食品级木聚糖酶黑曲霉工程菌的构建

Construction of food-grade xylanase engineering strain of Aspergillus niger

从黑曲霉CICC2462中扩增得到木聚糖酶基因xynB的成熟肽编码序列(746 bp),并针对黑曲霉CICC2462中高表达的糖化酶基因位点,通过重叠延伸PCR将xynB基因片段与糖化酶基因的5'同源臂、3'同源臂进行拼接得到同源重组表达框GBG,将其定向插入表达载体pSZH-CYM中,构建黑曲霉表达载体pSZH-xynB。将载体pSZH-xynB通过冻融法转化农杆菌AGL1,通过农杆菌介导法转化黑曲霉CICC2462菌丝体。经潮霉素筛选和PCR鉴定获得47株重组菌株,并从其中12株中筛选出在糖化酶基因位点发生基因置换的同源重组菌株9株。而后通过连续传代、筛选和PCR检测,获得消除潮霉素基因的纯合的同源重组菌株,获得符合食品级生产要求的工程菌。对工程菌进行发酵培养、酶活性检测和SDS-PAGE分析,表明在糖化酶摇瓶发酵条件下,工程菌培养液上清的木聚糖酶活性高达4 495.8975 U·mL-1。研究结果为生产低木糖苷酶活性的内切木聚糖酶提供新途径,为利用黑曲霉表达系统安全高效地生产其他酶制剂和重组蛋白提供参考。

The mature peptide coding sequence of xylanase gene xynB （746 bp）was amplified from the Aspergillus niger CICC2462,for high expression of glucoamylase gene locus in Aspergillus niger CICC2462, Splicing xynB gene fragment, glucoamylase gene 5 ＇homology arm and 3＇ homologous arm for homologous recombination expression cassette GBG by overlap extension PCR,The whole gene was inserted into expressional vector pSZH-CYM form the Aspergillus niger expressional vector pSZH-xynB , Using freeze thawing method pSZH-xynB expressional vector was transformed into Agrobaoterium tumefaciens AGLI, Thus transformed Aspergillus niger CICC2462 mycelium by agrobacterrium-mediated transformation. Detection 47 positive transgenic strains by hygromycin selection and PCR,Screening 9 homologous recombinant strains which occurrence gene replacement at the glucoamylase gene locus from 12 strains. Then through successive generations, screening and PCR to obtain eliminate hygromycin gene homozygous homologous recombination strains, thereby obtain the production requirements of thefood-grade engineering strain. Engineering strain showed that glucoamylase fermentation condition of the engineering strain culture supematant xylanase activity up to 4495.8975 U · mL-1 by fermentation culture, detection of enzyme activity and SDS-PAGE analysis. The results of this study provide a new way for production Iow-xylosidase activity of endo-xylanase, and also provide a valuable reference for the safe and efficient production other enzymes and recombinant protein expression system to take advantage of Aspergillus niger.