内切葡聚糖酶基因在黑曲霉中的同源表达  

Endo-glucanase gene homologous expressed in Aspergillus niger

作　　者：李杰[1,2] 高博[1,2] 江连洲[3] 刘君[1,2] 邓晨旭 陈璐璐[1,2] 张会[1,2] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]黑龙江省高校农业生物功能基因重点实验室,哈尔滨150030 [3]东北农业大学食品学院,哈尔滨150030

出　　处：《东北农业大学学报》2014年第9期56-61,共6页Journal of Northeast Agricultural University

基　　金：国家863高技术研究发展计划(2013AA102104)

摘　　要：内切葡聚糖酶作用于纤维素的非结晶区,随机水解β-1,4-糖苷键,将长链的纤维素截断,对纤维素的整体降解起重要作用。研究从黑曲霉CICC2462中扩增得到内切葡聚糖酶基因Eng1,并针对黑曲霉中高表达的糖化酶基因glaA位点,构建Eng1基因表达载体pSZHG-Eng1,进一步通过农杆菌介导法转化黑曲霉CICC2462。经潮霉素筛选和PCR鉴定获得2株在glaA基因位点发生基因置换的同源重组转化子。在摇瓶发酵条件下,发酵液上清中的内切葡聚糖酶活力最高可达到272 U·mL-1,是出发菌株5.2倍。SDS-PAGE分析显示,两株重组菌株都有约36 ku目的蛋白条带,表达量为165~193μg·mL-1。结果表明,研究实现内切葡聚糖酶在黑曲霉中的同源表达,可为食品级内切葡聚糖酶的大规模工业化生产奠定基础。An endo-glucanase can truncate long chains of cellulose in cellulose non-crystal ine region through random hydrolysis of β-1, 4-glycosidic linkage, which plays an important role in the overal degradation of cellulose. In order to improve the endo-glucanase activity of Aspergil us niger, a gene designated Eng1 encoding an endo-glucanase from Aspergil us niger CICC2462 was cloned by using the reverse transcription PCR method. Using the highly expressed elment of glaA encoding a glucoamylase, the recombinant expression vector pSZHG-Eng1 was constructed and transformed into the strain CICC2462 via the Agrobacterium-mediated method. Two strains were obtained by screening hygromycin resistant mutants and base pair substitution based on homologous recombination was established by PCR to exist in glaA gene loci gene. Under the shaking flask fermentation condition, the endo-glucanase activity of the A.niger transformant reached 272 U·mL-1 in the fermentation solution, which was 5.2-fold as high as that of the original strain. The approximately 36 ku protein band from two strains was observed on the SDS-PAGE, and the expressed enzyme was quantitated to be 165-193μg·mL-1. This results showed that the endo-glucanase gene was successful y transformed and homologously expressed in Aspergil us niger, which laid the foundation for the construction of an engineered strain to produce a food-grade endo-glucanase in large-scale industrial production.

关 键 词：内切葡聚糖酶 黑曲霉 同源重组 表达 

分 类 号：TQ925[轻工技术与工程—发酵工程] Q786[生物学—分子生物学]