一种新家族琼胶酶的酶学性质、水解机理与分子特征的研究

Flammeovirga sp. MY04 is a polysaccharide-degrading marine bacterium with a strong agarose-liquefying capability. The MY04 genome contains 15 predicted agarase genes, most of which encode proteins belonging to the glycoside hydrolase (GH) family 16 or 86. Recently we have reported the novel characteristics and molecular modifaction of a GH16 agarase AgaG4(Han WJ, et al. J. Biol. Chem. 2013). However, another agarase gene "agao" encodes a unique agarase AgaO, which shares the highest identity with the eluciated GH50 agarases but still lower than 30%. The protein sequence of AgaO shares few conservative region with all the eluciated agarases of the GH50, GH86, GH96 or the GH118 family. The recombinant protein of AgaO (rAgaO) is a capable of degrading agarose into oligosaccharides. Thererfore, it is suggested that the agarase AgaO belongs to a novel GH family undefined yet. In this project, further studies on the recombinant agarase rAgaO will be performed as following to construct a new GH family： (1) To learn the biochemical characteristics of Agao, the recombinant agarase rAgaO with high purity will be prepared and applied in the studies on its enzymatic characteristics, substrate-degrading patterns, and the type of hydrolysis mechanism. Agarose oligosaccharides produced by rAgaO will be purified into monomers and chemically characterized. (2) To define the location and the minimal border of the catlytic module or the carbohydrate-binding module in AgaO, the full-length gene of AgaO will be truncated using PCR primer pairs. The resulted gene fragments will be heterologously expressed,and the truncated proteins will be purified and characterized. (3) To further define the typical molecular properties of the novel GH family represented by AgaO, protein sequences will be aligned among the agarase AgaO, other hyperthecial members of the novel family, and agarases of the eluciated GH families. Candidate active site residues of AgaO will be characterized through site-directed mutagenesis strategies. This project will furtherly benefit the discovery of the structural properties and the catalytic mechanism of agarases within the novel GH family.

火色杆菌属海洋细菌MY04是课题组分离的1株高效多糖降解菌,琼脂糖液化能力强.MY04基因组共编码15个琼胶酶,主要归类于糖基水解酶(GH)16或86家族.我们已报道了GH16家族琼胶酶AgaG4的新颖结构特征与分子酶学改造研究(Han WJ,et al. J. Biol. Chem. 2013).序列分析表明，与AgaG4等14个琼胶酶不同,琼胶酶AgaO蛋白序列新颖,进化地位独特,不能直接进行家族归类.实验表明,重组蛋白(rAgaO)粗酶的琼胶酶活性高.本项目拟参照糖基水解酶新家族的设立标准,进行如下深入研究:(1)纯化、制备rAgaO,阐明常规酶学性质、水解机理类型与底物降解特性;(2)进行基因截短研究,鉴定AgaO的功能模块与组成特征;(3)分析以AgaO为代表的糖基水解酶新家族的典型序列,探讨候选活性位点的贡献.本项目还将为揭示这一琼胶酶新家族的催化机制与高级结构特征奠定基础.

近年来，与琼胶酶的功能、性质、机制及分子改造相关的研究聚焦于 GH16 家族 β-琼胶酶。现有研究已涉及到琼胶酶的 三维立体结构、功能模块、功能基序、关键活性位点及其残基的结构等多个层面，这为新型琼胶酶的鉴定、琼胶酶新家族分类单元的建立提供了一定的借鉴。火色杆菌属海洋细菌 MY04 是课题组分离的 1 株高效多糖降解菌，琼脂糖液化能 力强。MY04 基因组共编码 15 个可预测的琼胶酶，主要归类于糖基水解酶(GH)16 或 86 家族。课题组最近曾报道了MY04来源的GH16家族的内切型琼胶酶AgaG4。. 与已知琼胶酶不同，AgaO与GH50家族β-琼胶酶、GH42家族β-糖苷酶序列相似性最高，介于30-40%，无法直接进行家族归类。重组酶rAgaO在50oC、pH 7.0时琼胶酶活性最高,无糖苷酶活性。结合TLC、HPLC等色谱分析，以及MS、13C-NMR等波谱分析，证实rAgaO降解琼脂糖时以新琼二糖为唯一主产物，且最小底物是新琼四糖。以邻氨基苯甲酰胺标记的新琼六糖为底物，通过荧光HPLC分析，证明AgaO是从该底物的非还原性末端逐步切割产生新琼二糖的。这提供了关于外切型琼胶酶底物生成方式的首个可视的生化证据。结合寡糖链的邻氨基苯甲酰胺标记与MS/MS分析, 提供了一个寡糖链的测序方法。该方法可以替代传统的13C-NMR分析，且所需样品量为微克级、无需精细纯化。研究还表明，在降解琼脂糖过程中产生的微量的、疑似寡糖片段的副产物不是新琼四糖、六糖等偶数糖，且因AgaO不具有糖苷酶活性，推测不是奇数糖片段，而是含有硫酸根的系列寡糖片段。本研究还表明，与已知的二糖外切酶相似，该酶的整体结构与催化活性关系密切，分子截短极易造成截短体蛋白质丧失琼胶酶活性。遵照审稿人的建议，我们未直接定义酶的家族归类，留待晶体结构分析。. 此外，我们拓展地鉴定了一个Flammeovirga sp. MY04来源的新型古罗糖醛酸专一性内切型褐藻胶裂解酶Aly5，参与鉴定了Vibrio sp. FC509来源的硫酸软骨素4-O-内切型硫酸酯酶，阐明了它们的生化特征、寡糖生成特性及底物切割模式等酶学特性，分析了分子改造的影响与机制。本项目的研究为深入研发多糖降解的工具酶及酶的精确应用提供了有益借鉴。