嗜热菌Anoxybacillus sp．DL3产蛋白酶条件及其酶学性质研究

目的：对Anoxybacillussp．DL3的产蛋白酶条件及其酶学性质进行研究，为下一步进行蛋白酶基因的克隆、表达提供依据。方法：应用常规方法液体培养细菌，研究温度、pH、培养基中碳源、氮源对菌株产蛋白酶的影响，硫酸铵盐析的方法提取酶液，并采用Folin法测酶活性。用紫外分光光度计在OD680hi／1测吸光值。并对提取的蛋白酶液进行酶的最适温度、pH以及酶的热稳定性和pH稳定性研究，向酶液中添加金属离子和EDTA、PMSF，研究其对酶活性的影响。结果：在培养基初始pH是6．5，培养温度为40℃时菌株产酶酶活性最大；培养基中以乳糖为碳源，酵母膏和硫酸铵为氮源，碳源与氮源的比例为1：2时，酶活最大。酶学性质研究结果显示：该酶的最适反应温度是55℃，最适反应pH是7．0；在50℃保温20min-80min内，酶活力下降幅度较小。60℃保温60min后，仍保持约60％的酶活。70℃保温60min后，残余酶活为30％。该酶在pH为6．0～8．0范围内，相对酶活差别不是很大，下降趋势大致相同。在强碱条件下，相对酶活下降很明显。Fe2＋、Cu2＋和Hg2＋对酶活性有明显的抑制作用；Ca2＋、Mg^2＋、Mn2＋等金属离子对酶活性有明显的促进作用；乙二胺四乙酸（EDTA）和苯甲基磺酰氟（PMSF）对酶活性也有一定抑制作用。结论：Anoxybacillussp．DL3所产的蛋白酶为嗜热中性蛋白酶，此酶具有较好的热稳定性和pH耐受性，该菌株具有进一步开发、利用的价值。

Protease Production Conditions and Enzyme Characterizations of Protease-Producing Strain Anoxybacillus sp.DL3

Objective： To provide the theoretical basis for protease gene cloning and express,we studied the fermentation conditions and protease enzymatic properties of Anoxybacillus sp. DL3. Methods： we used conventional method to culture bacteria and the effect of temperature, pH, carbon source of the medium, the influence of the nitrogen concentration on the strains produced proteinase were studied, we salted the purified enzyme solution and used the Folin method to measure enzyme activity. We measured absorbance at OD680 nm with UV spectrophotometer. Protease solution and the extracted enzyme optimum temperature, pH and the thermal stability and pH stability of the enzyme were added to the enzyme solution, the metal ions and EDTA, PMSF, to study its influence on the enzyme activity. Results： With the initial pH 6.5, culture temperature 40~C strains had the highest enzyme activity. Cultured medium with lactose as the carbon source, yeast extract and ammonium sulfate as the nitrogen source, the carbon source and nitrogen source in the ratio of 1：2, it had the highest enzyme activity, the results showed that the optimum temperature was 55℃. The optimum pH was 7.0. At 50℃ for 20 min-80min, the enzyme activity decreased less. 60 min at 60℃, still kept about 60% of the enzyme activity. At 70℃ for 60 min, the residual enzyme activity was 30%. When the was pH was in the range 6 to 8, the relative enzyme activity was not much different, declining of roughly the same. In alkaline conditions, the relative enzyme activity decreased obviously. Fe2＋, Cu2＋ and Hg2＋ had significantly inhibition on the enzyme activity; Ca2＋, Mg~, Mn~＋ had an active obvious facilitation effect on enzyme; EDTA and PMSF had certainly inhibitory effect on the activity. Conclusion： The protease products of Anoxybacillus sp. DL3 is a thermophilie neutral protease, which enzyme has the thermal stability and pH well tolerated, so the bacteria is a thermophilic protease strain which has further development and utilization value yield.