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DH5a, 筛选出阳性转化子DH5 a -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-P

HEREDITAS (Beijing)

2008年5月, 30(5): 649―654 ISSN 0253-9772 www.1mpi.com

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-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-PAGE). 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 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.

Keywords: Bacillus megaterium; endoglucanase; gene expression; enzyme properties

纤维素是世界上最大的可再生资源。纤维素通过降解, 可转化为人类急需的能源、食物、化工原料、对于人类社会解决食物短缺和能源危机具有重大现实意义。利用微生物产生的纤维素酶(cellulase)

来分解和转化纤维素则是纤维素利用的有效途径。其中, 细菌产生的纤维素酶一般是中性或偏碱性, 这使它在棉织品水洗整理工艺及洗涤剂工业中有着独特的使用性能和巨大的经济价值。葡聚糖内切酶

收稿日期: 2007 10 27; 修回日期: 2008 01 04

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