A STUDY OF D-HYDANTOINASE PRODUCTION FOR IN VIVO IMMOBILIZATION

• Main Authors: yi-wen chien, 簡奕文
• Other Authors: 陳姍玗
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/33587167043356909046

碩士 === 元智大學 === 生物科技與工程研究所 === 98 === Optically pure amino acid have been widely used in the medical field and is economically attractive. N-carbamoyl-D-p-hydroxyphenylglycine (CpHPG) is a precusor of D-amino acid used in the synthesis of cephalosporins, such as amoxicillin and cefadroxil. This study demonstrated enzyme immobilization by a single step to produce CpHPG. The gene encoded D-hydantoinase was separately C-terminal and N-terminal fused to the PHA phasin phaP and expressed in recombinant strain Escherchia coli starin to produce immobilized D-hydantoinase on PHA granule. The results showed that the recombinant E. coli which produced phaP-hydantoinase fusion protein coversed more CpHPG than the other. SDS-PAGE analysis showed that the D-hydantoinase immobilization was carried out. As results, the effect IPTG and arabinose induction concentration on immobilized D-hydantoinase system is 0.2 mM and 0.5%. The immobilized D-hydantoinase displayed feasible activity at pH 9 and 60OC. In comparison with free D-hydantoinase, the immobilized enzyme exhibited higher activity in CpHPG production and 3 fold increase stability. Futhermore, the immobilized D-hydantoinase could be reused for 7 times to achieve the conversion yield exceeding 90%. Overall, it suggests a potential economically application of in vivo enzyme immobilizatio