木聚糖酶XynB分子Ser/Thr平面导入精氨酸对酶热稳定性的影响  

Thermostability of xylanase( XynB) by introducing Arg into its Ser /Thr surface

作　　者：李飞[1,2] 解静聪[1] 李琦[1] 张雪松[3] 赵林果[1,4] 

机构地区：[1]南京林业大学化学工程学院,江苏南京210037 [2]南京师范大学泰州学院生物技术与化学工程学院,江苏泰州225300 [3]江苏农林职业技术学院生物工程系,江苏镇江212400 [4]江苏省生物质绿色燃料与化学品重点实验室,江苏南京210037

出　　处：《南京林业大学学报（自然科学版）》2014年第4期107-112,共6页Journal of Nanjing Forestry University：Natural Sciences Edition

基　　金：江苏省高校自然科学研究重大项目(13KJA220004);江苏省普通高校研究生科研创新计划资助项目(CXLX12_0529);江苏高校优势学科建设工程资助项目(PAPD)

摘　　要：为提高木聚糖酶的热稳定性,通过定点突变的方法,在来源于黑曲霉Aspergillus niger nl-1的木聚糖酶XynB分子Ser/Thr平面上引入5个精氨酸,实现了突变酶在毕赤酵母中的表达。通过酶学性质的研究表明,突变酶XynB-104和XynB-77的最适反应温度为50℃,且与原酶相比相对酶活力得到显著提高。在存在底物的情况下,50℃热处理2 h,突变酶XynB-104和XynB-77残余酶活力较原酶的相对转化效率由26%提高到80%左右。突变酶XynB-104的最适反应pH与原酶一致,而突变酶XynB-77最适pH由5.0提高到5.5。结果表明,在木聚糖酶分子Ser/Thr平面不同折叠片引入精氨酸可以增强酶结构的稳定性,特别是能显著提高热稳定性。Replacing several serine and threonine residues in the Ser /Thr surface of xylanase（ XynB） with arginines effectively increased the thermostability of the enzyme by site-directed mutagenesis. The mutated enzymes XynB-104 and XynB-77 were expressed in Pichia pastoris and their enzymatic properties were determined. The optimal temperature of the mutations XynB-104 and XynB-77 were 50 ℃,and the relative activity of mutations were dramatically increased. Both mutated xylanases showed about 80% of maximal activity after incubating in xylan substrate for 2 h at 50 ℃ compared to only 26%activity of the native enzyme. The optimal pH of XynB-104 enzyme had no change,but the pH optimum of the XynB-77 enzyme was increased from 5. 0 to 5. 5. The results of the mutated enzymes indicated that properly arginines residues introduced into Ser /Thr surface of xylanase XynB might be effective to improve the enzyme thermostability and hydrolysis capacity.

关 键 词：定点突变 木聚糖酶 精氨酸 热稳定性 

分 类 号：TQ936[化学工程]