Mutation strategies for obtaining chitooligosaccharides with longer chains by transglycosylation reaction of family GH18 chitinase.
Mutation strategies for obtaining chitooligosaccharides with longer chains by transglycosylation reaction of family GH18 chitinase.
复制标题
通过GH18几丁质酶家族的转糖基反应获得更长链的几丁寡糖的突变策略。
DOI：
10.1080/09168451.2014.948373
复制
发表时间：
2014
期刊：
影响因子：
--
通讯作者：
T
中科院分区：
文献类型：
--
作者：
Sirimontree;P.;Suginta;W.;Srinrho;N.;Kanda;Y.;Shinya;S.;Ohnuma;T.;Fukamizo;T
提高糖苷水解酶的转糖基化（TG）活性并不总是导致具有较长链的寡糖的产生，因为TG产物通常被分解成较短的寡糖。本文研究了利用哈氏弧菌GH18几丁质酶A（VhChiA）催化TG反应制备长链壳寡糖的诱变策略。HPLC分析的TG产品从孵育的几丁寡糖底物，GlcNAcn，与几个muplasma VhChiAs表明，突变体W570 G（突变的Trp570到Gly）和突变体D392 N（突变的Asp392到Asn）显着增强TG活性，但TG产品立即水解成较短的GlcNAcn。另一方面，从突变体D313A和D313N（Asp313分别突变为Ala和Asn）获得的TG产物没有进一步水解，导致具有较长链的寡糖的积累。从多聚体VhChiAs中获得的数据表明，将DxDxE催化基序的中间天冬氨酸残基Asp313突变为Ala和Asn对于获得具有较长链的壳寡糖是最有效的。
Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A fromVibrio harveyi(VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAcn, with several mutantVhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAcn. On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutantVhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.