镰刀菌Q7-31T菌株植物细胞壁降解酶系的结构
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摘要
为探究植物细胞壁降解酶高效降解细胞壁的机制,用RT-PCR方法对Q7-31T菌株植物细胞壁降解酶基因片段进行克隆,使用Prot-Param、SOPMA、ProtScale Server等软件对质谱鉴定结果进行生物学分析。结果表明:将Q7-31T菌株植物细胞壁降解相关酶归为GH5家族内切葡聚糖酶、GH7家族内切葡聚糖酶、GH7家族外切葡聚糖酶和GH10家族内切木聚糖酶4类酶。这些相关酶类为中等分子量大小,二级结构由α-螺旋、延伸链、β-转角和无规则卷曲4种元件构成,其中无规则卷曲的数量最高;均为亲水性酶,磷酸化位点比例较高,存在1~2个糖基化位点;均存在较高比例的催化结构域,三级结构呈中空的C字形。结论:少量的糖基化位点、高比例的催化结构域、多变的三维构象、大量的磷酸化位点与高效的协同作用方式可能决定Q7-31T菌株的植物细胞壁降解酶对细胞壁的高效降解。
For exploring the mechanism of high-efficiency plant cell wall degradation process based on plant cell wall degrading enzymes from Fusarium strain Q7-31 T,for the gene cloning and biological analysis,the methods respectively were PCR amplification and some bioinformatic softwares such as ProtParam,SOPMA and ProtScale Server.Results:The plant cell wall degrading enzymes in Q7-31 Tare classified as:GH5family endoglucanase,GH7 family endoglucanase,GH7 family exo-glucanase and GH10 family endoxylanase,which are consistent with mass spectrometry.The enzymes are medium molecular size,and the secondary structure consists of four elements:α-helix,extended strand,β-turn and random coil,of which random coil is most abundant;four enzyme classes are hydrophilic enzymes,and have higher proportion of phosphorylation sites.There are 1~2glycosylation sites.Four enzymes are present in a higher proportion of catalytic domains and the tertiary structure of a hollow "C"shape.Conclusion:A small number of glycosylation sites,high proportion of catalytic domains,changeable threedimensional conformations,large number of phosphorylation sites and high-efficiency synergism probably determine the high-efficiency degradation of plant cell wall degrading enzymes in strain Q7-31 T.
For exploring the mechanism of high-efficiency plant cell wall degradation process based on plant cell wall degrading enzymes from Fusarium strain Q7-31 T,for the gene cloning and biological analysis,the methods respectively were PCR amplification and some bioinformatic softwares such as ProtParam,SOPMA and ProtScale Server.Results:The plant cell wall degrading enzymes in Q7-31 Tare classified as:GH5family endoglucanase,GH7 family endoglucanase,GH7 family exo-glucanase and GH10 family endoxylanase,which are consistent with mass spectrometry.The enzymes are medium molecular size,and the secondary structure consists of four elements:α-helix,extended strand,β-turn and random coil,of which random coil is most abundant;four enzyme classes are hydrophilic enzymes,and have higher proportion of phosphorylation sites.There are 1~2glycosylation sites.Four enzymes are present in a higher proportion of catalytic domains and the tertiary structure of a hollow "C"shape.Conclusion:A small number of glycosylation sites,high proportion of catalytic domains,changeable threedimensional conformations,large number of phosphorylation sites and high-efficiency synergism probably determine the high-efficiency degradation of plant cell wall degrading enzymes in strain Q7-31 T.
引文
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[2]汪维云,朱金华,吴守一.纤维素科学及纤维素酶的研究进展[J].江苏理工大学学报,1998,19(3):20-28.
[3]高凤菊,李春香.真菌与细菌纤维素酶研究进展[J].唐山师范学院学报,2005,27(2):7-10.
[4]高培基.纤维素酶降解机制及纤维素酶分子结构与功能研究进展[J].自然科学进展,2003,13(1):21-29.
[5]Lenting H B M,Warmoeskerken M M C G.Mechanism of interaction between cellulase action and applied shear force,an hypothesis[J].Journal of Biotechnology,2001,89(2):217-226.
[6]Henrissat B.Cellulases and their interaction with cellulose[J].Cellulose,1994(1):169-196.
[7]Eveleigh D,Mandels M,Andreotti R,et al.Measurement of saccharifying cellulose[J].Biotechnology For Biofuels,2009(2):21-23.
[8]Sambrook J,Russell D.分子克隆实验指南[M].3版.北京:科学出版社,2002.
[9]王楠,王养民,张斌.普通PCR与TD-PCR在临床医学科研中应用价值的比较[J].临床军医杂志,2008,36(2):280-282.
[10]Xie Z L,Gao H Y,Zhang Q,et al.Cloning of a novel xylanase gene from a newly isolated Fusarium sp.q7-31and its expression in escherichia coli[J].Brazilian Journal of Microbiology,2012,43(1):405-417.
[11]王禄山,张玉忠,高培基.纤维二糖水解酶I吸附结构域的新功能[J].中国科学C辑:生命科学,2008,38(7):678-686.
[12]汪天虹,邹玉霞,石屹峰,等.微紫青霉CBHI酶纤维素结合结构域在大肠杆菌中的分泌型表达及性质研究[J].中国生物化学与分子生物学报,2000,16(5):644-649.
[13]肖志壮.瑞氏木霉内切葡聚酶基因的亚克隆及表达[D].山东:山东大学,2001.
[14]阎伯旭,孙迎庆,高培基.有限酶切拟康氏木霉植物细胞壁降解酶分子研究其结构域的结构与功能[J].纤维素科学与技术,1998,6(3):1-9.
[15]梁前进,王鹏程,白燕荣.蛋白质磷酸化修饰研究进展[J].科技导报,2012,30(31):73-79.
[16]Junhua Zhang,Liisa Viikari.Impact of xylan on synergistic effects of xylanases and cellulases in enzymatic hydrolysis of lignocelluloses[J].Appl.Biochem.Biotechnol.,2014,174:1393-1402.