糖基化对Caldicellulosiruptor sp.F32中内切型木聚糖酶热稳定性的影响  

Effect of glycosylation on a thermophilic β-1,4-endoxylanase from Caldicellulosiruptor sp. F32

作　　者：王灵[1,2] 韩文杰[3] 孟冬冬[3] 朱静[1] 李福利[2] 吕明[2] 

机构地区：[1]西南林业大学生命科学学院,云南昆明650224 [2]中国科学院青岛生物能源与过程研究所,山东青岛266101 [3]青岛大学环境科学与工程学院,山东青岛266071

出　　处：《生物技术》2016年第3期300-306,共7页Biotechnology

基　　金：山东省自然科学杰出青年基金项目("糖苷水解酶的结构与功能";No.JQ201507);国家自然科学基金项目("极端嗜热细菌Caldicellulosiruptor中双功能纤维素水解酶作用机制的研究";No.31400060);山东省科技重大专项(新兴产业)("先进酶制剂生产技术的研究及应用;No.2015ZDXX0403A01)

摘　　要：[目的]探讨糖基化对来自Caldicellulosiruptor sp.F32的GH11家族木聚糖酶的影响,揭示其工业应用价值。[方法]在大肠杆菌和毕赤酵母中表达木聚糖酶Xyn A及其催化模块Tm1。通过酶学性质对比,论证糖基化对酶学特征的影响。[结果]毕赤酵母分泌的p-Xyn A和p-Tm1可被糖基化修饰,而大肠杆菌表达的e-Xyn A和e-Tm1无法被修饰。糖基化未明显影响Xyn A的催化效率和热稳定性,但可提高Tm1的催化效率约1.5倍,而且在80℃、85℃下,pTm1的半衰期相对于e-Tm1分别提高了约1.5、2倍。另外,Tm1的热稳定性和酶活均显著高于Xyn A。通过对Tm1同源建模分析,两个糖基化位点（天冬酰胺N23、N189）可能对热稳定性有促进作用。[结论]酵母对Tm1的糖基化修饰可热稳定性和催化效率提高1.5~2倍,相比于Xyn A更适合工业应用。[ Objective] In this present study,we explored the effect of glycosylation on an endo - β -xylanase XynA from Cal- dicellulosiruptor sp. F32 and the industrial value of this xylanase. [ Methods ] Both XynA and its mutant Tml without CBM were cloned and expressed in Pichia pastoris and E. coil Enzyme properties of p - XynA and p - Tml from Pichia pastoris and e - Tml and e - XynA from E. coli were analyzed and compared. [ Resnits] Results of glycosylation staining gel indicated that p - XynA and p -Tml could be glycosylated,but e -Tml and e -XynA could not. The catalytic efficiencies of p -XynA and e - XynA were similar,while the catalytic efficiency ofp -Tml was 1.5 times higher than that of e -Tml. The thermostability ofp - Tml at 80 ℃ and 85 ℃ were approximately by 1.5 - and 2 - fold higher than that of the e - Tml. Furthermore,the thermo- stability and specific activity ofp - Tml and e - Tml were greater than which ofp - XynA and e - XynA. The analysis of crys- tallographic structure and its amino acid sequence revealed that two potential glycosylated sites were in the back of the catalytic cleft. [ Conclusion] The N - glycosylation can improve the thermostability and catalytic efficiency of Tml by 1.5 - 2 times. According to our results,p -Tml shows potential applications in food, pulp and feed industries.

关 键 词：内切型β-1 4木聚糖酶 毕赤酵母 大肠杆菌 糖基化 热稳定性 

分 类 号：Q784[生物学—分子生物学]