内切葡聚糖酶基因在巨大芽孢杆菌中的表达及其酶学性质研究.pdfVIP

HEREDITAS (Beijing) 2008 年5 月, 30(5): 649―654 ISSN 0253-9772 研究报告 DOI: 10.3724/SP.J.1005.2008.00649 内切葡聚糖酶基因在巨大芽孢杆菌中的表达及其酶学 性质研究 陈惠, 胥兵, 廖俊华, 官兴颖, 吴琦 四川农业大学生命科学与理学院, 雅安 625014 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DH5 α -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 889 U, 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pH 6.0, 在pH 4.5~10.0 范围内50 ℃保温30 min 可保持在最高酶活的80% 以上。 关键词: 巨大芽孢杆菌; 内切葡聚糖酶; 基因表达; 酶学性质 Expression of endoglucanase gene in Bacillus Megaterium and char- acterization of recombinant enzyme CHEN Hui, XU Bing, LIAO Jun-Hua, GUAN Xing-Ying, WU Qi College of Biology and Science, Sichuan Agricultural University, Ya’an 625014, China Abstract: 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 coli 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 poly- acrylamide gel electrophoresis (SDS). WH320-pHIS1525-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 deter- mined. The optimum tem