红树林微生物DH-2胞外蛋白酶的性质及产酶条件优化

Properties of Extracellular Protease of Microbe DH-2 from Mangrove and Optimization of Enzyme Producing Conditions

从大亚湾红树林土壤样品中分离得到产蛋白酶菌株,鉴定所产胞外蛋白酶的酶学性质以及菌株的最佳发酵培养条件。采用平板透明圈法筛选菌株,福林酚显色法测定蛋白酶的酶活,通过单因素和正交试验确定其最佳发酵培养基以及发酵条件。从壤样品中分离得到一株产蛋白酶的枯草芽孢杆菌DH-2,该菌株分泌的蛋白酶最适反应pH和温度分别为8.0和65℃,50℃保温处理60 min后,剩余酶活仍保留80%以上。该蛋白酶对多种金属离子、有机溶剂及表面活性剂均有较好的耐受性。确定该菌株产蛋白酶的最适条件:1%(m/V,下同)可溶性淀粉,1%胰蛋白胨、1%NaCl,初始pH 5.5及7%的接种量,40℃培养36 h。在最适条件下测得其发酵液的酶活为236.30 U/mL,约为初筛时的酶活的8倍。该蛋白酶具有较为广阔的作用温度和pH范围,金属离子、有机溶剂及表面活性剂耐受性好,酶的性质比较稳定。

This work is aimed to isolate the strains producing proteases from Daya Bay mangrove and to characterize the enzymatic properties and the optimal fermentation conditions. The clear zone method was used to screen strains,the Folin-phenol reagent method to determine the activity of protease,and the single factor experiments and the orthogonal experiments to determine the optimal fermentation medium and the fermentation conditions. Protease-producing Bacillus subtilis DH-2 was isolated from soil samples,and the optimal fermentation pH and temperature of the protease secreted by DH-2 were 8.0 and 65℃,respectively. The protease remained over 80% of its original activity after treatment for 60 min at 50℃. The protease tolerated multiple metal ions,organic solvents and surfactants. The optimal fermentation conditions for the protease were：1%（m/V）soluble starch,1% tryptone,1% NaCl,the initial pH of 5.5,and the inoculation amount of 7%,36 h culture at 40℃. Under optimal fermentation conditions,the enzyme activity of the fermented liquid was 236.30 U/mL,which was about eight times of the activity in initial screening. Conclusively,the protease has a relatively wide range of action temperature and pH,and exhibits fine resistance to multiple metal ions,organic solvents and surfactants,and is characterized stably.