【作者】 黄鹭强；

【导师】 谢必峰；

【作者基本信息】 福建师范大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 芽孢杆菌属产生的脂肪酶具有可耐受较高的温度、较高的pH稳定性和良好的有机溶剂耐受性等特点，非常适用于非水相催化和手性化合物拆分等领域。关于该属产生的脂肪酶，国外有一定文献报道，国内研究很少。 本文对本单位分离保存的一株杆菌进行初步的分类鉴定，确定为Bacillus sp．。采用均匀试验设计对产酶培养基组成进行优选，结合DPS软件辅助计算，确定最佳产酶培养基组成为：玉米淀粉为0.78％，豆饼粉4.0％，K₂HPO₄ 0.4％，NaNO₃ 0.2％，pH值为10.0。 优化最佳摇瓶培养条件为：温度32℃，摇瓶装量20ml，摇瓶转速225r／min，种龄24h，接种量10％，发酵周期28h。 该酶经70％硫酸铵沉淀、阳离子CM-Sepharose FF交换和Sephacryl S-200柱层析等步骤处理，脂肪酶最终被纯化了9.45倍，并达到电泳纯。SDS—PAGE电泳确定该酶蛋白分子量为32.08KDa。 酶学性质研究发现： 1．该酶的最适温度为50℃，30℃～50℃下温度稳定性良好； 2．该酶的最适pH为9.0．在pH为7.0～11.0的范围内均表现较高的活性； 3．Ca²⁺对该酶没有促进作用，说明该酶不是钙依赖型脂肪酶： 4．非离子型表面活性剂在低剂量时对该酶有促进作用，而高剂量时有抑制作用；离子型表面活性剂无论剂量高低，均有不同程度的抑制。 由Bacillus sp．产生的脂防酶具有较高的温度耐受性和较高的pH适应范围，是具有良好开发前景的脂肪酶新酶种。更多还原

【Abstract】 Lipases from Bacillus show high thermostability of their lipase activities, stabilities of wide pH range and good organic solvent tolerant, which were applied in biocatalysis nonaqueous media and optically active substance resolution.We studied a new isolated which was conserved by our institute. It was identified as Bacillus sp. by physiological test and biochemical test. A uniform experimental design was used to determine optimum fermentation conditions and medium compositions in shake flask cultivation, optimization data were calculated with DPS software-aid. The optimal lipase-producing conditions of Bacillus sp. were as follows: 0.78% corn starch as best carbon source , 4.0% soy bean meal as best nitrogen source and K2HPO4 0.4%,NaNO3 0.2%, initial pH 10.0,culture temperature was 32C, shark flask capacity was 20ml,stirring rate was 225r/min,germ age was 24h,germ concentration was 10% and fermentation periods was 28h.The lipase was purified to homogeneity using ammonium sulfate precipitation, cation-exchange chromatography (CM-Spephrose FF) and Sephacryl S-200,and its specific activity was improved 9.45-fold.The molecular mass was shown to be 32.08 KDa by SDS-PAGEProperties of the lipase showed1 The optimum temperature was 50C It shows thermostability under 50C2 The optimum pH was 9 0 It showed high activity in a pH range from 7 0 to 11.0.13 There was not increased to enzymatic activity with Ca2+, which indicated lipase from Bacillus sp. was not a Ca2+ -depended lipase.4Nonionic detergent increased enzymatic activity with low concentration, yet it was decreased with high concentration.Inoic detergent showed varied decreased effects to the lipase with either high or low concentrationIt indicated that lipases from Bacillus sp. would be applied widely as it showed high thermostabilities and stabilities of wide pH range更多还原