Development and in vivo immobilization of enzyme on PHA granules

• Main Authors: Teng-Yi Huang, 黃騰毅
• Other Authors: 陳姍玗
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/31063296732995047724

碩士 === 元智大學 === 生物科技與工程研究所 === 99 === The β-lactam-based antibiotics are widely used in the pharmaceutical market. D-p-hydroxylphenylglycine (D-HPG) is the most important precursor of optically active D-amino acid used for the synthesis of semisynthetic antibiotics. Production of D-HPG with a bioconversion process consists of two hydrolytic steps, which requires D-hydantoinase and amidohydrolase. First, D-hydantoinase (HDT) converts DL-hydroxyphenylhydantoin (DL-HPH) to N-carbamoyl-D-p- hydroxylphenylglycine (CpHPG). Secondly, perform hydrolysis of CpHPG to D-HPG by carbamoylase. Polyhydroxyalkanoates (PHAs), commonly known as bioplastics, are biodegradable polymers and produced by certain bacteria. They are accumulated as reserve granules in the cytoplasm when the culture conditions are not optimal for cellular growth. It is based on the production of PHA as an immobilization matrix in bacteria. In the previous study, phasin-HDT was overexpressed in Escherichia coli, and the immobilized D-hydnatoinase was utilized to enhance the hydrolysis reaction of DL-HPH by raising the PHA content. In this study, hybrid proteins consisting of D-hydantoinase and PHA granules surface protein (phasin) or PHA synthase (PhaP -HDT and PhaC-HDT) were constructed and expressed in the PHA-producing bacteria Ralstonia eutropha H16. Through the binding of phasin or PHA synthase to PHA, HDase immobilized on the PHA granules in vivo is then generated. The results indicated that the best IPTG (Isopropyl-beta-D-thiogalactoside) concentration for the expression of fusion protein in R. eutropha H16/ pBBR1MCS2-phaP-hdt and R. eutropha H16/pBBR1MCS2-phaC-hdt was 50 ?M and 100 ?M, respectively. To further characterize the immobilized D-hydantoinase, the dependence of enzymatic activity on different temperature was investigated. It was found that the optimal temperature on the immobilized PhaP-HDT and PhaC-HDT was 60oC and 55oC. As compared to its free counterpart, the immobilized PhaP-HDT and PhaC-HDT exhibited higher tolerance to heat and gained a half life of 135 h and 120 h at 40oC, which was 2.7 and 2.4 fold higher than free enzyme. In addition, the shlf life (defined as 50% if initial activity remained) of the immobilized enzyme stored at 4oC was found to reach 43 and 40 days. D-hydantoinase immobilized on PHA granules could be reused for 8 times to achieve the conversion yield exceeding 80%. It is expected that the immobilized enzyme PhaP-HDT or PhaC-HDT can be generally applied to the industrial for various production processes.