海洋细菌HW08的鉴定及其所产低温碱性蛋白酶Ps5的分离纯化

IDENTIFICATION OF BACTERIUM HW08 AND PURIFICATION OF ITS EXTRACELLULAR ALKALINE COLD-ADAPTED PROTEASE PS5

由黄海底泥分离得到1株能够稳定高产蛋白酶的菌株,命名为HW08。对该菌株进行形态特征、生理生化分析表明,其为革兰氏阴性杆菌,极生单鞭毛,非发酵型,严格需氧,细胞内不积累多羟基丁酸作为储藏碳源,氧化酶阴性。将该菌16S rDNA序列(GenBank登录号FJ999660)和假单胞菌属中模式菌株16S rDNA序列进行比对,构建系统发育树,将其确定为Pseudomonas lundensis。分离纯化得到该菌分泌的蛋白酶Ps5,SDS-PAGE电泳显示其分子量为46kDa。Ps5的最适作用条件为pH10.4,温度30℃;其活性被EDTA显著抑制,表明Ps5属于金属蛋白酶家族。以AAPF为底物,测定Ps5酶动力学参数,计算表明K_m为1.17 mM,最大反应速率V_(max)为0.32μmol/min。

A strain named HW08, secreted protease abundantly and steadily, was isolated from Yellow Sea sediment. HW08 is an aerobic bacterium, non-fermentative, gram negative, rods and polar-flagellated. It accumulated Poly-P-hydroxybutyrate as a carbon reserve material and had no oxidase activity. The phylogenetic tree was built using Neighbor-joining method after comparing the 16S rDNA of HW08 （Gen Bank accession number FJ999660） with other Pseudomonas spp. type strains. Considering the phenotypical characteristics as well, it was determined that HW08 belongs to Pseudomonas lundensis. Furthermore, the protease named Ps5 was purified to homogeneity. The apparent molecular mass is 46 kDa showing on SDS-PAGE. The optimum pH is 10.4 while the optimum temperature is 30℃. This protease is strongly inhibited by EDTA, showing that it belongs to the metaloproteases superfamily. The kinetics properties were calculated to be Km 1.17 mM and Vmax 0.32μmol/ min using AAPF as substrate.