1株产褐藻胶裂解酶海洋细菌的分离鉴定及其酶学性质  

Isolation and identification of an alginate lyase-producing marine bacterium strain and its enzymological characteristics

作　　者：汤海青[1,2] 欧昌荣[3] 郑晓冬[1] 

机构地区：[1]浙江大学生物系统工程与食品科学学院,杭州310058 [2]宁波出入境检验检疫局,浙江宁波315012 [3]宁波大学海洋学院,浙江宁波315211

出　　处：《浙江大学学报（农业与生命科学版）》2013年第4期387-395,共9页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家星火计划资助项目(2012GA701063);钱江人才计划资助项目(2011R10072)

摘　　要：利用以褐藻胶为唯一碳源的培养基分离纯化产褐藻胶裂解酶的海洋细菌,通过形态特征、生理生化和16SrRNA基因鉴定菌株,用紫外法分析纯化酶液的pH、温度作用范围及稳定性等酶学性质.结果表明:从青岛近海分离到1株产褐藻胶裂解酶的菌株QZ-4,革兰阴性杆菌,适宜生长温度和NaCl质量浓度范围分别为15～25℃和15～60g/L;16SrRNA基因序列分析显示,该菌与交替假单胞菌(Pseudoalteromonas tetraodonis)相似性为97%,GenBank登录号为HM130919;该酶具有同时降解高古罗糖醛酸聚合物和高甘露糖醛酸聚合物的能力,其最适作用pH和温度分别为7.5和40℃,Mg2+、Na+、Fe3+、Mn2+等对酶活力有促进作用,Zn2+有抑制作用,1.0mol/L乙二胺四乙酸可完全抑制其活性;由Lineweaver-Burk(L-B)作图法求得该酶的最大反应速度(Vmax)为0.541U/mL,米氏常数(Km)为0.051mg/mL.Alginate polysaccharide produced by marine brown algae or certain Gram-negative bacteria is a linear anionic binary copolymer ofβ-D-mannuronic acid（M）andα-L-guluronic acid（G）residues.Alginate lyase（EC 4.2.2.3;EC 4.2.2.11）,also known as alginase or alginate depolymerase,catalyzes the degradation of alginate at the non-reducing terminus byβ-elimination mechanism,forming unsaturated oligosaccharides with double bonds.Since the original description of alginate lyases about 50years ago,more than 50enzymes have been characterized from a variety of algae,marine invertebrates,terrestrial and marine microorganisms.As a tool enzyme to degrade alginate biologically,alginate lyase has been explored and utilized in broad fields.It has been reported that alginate lyase or its crude extract can play a pivotal role in decomposing brown seaweeds,producing alginate oligosaccharides,analyzing the fine structure of alginate,preparing protoplasts of seaweed,and reducing viscosity of alginate biofilm built up in the lungs of cystic fibrosis sufferers.To elucidate the unique properties of alginate lyase,great efforts have so far been made around enzyme production,purification,characterization,and gene recombinant and so on.In the present study,a wild marine strain capable of producing alginate lyase was screened and studied in order to develop apotential overproducing strain using a low-cost culture medium.Marine microorganisms producing alginate lyase were screened by agar plate using alginate as only carbon source.The morphological,biochemical and physiological characteristics and 16SrRNA gene were analyzed to identify the taxonomic position of strain QZ-4.The enzymological characteristics of alginate lyase from the strain QZ-4were determined by ultraviolet（UV） method,including the optimal temperature,pH,thermal and pH stability,metal ions and ethylene diamine tetraacetic acid（EDTA） tolerance,etc.The results showed that the strain QZ-4was a Gram-negative rod bacterium.Its suitable growth temperature ranged from

关 键 词：海洋细菌 交替假单胞菌 16SrRNA 褐藻胶裂解酶 酶学性质 

分 类 号：Q936[生物学—微生物学]