根据NCBI(GenBank Accession No.:D83732.1)中注册的米曲霉内切葡聚糖酶CelB基因序列设计引物,以本实验室自行筛选的天然米曲霉基因组DNA为模板,PCR高保真扩增出内切葡聚糖酶基因eg,将其定向插入到酵母表达载体pPICZαA上,转化酵母宿主菌X33,刚果红水解圈筛选结果表明已成功表达。SDS-PAGE分析表明,表达产物分子量约为65 ku。对酵母表达工程菌X33-eg进行发酵条件优化,结果显示最适甲醇诱导浓度为0.75%,用1 L三角瓶诱导培养5天达到最高酶活120 U/mL。对重组酶的酶学性质分析表明,其最适反应pH值和温度分别为pH4.0和45℃,在30～45℃和pH3.4～pH6.9范围内可保持内切葡聚糖酶最高酶活力70%以上。

Using primers designed from the sequence of endoglucanase CelB gene(GenBank Accession No.: D83732.1) from Aspergillus oryzae,endoglucanase eg was amplified through PCR with Aspergillus oryzae giF-10 genomic DNA as template.The Aspergillus oryzae giF-10 endoglucanase eg gene was inserted into the yeast expression vector pPICZαA,then the pPICZαA-eg plasmid was electroporated into yeast competent cells of X33 host strain and a lot of positive transformants were obtained.Hydrolysis cycle tests proved that the gene could be expressed successfully.The SDS-PAGE showed that the molecular weight was about 65 ku.In the condition of shake flask culture,fermentation conditions of yeast expression engineering strain were optimized.The results showed that the optimal concentration of methanol induction was 0.75%.After five-day induction culture with 1L flask,it reached the highest activity 120 U/mL.The optimal pH and temperature of the recombinant enzyme were pH 4.0 and 45 ℃,respectively.In the range of 30~45 ℃ and pH3.4~6.9,it could maintain more than 70% of the highest endoglucanase activity.