产双功能褐藻胶裂解酶菌株的筛选与初步研究

目的：双功能褐藻胶裂解酶既能降解聚β-D-甘露糖醛酸，又能降解聚α-L-古罗糖醛酸，可以用一种酶来制备不同结构的褐藻胶寡糖。本文的目的是筛选能产生双功能褐藻胶裂解酶的菌株，对其产酶曲线和降解产物作初步研究。方法：利用唯一碳源培养基筛选产生褐藻胶裂解酶的菌株，通过16SrDNA序列比对进行菌种鉴定，通过在凝胶上检测褐藻胶裂解酶活性来判断发酵上清液中褐藻胶裂解酶的数量及分子量，利用薄层层析确定降解褐藻胶的终产物组成。结果：从褐藻上筛选到一株海洋细菌QY107，鉴定为弧菌属细菌。发酵120h时褐藻胶裂解酶产量为12．32U／mL，其发酵液上清中只含有一种褐藻胶裂解酶，分子量在28kDa左右，并且对聚β—D-甘露糖醛酸和聚α-L-古罗糖醛酸都能降解，降解褐藻胶的终产物主要为三糖。结论：本文筛选到一株弧菌QY107，其发酵液上清中只有一种双功能褐藻胶裂解酶，可用于大量制备褐藻胶三糖。推测该酶具有特殊的催化腔结构，对其结构与功能相互关系的研究可能会发现新的底物结合与催化机制。酶解制备褐藻胶寡糖因其环保高效而越来越受到人们的重视，因此该菌株能促进海洋寡糖类生物制品的开发，在医药、食品、农业、生物燃料等领域具有广阔的应用前景。

Screening and Characterization of a Bifunctional Alginate Lyase-Producing Bacterium Strain

Objective： Bifunctional alginate lyases, which could degrade both poly tx-L-guluronic acid and poly 13 -D-mannuronic acid, could be used to prepare alginate oligosaccharides with different structure. The aim of this research is to find and characterize a bifunctional alginate lyase-producing bacterium strain. Methods： Alginate lyase-producing bacteria were screened on the selective medium using alginate as the sole carbon source and identified by 16S rDNA sequence analysis. Detection of alginate lyase activity after SDS-PAGE was carried out to determine the number and molecular weight of alginate lyase in the culture supernatant of strain QYI07. The enzymatic product of alginate was analyzed by Thin-Layer Chromatography. Results： A marine bacterium strain QY107 was screened from brown algae and identified as Vibrio sp. QY107. The highest alginate lyase activity （12.32 U/mL） occurred at 120 h. There was only one alginate lyase with a molecular weight of about 28 kDa in the culture supernatant of strain QY107. The supernatant showed activities toward both poly α-L-guluronic acid and poly β-D-mannuronic acid, and released mainly trimers from alginate. Conclusions： Vibrio sp. QY107, isolated because of its high alginate lyase production, secreted only one alginate lyase into the culture supernatant. This enzyme is a bifunctional alginate lyase and could be used to prepare trisaceharides from alginate. This enzyme might have special active site structure and be a good sample for the study of structure-function relationship to find new substrate binding and catalytic mechanisms. Preparation of alginate oligosaccharides using alginate lyase have received more and more attention because of its high efficiency and low pollution to the environment. Strain QY107 might accelerate the exploitation of marine oligosaccharides and be applied in medicine, food, agriculture and biofuel industries.