新型GH5家族多结构域纤维素酶的结构与功能研究  

Structure and Function Analysis of Novel GH5 Multi-domain Cellulase

作　　者：杨俊钊 张新蕊 赵国柱[1] 郑菲[1] YANG Jun-zhao;ZHANG Xin-rui;ZHAO Guo-zhu;ZHENG Fei(College of Biological Sciences,Beijing Forestry University,Beijing 100083)

机构地区：[1]北京林业大学生物科学与技术学院,北京100083

出　　处：《生物技术通报》2023年第4期71-80,共10页Biotechnology Bulletin

摘　　要：纤维素酶能够将纤维素转化为可发酵的糖类,为丰富纤维素酶的序列与结构资源、揭示纤维素酶结构与功能之间的关系,本研究对两个新型GH5家族多结构域内切纤维素酶TlCel5和ReCel5进行了克隆表达和酶学性质测定,并对其结构域开展了突变研究。序列和结构分析显示,Tlcel5和Recel5分别编码了655个和632个氨基酸,理论分子量分别为68.3 kD和65.9 kD,均包含了CBM1区、CD区、CBMX2区和一个未知结构域,这与以往报道的多数单一结构域或双结构域纤维素酶显著不同。为了解附加结构域对酶功能的影响效果,以ReCel5为研究对象,分别构建了N端CBM1结构域的截断突变体TM1,和C端未知结构域的截断突变体TM2。酶学性质分析显示,TlCel5和ReCel5的最适作用pH和最适作用温度分别为pH 3.0、50℃和pH 4.0、70℃,在50℃和70℃下能够保持良好的稳定性,并且对多种类型的纤维素类底物、半纤维素类底物表现出水解能力。虽然突变体TM1和TM2的酶学性质较野生型没有发生显著变化,但其对羧甲基纤维素钠、大麦葡聚糖、地衣多糖的水解比活值降低了23%-68%,由此说明,在多结构域酶中,附加结构域与酶的水解能力之间存在密切关系。Cellulase can convert cellulose into fermentable sugars.In order to enrich the sequences and structure resources of cellulase and reveal the relationship between cellulase structure and function,two novel multi-domain endoglucanases belonging to glycosyl hydrolases(GH)family 5 TlCel5 and ReCel5 were cloned and expressed.Their enzymatic properties were determined and the mutation of structure domain was studied.Sequence and structure analysis showed that Tlcel5 and Recel5 encoded 655-residue and 632-residue polypeptides with theoretical molecular weights of 68.3 kD and 65.9 kD respectively.They wee both multi-domain cellulases,comprising a carbohydrate-binding module grouped into family 1(CBM1)and a catalytic module of family 5 glycoside hydrolase(CD)at N-terminal,a family X2 carbohydrate binding module(CBMX2)and an unknown domain(UM)at the C-terminal,which made them significantly different from most singledomain or dual-domain cellulases reported previously.With the purpose of understanding the effect of additional domains on enzyme function,the truncated mutant without the N-terminal CBM1 domain(TM1)and the truncated mutant without the C-terminal unknown domain(TM2)were constructed based on wild-type ReCel5.The assays of enzymatic properties demonstrated that the optimal pH and temperature of TlCel5 and ReCel5 were pH 3.0,50℃and pH 4.0,70℃,respectively.They remained fine stability at 50℃and 70℃,and showed certain hydrolytic ability to a variety of cellulose and hemicellulose substrates.Their hydrolysis specific viabilities to sodium carboxymethyl cellulose,barleyβ-glucan,and lichenan reduced by 23%to 68%although the enzymatic properties of TM1 and TM2 did not change significantly compared with the wildtype proteins.These results suggested that there was a close relationship between the additional domains and the hydrolysis ability of multidomain enzymes.

关 键 词：多结构域内切纤维素酶 克隆表达 酶学性质 结构与功能 

分 类 号：Q55[生物学—生物化学]