绿色木霉产双功能酶二级结构的FT-IR光谱法分析

食品科学 ›› 2012, Vol. 33 ›› Issue (17): 150-153.

绿色木霉产双功能酶二级结构的FT-IR光谱法分析

刘萍1，刘靖1，祁兴普1，夏文水2,*

1.江苏畜牧兽医职业技术学院食品科技学院，江苏省畜产品深加工工程技术研究开发中心 2.江南大学食品学院，食品科学与技术国家重点实验室

收稿日期:2011-08-25 修回日期:2012-07-19 出版日期:2012-09-15 发布日期:2012-11-09
通讯作者: 刘萍 E-mail:yaoyao154@yahoo.com.cn
基金资助:
具有水解壳聚糖活性的双功能酶的结构与功能关系研究;绿色木霉产双功能酶降解壳聚糖的催化机制研究

Secondary Structure of Bifunctional Chitosanase-Cellulase from Trichoderma viride as Determined by FT-IR Spectrometry

Received:2011-08-25 Revised:2012-07-19 Online:2012-09-15 Published:2012-11-09
Contact: ping liu E-mail:yaoyao154@yahoo.com.cn

摘要/Abstract

摘要： 采用傅里叶红外光谱(FT-IR)的酰胺Ⅰ带和酰胺Ⅲ带测定绿色木霉产双功能壳聚糖酶-纤维素酶(CCBEs)的天然二级构象，结果显示：FT-IR光谱酰胺Ⅰ和Ⅲ带可以用来对绿色木霉所产的双功能酶二级结构进行预测，对两种双功能酶的主要二级结构组分α-螺旋和β-折叠的测定误差分别在2%和4%范围内，CCBEⅠ的二级构象以β-折叠为主，高达70%，与其圆二色谱和Garnier-Robson预测结果基本一致；而CCBEⅡ的二级构象中的β-折叠和α-螺旋含量分别为53.6%和14.6%。

关键词: FT-IR光谱, 二级结构预测, 壳聚糖酶, 纤维素酶, 绿色木霉

Abstract: The secondary structure of two bifunctional chitosanase-cellulase enzymes (CCBEⅠ and CCBEⅡ) from Trichoderma viride was explored by FT-IR spectroscopy. Amide Ⅰ and Ⅲ FT-IR bands allowed prediction of the secondary structure of CCBE. The errors of determination for α-helix and β-sheet contents in CCBEs were less than 2% and 4%, respectively. The quantitative analysis of CCBEⅠ revealed that the amino acid sequence was consistent with the results of circular dichroism analysis and Garnier-Robson prediction. The β-sheet content was approximately 70% in CCBEⅠ, while the β-sheet and α-helix contents of CCBEⅡ were approximately 53.6% and 14.6%, respectively. The measurement errors of α-helix and β-sheet of CCBEs were less than 2% and 4%, respectively.

Key words: FT-IR spectroscopy, secondary structure prediction, chitosanase, cellulase, Trichoderma viride

中图分类号:

TS201.3

刘萍1，刘靖1，祁兴普1，夏文水2,*. 绿色木霉产双功能酶二级结构的FT-IR光谱法分析[J]. 食品科学, 2012, 33(17): 150-153.

参考文献

[1]Surewicz WK，Mantsch HH．New insight into protein secondary structure from resolution-enhanced infrared spectra[J]．Biochim Biophys Acta，1988，952(2)：115-130．
[2]Surewicz WK，Mantsch HH，Chapman D．Determination of protein secondary structure by fourier transform infrared spectroscopy：A Critical Assessment[J]．Biochemistry，1993，32(2)：389-394．
[3]谢孟峡，刘媛．红外光谱酰胺Ⅲ带用于蛋白质二级结构的测定研究[J]．高等学校化学学报，2003，24(2)：226-231．
[4]曹栋，史苏佳，张永刚，等．酰胺I 带和酰胺Ⅲ带测定花生磷脂酶D 的α-螺旋和β-折叠含量[J]．化学通报，2008，71(11)：877-880．
[5]Goormaghtigh E, Ruysschaert JM, Raussens V. Evaluation of the Information Content in Infrared Spectra for Protein Secondary Structure Determination[J]. Biophys J,2006,90:2946–2957.
[6]刘靖,夏文水.纤维素酶中具有壳聚糖水解酶活性成分的鉴定[J].中国生物化学与分子生物学报, 2005, 10 (5) : 713～716.
[7] Liu J， Xia WS. Purification and Characterization of a Bifunctional Enzyme with Chitosanase and Cellulase Activities from Commercial Cellulase[J]. Biochemical Engineering Journal, 2006,30：82-87
[8]刘萍，夏文水,刘靖,高勤学.绿色木霉产双功能壳聚糖酶-纤维素酶的基因克隆、序列分析及表达[J].中国科技论文在线精品论文,2009,2(16): 1736-1742.
[9]Fabian H， Naumann D. Methods to study protein folding by stopped-flow FT-.IR[J]. Methods, 2004，34：28-40.
[10]Hu Y, Li W， Hu J. Resolving overlapped spectra with curve fitting[J]. Spectrochim. Acta. A Mol. Biomol. Spectrosc,2005，62：16–21.
[11]Petibois C, Gouspillou G, Wehbe K. Analysis of type I and IV collagens by FT-IR spectroscopy and imaging for a molecular investigation of skeletal muscle connective tissue[J].Anal Bioanal Chem, 2006, 386:1961–1966.
[12]刘刚，邢达，王海珉，等．胆结石中蛋白质的傅里叶变换红外光谱和表面增强拉曼光谱研究[J]．光学学报，2002，22(4)：441-446．
[13]黄金凤，沈文洁，刘能盛，等.FT-IR光谱法测定蔗糖酶的二级结构[J].广东化工, 2010 , 37（7）：254-255.
[14]刘萍，顾雨辰，刘靖，等. 绿色木霉非专一性产双功能酶的鉴定[J].安徽农业科学，2010,12：20542-20546.
[15]刘靖.纤维素酶作用壳聚糖的机理研究[D].江南大学食品科技学院，无锡，2006.
[16]刘萍.绿色木霉产双功能酶的结构与功能研究[D].江南大学食品科技学院，无锡，2009.