| Summary: | 碩士 === 逢甲大學 === 化學工程學系 === 87 === The increasing demand for antibacterial drugs has called for a mass production of semisynthetic antibiotics. Among the optically active D-amino acids, D-p-hydroxyphenylglycine (D-HPG) and D-phenylglycine are the most important precursors used for the synthesis of semisynthetic cephalosporin and penicillin. D-HPG can be produced in a two-step reaction.First, D-htdantoinase converts DL-hydroxyphenylhydantoin(DL-HPH) to N-carbamoyl-D-p- hydroxyphenylglycine(CpHPG) Secondly, either a chemical method or an enzymatic route enables to perform subsequent hydrolysis of CpHPG to D-HPG by N-carbamoyl-D-amino acid amidohydrolase (hereinafter carbamoylase).
In this study, we successfully cloned the gene encoding D-hydantoinase and carbamoylase from A. radiobcter NRRL B11291. By highly expressing both D-hydantoinase and carbamoylase, recombinant E. coli strains were able to convert substrate DL-HPH to D-HPG with a conversion yield of 97%, accounting for 5 times productivity higher than that obtained by A. radiobcter NRRL B11291. Immobilizing the recombinant cells with k-carrageenan could also achieve a conversion of 93% while A. radiobcter NRRL B11291 attained 20% within the same period of reaction time.
An amplified yield of foregin proteins may impair the cell growth and in turn reduce protein production as a result.We attempted to solve this problem by using the promoter of ribosome modulation factor(rmf) . To characterize the rmf promoter,we choosed LacZ as a model system.Under different culture condition ,We found that the expression of the rmf promoter heavily relatived on the specific growth rate(m).When the cell gains a higher growth rate,the protein production is lower.Inaddition the induction ratio can vary between 15 to 40-fold.
Overall,we have successfully cloned hydantoinase and carbamoylase in Escherichia coli strains.The constract of expression vectors based on the rmf promoters is capable of being controlled by adjusting the specific growth rate of the cell.
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