内切葡聚糖酶基因在穿梭载体中的表达研究

将无信号肽编码序列的内切葡聚糖酶基因(GenBankNo.DQ782954)与表达载体PMK4连接后转化大肠杆菌DH5α,筛选出阳性转化子DH5α-PMK4-egls,并将提取的质粒转化到枯草芽孢杆菌WB600原生质体内,获工程菌WB600-PMK4-egls。同时,通过刚果红染色和SDS-PAGE分析表明,该基因在枯草芽孢杆菌中得到了表达。通过优化培养基因工程菌,胞外上清液中的酶活力达998U,该酶促反应的最适温度为60℃,最适pH为6.0,且pH在4.5～7.5,温度在30℃～65℃范围内,可保持最高酶活的70%以上。

Expression of Endoglucanase Gene in Shuttle Expression Vector and Characterization of Recombinant Enzyme

The endoglucanase gene with the removed signal peptide-encoding sequence,（GenBank No.DQ782954） was ligated with the expression plasmid PMK4.The recombination plasmid PMK4-egls was then transformed into Escherichia coli DH5α and the transformant was designated DH5α-PMK4-egls.The plasmid from the recombinant DH5α-PMK4-egls was further transformed into the protoplasts of Bacillus megaterium strains WB600,and the genetically engineered bacterium,known as WB600-PMK4-egls,was obtained.The effective expression of the gene in the recombinant was detected by Congo-red dyeing and SDS polyacrylamide gel electrophoresis（SDS-PAGE）.WB600-PMK4-egls was cultured in optimal condition.The activity of the endoglucanase was as high as 998U.The optimal temperature and pH value were 60℃ and pH 6.0,respectively.The enzyme maintained over 70% of the original activity between pH 4.5 and pH 7.5 when incubated in 30℃-65℃.