易错PCR技术提高中性内切葡聚糖酶活性

Improving Endoglucanase Activity from Bacillus subtilis by Error-Prone PCR

DOI：10.3969/j.issn.1673-1689.2013.07.013

中文关键词: 枯草芽胞杆菌 内切葡聚糖酶 易错PCR 酶学性质

英文关键词: Bacillus subtilis endoglucanase error-prone PCR enzymatic properties

基金项目:四川省科技支撑计划项目(2008Z0150)

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中文摘要:

近年来随着纤维素酶在食品加工工业中具有广阔应用前景,而纤维素酶的工业应用一直受酶活性低,成本高的限制。为了提高中性内切葡聚糖酶的活性,作者利用易错PCR技术对来自枯草芽胞杆菌(Bacillus subtilis)C-36的内切葡聚糖酶基因进行定向进化研究,构建了在大肠杆菌中的突变体库。通过刚果红平板筛选,获得了酶活性提高的突变株F-10,活力比亲本酶提高4.2倍。序列分析表明:F-10有5个碱基发生突变,两个氨基酸突变:D212V和T307S。SDSPAGE条带,表明基因表达正确,而表达量较原始菌株显著增加。酶学性质分析,表明该酶的最适反应pH为5.6,最适反应温度为45℃,在pH 4.5~10.0范围内50℃保温30 min可保持80%的剩余酶活,在60~75℃酶活力相对稳定,可保持在最高酶活的80%以上,当温度高于75℃时,酶开始变性失活,酶活力迅速下降。研究获得了酶活性提高的内切葡聚糖酶菌株,为进一步在分子水平研究内切葡聚糖酶的功能和其应用打下了基础,也为高酶活酶分子在其他高表达系统的表达提供了基础材料。

英文摘要:

Endoglucanase cooperates with cellobihydrolase and β-1,4-glucosidase to translate cellulose to glucose completely.Now lots of researchers found that low enzymatic activity and high cost are the two main problems that restrict the industrial applications for cellulase.In order to enhance the enzymatic activity of the neutral endoglucanase activity,error-prone PCR strategy was conducted on Bacillus subtilis C-36 endoglucanase gene.The mutant F-10 with endoglucanase activity improved 4.2 folds respectively were obtained.The sequence of F-10 showed five nucleotides substitutions to two mutated amino acids.SDS-PAGE showed genes are correctly expression.Enzymatic properties of mutant F-10 showed that:The optimal temperature and pH value changed compared with the original enzyme,it’s optimal temperature and pH is 45 ℃ and 5.6,respectively.The enzyme maintained over 80% of the original enzymatic activity after incubated from pH 4.5 to 10.0 at 50 ℃ for 30 min and the enzyme maintained over 80% of the original enzymatic activity in 60~75℃,but when temperature upon 75 ℃,then enzymatic activity decreased sharply.

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