p-hydroxyphenylhydantoin (DL-HPH) In this two-step reaction, D-hydantoinase first converts DL-HPH to N-carbamoyl-D-p-hydroxyphenylglycine (CpHPG), and subsequent hydrolysis of CpHPG to HPG is mediated D-stereospecifically by amidohydrolase. In light of establishment of the process for preparation of D-HPG from CpHPG, we have attempted to clone amidohydrolase from Agrobacterium radiobacter NRRL B11291 into a variety of cloning vectors and expressed in distinct E. coli strains. In addition, to overproduce amidohydrolase, we have tried to set up fermentation conditions for the optimum production. In an effort to investigate D-HPG production by D-carbamoylase-catalyzed reaction, we demonstrate the feasibility of using recombinant E. coli cells for this transformation. The result showed that with recombinant cells a conversion yield of 98% in 5 hours and productivity of 1.9 g/l/hr could be obtained, which accounted for 55-fold increase compared to A. radiobacter. Employing the neural-network experimental design, we have formulated culture medium for amidohydrolase production (increasing enzyme activity to 0.171 U/mg-DCW from 0.121 U/mg-DCW). In general, the production of amidohydrolase can be influenced by the seeding time and dissolved oxygen (DO) strength. The best strategy for high production of amidohydrolase is to employ the seeding culture at growth phase and the DO concentration should be maintained above 45% staturation air.By applying the fed-batch fermentation, the production yield of amidohydrolase can be increased to 810 U/L, which accounted for 3.5-fold increase as compared to that obtain in the batch fermentation.