定向引入N-糖基化位点改进黑曲霉β-1,4-内切木聚糖酶热稳定性  

Improvement of thermal stability of endo-β-1,4-xylanase from Aspergillus niger by introducing N-glycosylation site

作　　者：吴凤梅 周燕霞 姚冬生[1,2] 谢春芳[3] WU Feng-Mei;ZHOU Yan-Xia;YAO Dong-Sheng;XIE Chun-Fang(Institute of Biomedicine,Jinan University,Guangzhou,Guangdong 510632,China;National Engineering Research Center of Genetic Medicine,Jinan University,Guangzhou,Guangdong 510632,China;Department of Bioengineering,College of Life Science and Technology,Jinan University,Guangzhou,Guangdong 510632,China)

机构地区：[1]暨南大学生物医药研究院,广东广州510632 [2]暨南大学基因工程药物国家工程研究中心,广东广州510632 [3]暨南大学生命科学技术学院生物工程学系,广东广州510632

出　　处：《微生物学通报》2020年第7期2128-2139,共12页Microbiology China

基　　金：国家高技术研究发展计划(2013AA102801);广东省科技计划(2013B090600141);广东省级财政技术研究开发补助项目。

摘　　要：【背景】木聚糖是生物圈中仅次于纤维素的第二大多糖,其结构复杂,完全降解需要多种木聚糖酶协同作用。β-1,4-内切木聚糖酶是木聚糖主链水解过程中最关键的酶,已广泛应用于饲料、造纸、能源、食品和医药等行业。但在实际应用中,由于真菌木聚糖酶的热稳定性较差,限制了其在工业中的应用。【目的】提高来源于黑曲霉(Aspergillusniger)的β-1,4-内切木聚糖酶(xynB)热稳定性。【方法】采用氨基酸虚拟突变技术对xynB定向引入一个N-糖基化位点,将虚拟突变后筛选获得的候选突变体和野生型在毕赤酵母SMD1168中表达,并对纯化后的野生型和突变体酶进行酶学性质和稳定性分析。【结果】经虚拟突变和筛选获得5个候选突变体,在毕赤酵母SMD1168中成功表达了4个突变体,其中3个突变体发生了糖基化。突变体和野生型酶均表现出宽范围的酸碱耐受性,且突变体xynBA92N/D94T在pH4.0–11.0条件下的稳定性明显优于野生型;糖基化突变体xynBA92N/D94T、xynBG66N/A68T和xynBG66F/D67N/G69T在温度为60–80°C时热稳定性明显高于野生型,xynBG66N/A68T在80°C保温30 min后的残留酶活比野生型提高了约30%。【结论】本研究方法可为其他来源木聚糖酶和其他工业酶的热稳定分子改造提供参考。[Background]Xylan is the second most abundant polysaccharide in nature,and second only to cellulose.The structure of xylan is complex,and its complete degradation needs many xylanases.Endo-β-1,4-xylanase is the key enzyme in the hydrolysis process of xylan main chain.It has been widely used in feed,papermaking,energy,food and medicine industries.However,in practical application,due to the poor thermal stability of fungal xylanase,its application in industry is limited.[Objective]The purpose of this study is to improve the thermal stability of endo-β-1,4-xylanase(xynB)from Aspergillus niger.[Methods]Firstly,an N-glycosylation site was introduced into xynB by amino acid virtual mutation technology to obtain the mutants.Then the mutants and wild-type enzymes were expressed in Pichia pastoris SMD1168.Finally,the purified wild-type and mutant enzymes were characterized by enzymatic properties.[Results]Five candidate mutants were obtained by virtual mutation and screening.Four mutants were successfully expressed in Pichia pastoris SMD1168,and three of which were glycosylated.The mutants and wild-type enzyme showed wide pH tolerance,and the stability of xynBA92 N/D94 T at pH 4.0–11.0 was significantly better than that of the wild-type;xynBA92 N/D94 T,xynBG66 N/A68 T and xynBG66 F/D67 N/G69 T,which were glycosylated,showed significantly higher thermal stability at 60–80°C than that of the wild-type,and the residual enzyme activity of xynBG66 N/A68 T at 80°C after incubation for 30 min was about 30%higher than that of the wild-type.[Conclusion]The method of this study can provide reference for the thermal stability molecular modification of other xylanases and other industrial enzymes.

关 键 词：β-1 4-内切木聚糖酶 N-糖基化 氨基酸虚拟突变 酶学性质 

分 类 号：Q936[生物学—微生物学]