北黄海沉积物可培养产蛋白酶细菌分离鉴定

摘要

[目的]揭示北黄海沉积物中可培养产胞外蛋白酶细菌及蛋白酶多样性,增加人们对北黄海生态系统中产蛋白酶菌多样性的认识,为海洋产蛋白酶微生物的挖掘提供菌群资源.[方法]分别将5个北黄海沉积物样品梯度稀释涂布至酪蛋白明胶筛选平板,选择性分离产蛋白酶细菌;并通过分析基于16S rRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;分别测定胞外蛋白酶活性并对酶活较高的39株菌进行基于苯甲基磺酰氟(PMSF,丝氨酸蛋白酶抑制剂)、邻菲罗啉(o-phenanthroline,O-P,金属蛋白酶抑制剂)、E-64(半胱氨酸蛋白酶抑制剂)和pepstatin A(天冬氨酸蛋白酶抑制剂)4种抑制剂的酶活抑制实验以及所有菌株对3种底物(酪蛋白、明胶、弹性蛋白)的水解能力;分析这些细菌所产胞外蛋白酶的特性及多样性.[结果]从5个北黄海沉积物样品中分离获得66株产蛋白酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria、Actinobacteria和Firmicutes 4个门的7个属,其中Pseudoalteromonas (69.9％)、Sulfitobacter (12.1％)和Saleg entibacter (10.6％)是优势菌群;沉积物中可培养的产蛋白酶细菌的丰度为104 CFU/g;蛋白酶酶活抑制实验表明所有测定菌株产生的胞外蛋白酶属于丝氨酸蛋白酶和/或金属蛋白酶,仅有少数菌株所产蛋白酶具有半胱氨酸蛋白酶或天冬氨酸蛋白酶活性.[结论]北黄海沉积物中可培养产蛋白酶细菌类群较为丰富,Pseudoalteromonas、Sulfitobacter 和Salegentibacter菌株是优势菌群,测定菌株所产胞外蛋白酶主要是丝氨酸蛋白酶和/或金属蛋白酶.%[Objective] To uncover the diversity of extracellular protease-producing bacteria and to expand our knowledge on protease-producing bacteria in the sediments of North Yellow Sea,and to screen efficient protease production strains that might provide flora resources for mining marine protease-producing microorganisms.[Methods] Protease-producing bacteria were isolated by using casein gelatin plate from 5 sediment samples of the North Yellow Sea.The bacteria diversity was evaluated through phylogenetic analyses based on 16S rRNA genes.The protease diversity was evaluated through the inhibition tests for 39 strains with higher enzymatic activities by using 4 inhibitors:phenyl methyl sulfonyl fluoride (PMSF,serine protease inhibitor),1,10-phenanthroline (O-P,metalloproteinase inhibitor),E-64 (cysteine protease inhibitor) and pepstatin A (aspartic protease inhibitor).Furthermore,degradation abilities for different protein substrates such as casein,gelatin and elastin were evaluated through observing hydrolytic zones.[Results] A total of 66 protease-producing strains were isolated from 5 sediment samples.These isolates were classified into 7 genera of 4 phyla including Bacteroidetes,Proteobacteria,Actinobacteria and Firmicutes,with Pseudoalteromonas (69.9％),Sulfitobacter (12.1％) and Salegentibacter (10.6％) as the dominant.The richness of the cultivable protease-producing bacteria reached 104 cells/g in all sediment samples.The inhibition tests indicated that all the tested strains produced serine protease and/or metal protease,only a few strains produced cysteine protease or aspartic protease.[Conclusion] The cultivable protease-producing bacteria in the North Yellow Sea are diverse with Pseudoalteromonas,Sulfitobacter and Salegentibacterbacteria as the dominant groups and the extracellular proteases belong to serine proteases and/or metalloproteinases.