锈凹螺褐藻胶裂解酶的分离纯化及性质研究  

Purification and charaterization of alginate lyase from Chlorostoma rustica

作　　者：孙丽萍[1] 薛长湖[1] 许家超[1] 李兆杰[1] 蔡跃飘[1] 赵雪[1] 

机构地区：[1]中国海洋大学食品科学与工程系,山东青岛266003

出　　处：《中国水产科学》2004年第3期266-271,共6页Journal of Fishery Sciences of China

基　　金：国家"863"高技术研究发展项目(2003AA625030)

摘　　要：采用硫酸铵沉淀、离心超滤、DEAE-SephadexA25、SephadexG-100和SephacrylS-300HR柱层析等方法从海洋食藻性锈凹螺(Chlorostomarustica)消化腺中分离纯化出一种褐藻胶裂解酶(Alginatelyase),得到电泳纯酶制品,并对此酶的酶学性质进行了研究。结果表明:此褐藻胶裂解酶的分子量为28kD,反应的最适温度为40℃,具有热不稳定性,最适pH为7.0;酶的反应受多种离子的影响,Mn2+、Co2+和Cd2+对此褐藻胶裂解酶活性具有明显的抑制作用,而Mg2+、Zn2+、Na+和K+则具有促进作用;采用透明圈法和底物法验证了此裂解酶对聚甘露糖醛酸和聚古洛糖醛酸的特异性,实验表明,此酶对聚古罗糖醛酸(Polyguluronate,PG)有特异性。Alginate is an acidic polysaccharide composed of (1→4)-linked α-L-guluronic acid (GulA) and β-D-mannuronic acid (ManA), arranged in three types of block structures, polyguluronate 〔poly(GulA)〕, polymannuronate〔poly(ManA)〕and hetero-polymeric random sequences〔poly (ManA/GulA)〕. Alginate lyase has recently attracted the attention, because the enzymatic degradation of alginate expands a potential use of this polysaccharide. Additionally, some of the alginate-derived oligouronic acids are gaining biochemical interest due to their physiological activities on plant growth. As alginate is a hetero-polysaccharide, the choices of enzyme sources and reaction conditions affect the end products. Thus, the elucidation of the substrate specificities of alginate lyase toward various kinds of oligouronic acids is important for the preparation of desired oligouronic acids by enzymatic degradation of alginate. Alginate lyases have been tentatively classified into two types based on their substrate specificity, defined as the preference for either polymannuronate lyase or polyguluronate lyase block. The alginate-degrading enzymes, polyM lyase and polyG lyase, cleave the glycosidic linkage of alginate by β-elimination reaction and produce oligouronic acids with a 4,5-unsaturated uronic acid residue at the non-reducing end. Many alginate lyase were purified, and their physicochemical properties were characterized. In these studies, some invertigators elucidated the substrate specificities of the enzymes toward each of the three types of blocks, and end-products of the reaction were identified using NMR, TLC, HPLC and paper chromatographic techniques. Alginate lyase activity has been detected from a wide variety of sources, including marine mollusks, bacteria, fungi and marine brown algae.The alginate lyase activity from Chlorostoma rustica was detected, and purified by using ammonium sulfate precipitations,and successive fractionation on DEAE-Sephadex A25,Sephadex G-100 and Sephacryl S-300 HR columm chromatography. T

关 键 词：锈凹螺 褐藻胶裂解酶 特异性 

分 类 号：S986.2[农业科学—捕捞与储运]