摘要
通过PCR方法将已克隆的内切葡聚糖酶基因(GenBank No.DQ782954)信号肽编码序列去除,然后与表达载体pHIS1525连接后转化大肠杆菌DH5α,筛选出阳性转化子DH5α-pHIS1525-G7并提取质粒进一步转化巨大芽孢杆菌WH320原生质体,获得基因工程菌WH320-pHIS1525-G7。刚果红染色和SDS-PAGE分析表明该基因在巨大芽孢杆菌中得到了有效表达。基因工程菌经优化培养后,胞外上清液中的酶活力可达889U,是出发菌株(即枯草芽孢杆菌C-36)的11.22倍。酶学性质研究表明:该酶的最适反应温度与pH值分别为65℃与pH6.0,在pH4.5～10.0范围内50℃保温30min可保持在最高酶活的80%以上。
The signal peptide-encoding sequence, which was included in the gene （GenBank No. DQ782954） encoding for endoglucanase, was removed by PCR. The gene without the signal peptide was then ligated with the expression plasmid pHIS1525. The recombination plasmid pHIS1525 was transformed into Escherichia coil DH5α and the transformant was designated DH5α-pHIS1525-G7. The plasmid from the recombinant DH5α-pHIS1525-G7 was transformed into the proto- plasts of Bacillus megaterium strains WH320, and the genetically engineered bacterium, known as WH320-pHIS1525-G7, was acquired. The effective expression of the gene in the recombinant was confirmed by Congo-red dyeing and SDS polyacrylamide gel electrophoresis （SDS-PAGE）. WH320-pHIS 1525-G7 was cultured in optimum condition. The activity of the endoglucanase was 899U, which was 11.22-fold higher than that of B.subtilis C-36. The properties of enzyme were determined. The optimum temperature and pH value were 65 ℃ and pH 6.0, respectively. The enzyme maintained over 80% of the original enzyme activity between pH 4.5 and pH 10.0 after incubated at 50℃ for 30 min.
出处
《遗传》
CAS
CSCD
北大核心
2008年第5期649-654,共6页
Hereditas（Beijing)
作者简介
陈惠（1962-），女，四川人，博士，教授，研究方向：微生物酶工程．Tel-0835—2886126；E—mail：chenhui@sicau．edu．cn
