【作者】 崔锦；

【导师】 马向东；

【作者基本信息】 河南农业大学 ， 微生物学， 2006， 硕士

【摘要】 本研究采用5-溴脱氧尿苷三磷酸（5-BrdUTP）掺入法，即在PCR反应体系中加入一定比例的5-溴脱氧尿苷三磷酸（5-BrdUTP）部分取代脱氧胸苷三磷酸（dTTP），对野油菜黄单胞菌α-淀粉酶基因（pHN8004）进行体外诱变。结果表明：5-BrdUTP浓度越高，诱变越强；浓度越低，诱变越弱。当5-BrdUTP浓度为dTTP的0.1％时，有利于筛选到高酶活的α-淀粉酶突变基因，过高的比例则产生过多的失活基因，比例太小又不足以形成一定的突变体库。用LBSP鉴别培养基进行筛选，经过3轮诱变和筛选，获得一编码α-淀粉酶且酶活力提高10倍的基因，将其克隆到pBluescript KS（+）载体中，重组质粒命名为pHNh301。序列分析发现pHNh301有三个位点发生碱基突变，其中一个位于-35区，是A→G的转换。另外两个位于编码区，是G→A和C→T的转换，这两个碱基的突变造成两个氨基酸的改变，第一个是S263N（Ser→Asn），在紧靠保守区3区下游。第二个是R380C（Arg→Cys），在保守区4区下游。 将重组质粒pHNh301的α-淀粉酶基因克隆到毕赤酵母表达载体pHBM905A中，重组载体命名为pHBM905AM，将其用Sal Ⅰ酶切线性化后转化毕赤酵母GS115且实现了分泌表达。对重组酵母进行诱导，在培养温度28℃、甲醇流加量为1.0％（v／v）的情况下，第7d其分泌表达的α-淀粉酶的酶活最高，最高值为1081.30u.mL^-1。该酶的最适反应pH值和最适温度与出发菌株相同，分别为pH5.9和30℃。更多还原

【Abstract】 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.更多还原