定点突变技术提高内切葡聚糖酶基因F-10酶活性

Improving the Character of the F-10 Endoglucanase Gene by Site-Directed Mutation

摘要:

利用高酶活F-10突变株内切葡聚糖酶基因进行定点突变，对91位 （K91E）和369位（K369R）氨基酸进行突变，构建突变质粒（K91E）；（K369R）和（K91E/K369R），转化大肠杆菌BL21，经筛选获得突变内切葡聚糖酶基因工程菌株K91E，K369R和K91E/K369R。经IPTG诱导后，分离纯化酶学性质分析。结果表明：蛋白质相对分子质量为53 000；突变前后最适pH值未发生变化，为pH 6.8；突变体K369R和K91E/K369R的最适温度为50 ℃，而突变体K91E最适温度发生了很大的变化，为40 ℃；酶的比活力分别为202、 162.8、77.9 U/mg，分别是原始酶（66 U/mg）的3.0倍，2.4倍和1.2倍；三个突变酶的热稳定性有较大提高，70 ℃处理1 h后，原始酶的活性急剧下降，剩余活力为12%，而K91E的剩余活力为36% ，K369R剩余活力为30%，K91E/K369R剩余活力为41%。当经80 ℃处理1 h后，K91E残余活力仍有22%。本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及应用打下了基础，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。

Abstract:

Endoglucanase is one of the most important cellulose enzymes, However, the large-scale industrialized application of these enzymes was restricted by the low activity and high cost price. Improving their activity through genetic engineering and protein engineering was considered an efficient approach to cut down the cost price. In this study the site-directed mutation technology was used to improve the activity of the F-10 endoglucanase gene. The cellulose of endoglucanase 91 lysine(AAA) was replaced by glutamic acid(GAA)(K91E), 369 lysine(AAA) was replaced by arginine(AGA)(K369R) and the 91 and 369 were replaced by arginine(AGA). Mutation plasmids were transfored into E. coli, the expression product was induced by IPTG. The result showed that protein molecular weight was 53 000. The optimum reaction pH unchanged, was pH 6.8. The optimum temperature of K369R and K91E/K369R were 50 ℃, the optimum temperature of the mutant K91E have great change, is 40 ℃. The specific activity reached 162.8、77.9、202 U/mg. The thermal stability of the three mutants increased, when the temperature reached 70 ℃, the activity of F-10 fell sharply, surplus energy is only 12%, 36%, 30% and 41%, when the temperature reached 80 ℃, the residual activity of K91E is still about 22%. By site-directed mutation technique, we obtain the efficient expression in E. coli and good thermal stability of endoglucanase gene engineering strains. This research can provide the basis for the further investigations of cellulose.