葡萄糖氧化酶基因在黑曲霉中的分泌表达

通过PCR扩增从一株葡萄糖氧化酶生产菌黑曲霉中克隆得到1818bp葡萄糖氧化酶基因GOD,将GOD基因与糖化酶基因的5’同源臂、3’同源臂进行重叠延伸,构建葡萄糖氧化酶基因的表达框。将此表达框插入载体pSZH中,构建黑曲霉同源重组表达载体pSZH-GOD。将载体pSZH-GOD通过冻融法转化农杆菌AGL1,进而通过农杆菌介导法转化高产糖化酶的黑曲霉。经潮霉素筛选和PCR鉴定获得8株重组菌株。对8株重组菌株的发酵液上清液进行酶活测定,结果表明葡萄糖氧化酶基因在高产糖化酶的黑曲霉中得到了分泌表达,静置培养6d后其分泌表达的葡萄糖氧化酶酶活最高可达1.166U/mL,而在出发菌株中检测不到葡萄糖氧化酶的分泌表达。同时,重组菌株胞内表达的葡萄糖氧化酶最高酶活可达11.45U/mL,是出发菌株的266倍。此研究结果为简化葡萄糖氧化酶发酵生产工艺、提高酶产量提供了新的途径。

Secretory expression of glucose oxidase gene in Aspergillus niger

One GOD(Glucose oxidase,1818bp) gene was cloned from Aspergillus niger and then overlapped extension to obtain 5’ homologous arm GLA(saccharifying enzyme) and 3’ homologous arm GLA of expressional box of the gene.The whole gene was inserted into expressional vector pSZH to form the homologous recombination expressional vector pSZH-GOD of Aspergillus Niger.Using freeze thawing method pSZH-GOD expressional vector was transformed into Agrobacterium AGLI which transformed into Aspergillus niger which could expressed highly saccharifying enzyme.8 positive transgenic strains were detected by hygromycin selection and PCR.The enzyme activity of eight positive transgenic strains were tested,the result showed that GOD gene was highly expressed in transforming strains whose exocellular enzyme activity were up to 1.166U/mL after 6d static cultivation,while enzyme activity of GOD gene was not detected in non-transformed strains.Meanwhile,endocellular enzyme activity of GOD gene was up to 11.45U/mL which was 266 times that of the starting strains.The result of this study provided a new approach to simplify the GOD fermentational technology to improve the enzyme production.