根据NCBI（GenBankAccessionNo．：D83732．1）中注册的米曲霉内切葡聚糖酶＆馏基因序列设计引物，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65ku。对酵母表达工程菌X33一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1L三角瓶诱导培养5天达到最高酶活120U／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 ge- nomic 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 successful- ly. The SDS-PAGE showed that the molecular weight was about 65ku. In the condition of shake flask culture, fer- mentation conditions of yeast expression engineering strain were optimized. The results showed that the optimal con- centration of methanol induction was 0.75 %. After five-day induction culture with 1L flask, it reached the highest ac- tivity 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. Key words Aspergillus oryzae, endoglucanase, gene cloning, yeast expression, enzymatic properties
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