生物工程學(xué)報(bào) 25 June 2009, 25(6):927～931
臺(tái)灣家白蟻內(nèi)切葡聚糖酶活性中心氨基酸的飽和突變
林麗華1, 秦國(guó)梅1, 韋宇拓1, 杜麗琴1, 龐宗文1, 黃日波1,2
1 廣西大學(xué)生命科學(xué)與技術(shù)學(xué)院, 南寧 530004
2 廣西科學(xué)院, 南寧 530007
摘 要: 對(duì)內(nèi)切葡聚糖酶的功能改進(jìn)一直是纖維素酶研究領(lǐng)域的焦點(diǎn)。本研究對(duì)臺(tái)灣家白蟻內(nèi)切葡聚糖酶(CfEG)的活性位點(diǎn)做了飽和突變,。首先, 以PDB數(shù)據(jù)庫(kù)中高山象白蟻內(nèi)切葡聚糖酶(NtEG)的三維結(jié)構(gòu)(PDB id=1ks8)為模板, 對(duì)CfEG進(jìn)行三維結(jié)構(gòu)同源建模, 二者序列一致性高達(dá)79%,。位于CfEG活性中心的D53、D56,、E411, 分別與NtEG的催化殘基 D54,、D57、E412重合,。用簡(jiǎn)并引物對(duì)CfEG的假定活性位點(diǎn)D53,、D56、E411進(jìn)行定點(diǎn)飽和突變,。在位點(diǎn)D53,、D56各篩選到羧甲基纖維素酶活有一定提高的突變子D53E、D56C, 其中D56C的 Km值減小為原始酶的三分之一 ,。雙突變子D53L/D56I的比活比原始酶提高了近2倍, 同時(shí)Km值減小至原始酶的一半,。而E411的飽和突變子庫(kù)均沒(méi)有活性, 進(jìn)一步將其替換為近似氨基酸的E411D,、E411Q定點(diǎn)突變子也喪失了酶活。由突變結(jié)果可推斷, 位點(diǎn)E411為該酶行使功能的必需殘基,。
關(guān)鍵詞: 臺(tái)灣家白蟻, 內(nèi)切葡聚糖酶, 定點(diǎn)飽和突變, 雙突變, 定點(diǎn)突變
Saturation mutagenesis of three amino acid positions consisting of the active site of an endoglucanase from termite Coptotermes formosanus
Lihua Lin 1, Guomei Qin 1, Yutuo Wei 1, Liqin Du 1, Zongwen Pang 1, and Ribo Huang1,2
1 College of life Science and Technology, Guangxi University, Nanning 530004, China
2 Guangxi Academy of Sciences, Nanning 530007, China
Abstract: Functional improvement to one component of the cellulase, endo- -1, 4-glucanase, has been a focus of the recent research in this area. We report here the saturation mutagenesis of the active site of an endoglucanase (CfEG) from termite Coptotermes formosanus. First, three dimensional structure of CfEG was built via homology modeling by using a close-related (79% homology in sequence) endo- -1,4-glucanase (NtEG, PDB id=1ks8) from higher termite Nasutitermes takasagoensis as a template. Second, we identified three corresponding amino acid positions at the active site of CfEG by structural superposition onto NtEG. These three putative amino acids for the active site of CfEG, i.e., Asp53, Asp56 and Glu411, were subjected to saturation mutagenesis using degenerate primers. Among the mutants, Asp53Glu and Asp56Cys showed somewhow higher activities than the wildtype, with the latter having more than 3-fold decrease in Km. Double mutation Asp53Leu/Asp56Ile showed nearly 2-fold increase in specific activity and in the same time 2-fold decrease in Km. Saturation mutagenesis to the position Glu411 produced no active mutant, even changing Glu411 explicitly into its similar amino acids such as Glu411Asp and Glu411Gln could not result in any active mutant. These imply that position Glu411 could be extremely important and therefore indispensable for CfEG functionality.
Keywords: Coptotermes formosanus, endoglucanase, site-saturation mutagenesis, combined mutation, site-directed mutagenesis
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