来源于土壤农杆菌PJD-1-1的碱性β-葡萄糖苷酶的生产、纯化及酶学性质研究（英文）

Production,Purification and Characterization of Alkalineβ-Glucosidase Isolated from Agrobacterium sp. PJD-1-1

为从环境中筛选新的β-葡萄糖苷酶生产菌株,利用筛选平板涂布法从腐败的甘蔗叶中筛选目的菌株,从中筛选到1株能产β-葡糖糖苷酶的菌株PJD-1-1,16S r DNA鉴定该菌株为新型的土壤农杆菌。该菌株在20℃,初始p H 7.0,以乳糖为碳源,NaNO_3为氮源的条件下,其酶活性最高为3.92 U/mg。经硫酸铵沉淀、葡聚糖凝胶过滤层析和离子交换层析纯化后(纯化倍数4.85,得率8.0%),SDA-PAGE分析表明得到一条分子量大小为40 k Da条带;该酶的最适反应温度和pH分别为50℃和8.0;在低于50℃条件下保有较好的稳定性;Hg^(2+)和Ag^+对酶活性有明显的抑制作用,表明该酶的催化活性中心可能含有硫醇基,Ba^(2+)、Ca^(2+)、Pb^(2+)、Co^(2+)、Zn^(2+)、Mn^(2+)、Na^+、K^+、EDTA和尿素对酶活性没有明显的影响。该酶的温度稳定性、碱性特征以及对金属离子的耐受性等使其在食品及其他领域具有广泛的应用潜力。

In order to screen novelβ-glucos0idase producing strains from environment,one targeted novel strain PJD-1-1producingβ-glucosidase were isolated from putrefied sugarcane leaves with screening and spreading plate.16S rDNA analysis revealed it was a novel grobacterium sp.When the strain was incubated at initial pH7.0,20℃with lactose as carbon and NaNO3as nitrogen sources,the maximum enzyme activity was3.92U/mg.β-Glucosidase from this strain was purified using(NH4)2SO4precipitation followed by dextran gel-filtration chromatography and ion-exchange chromatography.A purifying fold of4.85with gaining rate of8.0%was obtained.SDA-PAGE analysis of the purified enzyme showed that it was a clear and pure band with molecular mass of ca.40kDa.The most optimum activity of the enzyme was at50℃and pH at8.0.The enzyme could maintain stability under the conditions below50℃.Hg2+and Ag+heavily inhibited the enzyme activity suggesting that the active catalytic sites of the enzymes might possess thiol radical.Ba2+,Ca2+,Pb2+,Co2+,Zn2+,Mn2+,Na+,K+,EDTA,and urea had no obvious effects on the enzyme activity.It is concluded that the novel strain Agrobacterium sp.PJD-1-1producingβ-glucosidase was successfully screened from putrefied sugar cane leaves.The produced enzyme had thermal stability,alkaline feature and metal ions tolerance made it useful in the food and broad potential applications in other fields.