新疆阿勒泰和温泉县冷水鱼肠道产低温蛋白酶菌株的筛选及其酶学性质的研究

Screening of Strains Producing Cold-active Proteases from the Intestinal Tracts of Cold-water Fish from Wenquan County and Altay in Xinjiang and Study on Enzymatic Properties

[目的]从50株冷水鱼肠道产蛋白酶菌株中筛选优良产酶菌株,并研究其酶学性质,为高效低温蛋白酶技术的研发奠定基础。[方法]对比脱脂乳平板初筛结果确定供试菌株,对选定菌株进行表型及16S rRNA/26S rRNA序列同源性分析,确定产酶菌株的进化地位。用Folin酚显色法复筛,并研究最佳产酶菌株所产蛋白酶的酶学性质。[结果]从6株供试菌株中筛选出一株高产酶菌株P-D-10,初步鉴定隶属于Pseudomonas。酶学性质的初步研究显示,P-D-10产生的蛋白酶最适反应pH在8.5左右;在0～60℃均有催化活性;具有10和40℃2个最适酶活温度;热稳定性较差,在70℃水浴10 min后剩余酶活力仅为20.14%。除了Tween-80对该酶酶活力有促进作用外,其他金属离子、还原剂、表面活性剂和金属离子螯合剂对该酶酶活力均有不同程度的抑制作用。[结论]筛选获得的高产酶菌株P-D-10隶属于Pseudomonas,是一株具有研究与应用潜力的产低温蛋白酶菌株。

[Objective] The study aimed to screen high-yield cold-active protease strain and study its enzymatic properties,lay a foundation for development of high-efficiency cold-active proteases.[Method] Among 50 strains producing proteases,6 strains with larger diameter ratio of hydrolyzation halo and colony were selected as original strain.Morphological observation,enzyme activity determination and the phylogenetic analysis were carried out.[Result] The high-yield strain P-D-10,belonged to Pseudomonas,was finally obtained through the second screening with Foline-Phenol method.The extracellular proteases of strain P-D-10 were able to hydrolyze casein at 0-60 ℃,the optimum reaction pH and temperature was pH 8.5,10 ℃ and 40 ℃,respectively.The protease was susceptible to heat and retained only 20.14% enzyme activity at 70 ℃ for 10 min.With the exception of Tween-80,the rest of metal ion,surfactant,reductant and chelant all inhibited enzyme activity of the extracellular proteases of strain P-D-10 in different extent.[Conclusion] The high-yield enzyme strain P-D-10 belongs to Pseudomonas and is a potential strain in the research and application of cold-active proteases.