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

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 15to 25 ℃ and NaCl concentration ranged from 15to 60g / L.Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that the strain QZ-4was a member of Pseudoalteromonas tetraodonis.The GenBank accession number was acquired as HM130919.The alginate lyase was characterized as a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） and had the same decomposing ability on high guluronate and mannuronate polymers separately.The alginate lyase remained stable below 35 ℃ at the alkaline conditions.The optimal catalytic activity was showed at 40 ℃ and pH 7.5.The alginate lyase was activated by ions such as Mg 2＋,Na ＋,Fe 3＋ and Mn 2＋,but was inhibited by Zn 2＋ and EDTA.The results of kinetic studies showed that the V max（maximum reaction velocity） and Michaelis-Menten constant（K m） were measured individually as 0.541U / mL and 0.051mg / mL by Lineweaver-Burk（L-B） plot.In conclusion,the above results indicate that P.tetraodonis strain QZ-4has a consistent alginate lyase yield and biomass at the present culture condition,compared with other alginate lyase-producing strains among the genus Pseudoalteromonas sp.The simple nutrition demand of strain QZ-4 makes a low-cost culture medium possible,and the yield of alginate lyase can reach as high as 135 U / mL in shaking flask during a short fermentation period.Therefore,the strain QZ-4has the potential of producing alginate lyase on a large scale and further research is required to elucidate fermentation conditions and kinetics of this enzyme.