天山冻土中产低温蛋白酶菌株的筛选、鉴定及酶学性质

借助形态观察、生理生化指标测定及16S rDNA序列分析,对从新疆天山冻土中筛选得到的产低温蛋白酶菌株进行鉴定,确定其为Pseudomonas sp.(假单胞菌属)N-8。通过单因素试验和正交实验对其发酵条件进行优化,确定最佳发酵培养基配方为(g/L):葡萄糖10,酵母粉8,酪蛋白20,K+0.4,pH 7.0。酶学性质研究表明,该菌产生的低温蛋白酶最适作用pH为7.0,在pH 7.0～8.0呈现良好的稳定性;最适作用温度为25℃,在25、35、45℃下均呈现良好的稳定性;金属离子Cu2+、K+、Ca2+、Mn2+对酶活有一定的抑制作用,其中Cu2+抑制作用明显,Fe2+、Mg2+、Na+、Zn2+对酶活力有不同程度的激活作用。

Screening,Identification of Pseudomonas sp. N-8 producing Cold-adapted Protease and the Study on Enzymatic Properties

A strain producing the cold-adapted protease has been isolated from the frozen soil of the Tianshan Mountains,China.Based on the morphological,physiological,biochemical properties and 16S rDNA sequence analysis,the strain was identified as Pseudomonas sp.N-8.Fermentation conditions were optimized by single factor and orthogonal experiments.The optimal medium components were determined as follows:10 g/L glucose,8 g/L yeast powder,20 g/L casein,0.4 g/L KH2PO4,pH 7.0.The research on the properties of crude enzyme showed that the optimal pH and temperature for activity were 7.0 and 25 ℃,respectively.As to the thermal stability of the cold-adapted protease,it was stable over the range of 25～45 ℃.The test of pH stability indicated that the cold-adapted protease was more stable within pH 7.0～8.0.The protease activity was inhibited by Cu2+,K+,Ca2+,and Mn2+,but it was stimulated by Fe2+,Mg2+,Na+,and Zn2+.