| A new method of gene mutated in vitro was used in this study, which is the deoxythymidine triphosphate (dTTP) was partially replaced by 5-BrdUTP, mutation was obtained during PCR cycles. In this study, Xanthomonas compestris α-Amylase gene was mutated in vitro by PCR with dNTP in which deoxythymidine triphosphate (dTTP)was partially replaced by 5-BrdUTP. Results showed that the higher the concentration is, the stronger its inducing ability is in a certain range. and 0.1% 5-BrdUTP substitution ratio was optimal in mutagenesis in vitro, suitable for acquirement of high activity mutant α-amylase gene. Too high ratio would lead to too many inactive genes and too low ratio would not be enough to form a large mutant library. In this experiment the LBSP identification medium screening techniques was used. Using this method after 3 rounds of mutagenesis and screening, a high activity mutant of a -amylase gene pHNh301 was obtained, which showed 10 times a -amylase activity of the wild gene (pHN8004). Nucleotide sequence analysis revealed that due to 2 amino acids were changed, which are S263N (Ser→Asn) located in the downstream of conserved regions III and R380C (Arg→Cys) located in the downstream of conserved regions IV.The high activity α-amylase gene (pHNh301) was cloned into the Pichia pastoris expression vector pHBM905A, the recombinant plasmid was named pHBM905AM. The recombinant plasmid pHBM905AM was linearized, then transformed into P.pastori GS115. The recombinant P.pastoris GS115(pHBM905AM1) secreted functional α-amylase, the halo was formed clearly on 1% starch of BMMY substrate plate. The recombinant GS115 was fermented with high density liquid medium and induced by 1.0% (v/v) methanol at culture temperature 28°C. The enzymatic activities reached 1081u.mL-1. The optimal temperature and optimal pH value of the recombinant α-amylase was not changed, was 50 ℃and pH5.9 respectively.
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