68株北极产蛋白酶菌株的筛选、鉴定以及部分酶学性质

Isolation，identification and characterization of 68protease-producing bacterial strains from the Arctic

摘要:

摘要:【目的】从北极海水样品中分离产蛋白酶细菌，并对其进行初步的分类鉴定，为低温蛋白酶的低温适应性及其应用研究奠定基础。【方法】通过酪蛋白筛选培养基低温培养的方法从北极水样中分离出68株产蛋白酶细菌，采用16S rRNA基因PCR-RFLP(限制性酶切多态性)方法及传统的表型特性分析对所分离纯化的菌株进行分类，每种细菌类型各取1 株代表菌株进行16S rRNA基因序列测定、GenBank数据库blast分析以及通过DNAMAN软件进行系统进化树分析。对代表菌株的蛋白酶酶学性质进行初步研究。【结果】68 个菌株可归为3种类型(54.41%、42.65%和2.94%)，分别以菌株6、11和52为代表菌株。16S rRNA基因序列分析结果表明，菌株11与比目鱼黄杆菌(Chryseobacterium scophthalmum)具有98.24%的同源性;菌株52与嗜根寡养单胞菌(Stenotrophomonas rhizophila)具有98.55%的同源性;菌株6与Stenotrophomonas rhizophila具有96.50%的同源性，可能为该属的新物种。对3种类型代表菌株进行表型性状研究显示，菌株6、11和52为革兰氏阴性、直杆状、不产胞外脂肪酶和淀粉酶，具有强的蛋白酶活性。菌株6 的蛋白酶最适酶活温度为55℃，最适宜pH为6.7;菌株11的蛋白酶最适酶活温度为40℃，属于低温酶，最适酶活pH约为8.5;菌株52的蛋白酶最适酶活温度为65℃，最适酶活pH 为7.4。【结论】本文首次报道了Stenotrophomonas和Chryseobacterium的菌株在北极海水样品中的分布，充实了极地产蛋白酶菌的种属分布多样性，为后续低温蛋 白酶的研究和应用奠定了基础。

Abstract:

Abstract:［Objective］We screened and identified protease-producing bacterial strains from the Arctic，the results would help find cold-adapted protease．［Methods］In total 68 protease-producing strains were screened from the Arctic using the casein-agar plate under low temperature． All strains were classified using the 16S rRNA gene-restriction fragment length polymorphism (RFLP) and traditional phenotypic test． One strain was chosen to be the representative strain from each group respectively and identified using 16S rRNA gene sequence analysis，GenBank database Basic Local Alignment Search Tool (BLAST)，phylogenetic analysis and phenotypic features analysis．The enzymatic properties of the epresentative strains were determined．［Results］ The 68 strains belonged to 3 groups (54.41%，42.65% and 2.94%)，and strains 6，11 and 52 were the representative strains respectively．The results of the 16S rRNA gene sequence analysis showed that:Strain 11 was most closely related to Chryseobacterium scophthalmum with 98.24% sequence similarity; strain 52 was most closely related to Stenotrophomonas rhizophila with 98.55% sequence similarity; strain 6 was most closely related to Stenotrophomonas rhizophila with 96.50% sequence similarity，which might represent a novel species of the genus． The phenotypic study showed that: strains 6，11 and 52 were Gram negative，straight rodshaped，did not produce extracellular lipase and amylase，possessed strong proteolytic activity．The optimal temperature and pH for the enzyme activity were 55℃ and 6. 7 for the protease from strain 6，40℃ and 8. 5 for the protease from strain 11 and 65℃ and 7.4 for the protease from strain 52.［Conclusion］The research firstly introduced the distribution of Stenotrophomonas and Chryseobacterium specieses in the Arctic marine water，extended the diversity of the proteaseproducing bacteria from the Arctic，and provided a useful basis for further study of cold-adapted protease．