马克斯克鲁维酵母外切菊粉酶INU1的晶体结构研究  

The Crystal Structure Study of Exoinulinase INU1 from Kluyveromyces marxianus

作　　者：李龙 苏晓琴 方自安 周峻岗 胡小健[1,2] 吕红 LI Long;SU Xiaoqin;FANG Zhi'an;ZHOU Jungang;HU Xiaojian;Lü Hong(School of Life Sciences,Fudan University,Shanghai 200438,China;Shanghai Engineering Research Center of Industrial Microorganisms,Shanghai 200438,China)

机构地区：[1]复旦大学生命科学学院,上海200438 [2]上海工业微生物工程技术研究中心,上海200438

出　　处：《复旦学报（自然科学版）》2019年第4期482-488,共7页Journal of Fudan University：Natural Science

基　　金：国家重点基础研究发展计划(2013AA102803,2014AA021301);上海市科学技术委员会项目(13DZ2252000)

摘　　要：菊粉酶是一类能够将菊粉水解成果糖和低聚果糖的酶,它广泛应用于糖浆和果糖的工业生产.为了深入研究菊粉酶的结构与功能特征,我们解析了马克斯克鲁维酵母ATCC12424的外切菊粉酶INU1的晶体结构.通过毕赤酵母SMD1168(his4,pep4)对INU1进行了分泌表达,其体积酶活达到了1 671 U/mL.使用阴离子交换柱对INU1进行纯化并结晶解析了结构,INU1的晶体属于空间群I4 122,晶胞参数a=b=c=172.65,在上海同步辐射光源其衍射分辨率为2.8,一个非对称单位中有两个INU1分子以面对面方式堆积成二聚体.INU1的晶体结构与GH32(糖苷水解酶32)家族其他蛋白一致,主要由N端保守的5个β螺旋桨结构域和C端不保守的β夹层结构域组成.氨基酸序列和晶体结构对比显示,INU1的催化位点为Asp53,Asp182和Glu238.这个晶体结构为菊粉酶INU1的进一步改造以适应工业化生产提供了基础.Inulinase is an enzyme that hydrolysis inulin into fructose and oligo-fructose.It is widely used in industry to produce syrup and fructose.To investigate the structural and functional relationship of inulinase,here we study the crystal structure of an exoinulinase INU1 from Kluyveromyces marxianus ATCC12424.The INU1 was expressed in Pichia pastoris SMD1168(his4,pep4)and the maximum activity reached 1 671 U/mL.After purified through the anion exchange chromatography column,the INU1 was finally crystallized and the structure was solved using molecular replacement methods.The INU1 crystal belonged to space group I4 122,with unit-cell parameters of a=b=c=172.65.In one asymmetric unit two INU1 molecules packed face-to-face as a dimer.It diffracted to 2.8 resolution at Shanghai Synchrotron Radiation Facility.The crystal structure of INU1 is identical to other GH32 family members and it is composed of N-terminal conserved beta propeller domain and C-terminal non-conserved beta sandwich domain.The amino acid sequence and structure alignment suggested that the catalytic residues of INU1 were Asp53,Asp182 and Glu238.These results provide structural basis for the further enzyme engineer of INU1.

关 键 词：菊粉酶 外切菊粉酶 菊粉 马克斯克鲁维酵母 晶体结构 

分 类 号：Q518.3[生物学—生物化学]