产脂肪酶菌株的筛选、鉴定及产酶条件优化
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摘要
从海口市各地采集含油污土样10份,富集培养得到产脂肪酶细菌优势菌种,梯度稀释后,以花生油为唯一碳源采用油脂固体平板透明圈法进行分离纯化,得到145株产脂肪酶的菌株。再经摇瓶复筛,获得两株产脂肪酶活力较高的菌株,编号分别为C737-11和C7828-5。按照《常见细菌系统鉴定手册》和《伯杰氏细菌鉴定手册》(2004版)的方法对两株菌进行了生理生化鉴定,以及结合分子生物学鉴定,确定菌株C737-11为洋葱伯克霍尔德氏Ⅰ型菌,菌株C7828-5为铜绿假单胞菌。
菌株C737-11粗酶的最适作用温度为37℃,最适pH值为8.0,初步优化了C737-11的产脂肪酶条件,获得高达10.5 U mL-1的脂肪酶活性。
应用单因子试验和正交实验对铜绿假单胞菌C7828-5产脂肪酶发酵培养基进行了快速优化。首先应用单因子试验确定铜绿假单胞菌C7828-5产脂肪酶最适碳源和最适氮源分别是花生油和蔗糖组合及牛肉膏。在此基础上通过L9(34)正交试验,考察了花生油、蔗糖、牛肉膏、硫酸铵等四个因素对产酶的影响,获得优化的培养基组成。最后通过单因子试验确定最适发酵温度、最适起始pH、最适接种量及最适摇床转速等。试验获得的优化培养条件为：蔗糖5gL-1、牛肉膏20 g L-1、(NH4)2SO4 1 g L-1、MgSO4·7H2O 0.5 g/L、CaCl20.5gL-1,聚乙烯醇花生油乳化液120 mLL1,起始pH7.0,培养温度28℃,转速为200 r min-1,培养72h后,获得高达8.08 U mL-1的脂肪酶表达量。
对菌株的发酵粗酶液进行了酶学性质探讨,结果表明菌株C737-11和C7828-5脂肪酶的最适作用温度和最适作用pH分别都是37℃和8.0,都属于中温弱碱性酶。菌株C737-11脂肪酶热稳定性良好,50℃处理60 min后还有82%的酶活力；60℃处理40 min后还有55%的酶活力；70℃处理20 min后还有63%的酶活力；该脂肪酶在pH6.0-8.0范围内具有较好的稳定性,不过,在强碱性条件下(pH9-10),该酶稳定性较差。菌株C7828-5脂肪酶60℃处理1 h后酶活力几乎丧失,在pH 5.0-10.0范围内具有较好的稳定性,而且在中性条件下(pH 7.0),该酶酶活性仍保持不变,pHH10.0温浴1 h后仍有45%的酶活性。
Ten oil-contaminated soil samples were collected from Haikou city.145 lipolytic bacterial strains were isolated from soil samples rich in oil. The screening method is based on the formation of clearance zones on turbid solid media supplemented with emulsified peanut oil. Two strains with high ability of lipases production were obtained by second flask assay. These strains were characterized according to their conventional morphplogical, physiological, biochenmistrica characteristics and also with phylogenetic methods. The strains C737-11 and C7828-5 were identified as B. cepacia and P. aeruginosa, respectively.
The optimal reaction conditions of the lipase produced by C737-11 were 37℃, at a pH of 8.0. The optional medium for the induction or supporting of lipase from C737-11 was preliminarily determined, and the lipase activity reached the maximal level of 10.5 U mL-1.
Lipase-induction conditions of P. aeruginosa C7828-5 were optimized using monofactorial experiment and orthogonal test. The optimum carbon source and nitrogen source for P. aeruginosa C7828-5 were peanut oil, sucrose and beaf extract. Fours factors, peanut oil, sugar, beaf extract and (NH4)2SO4 were investigated by orthogonal test to obtain optimal medium composition. The effect of pH、temperature、inoculum and speed of shaker on the production of the lipase was also examined with the optimized medium by monofactorial experiment. The optimized condition was:sugar 5 g L-1, beaf extract 20 g L"1, (NH4)2SO4 1 g L-1, MgSO4·7H2O 0.5 g L-1, CaCl2 0.5 g L-1, emulsified peanut oil 120 mL L-1; the initial pH of fermentation was pH 7.0, and the temperature was 28℃; After 72 h, the alkaline lipase activity reached the maximal level of 8.08 U mL"1.
The enzymatic properties of the lipase produced by C737-11 and C7828-5 in the fermentation medium was studied. Result showed that they had the same optimal reaction pH and temperature, which were 8.0 and 37℃, respectively, suggesting that they were warm-adapted alkaline lipases. The lipase produced by C737-11 retained 97%,87.7%, and 62.9% of its activity after incubation at 50,60, and 70℃for 20 min, respectively; and 85.9%,55.3%, and 22.6% of its activity after incubation at 50,60, and 70℃for 40 min, respectively, similar to the lipase produced by C7828-5, which retained 97.8%,97%, and 54.3 % of its activity after incubation at 50,60 and 70℃for 20 min, respectively, and 87.2%, 61.5%, and 6.3% of its activity after incubation at 50,60, and 70℃for 40 min, respectively. The lipase produced by C7828-5 was stable between pH 5 and 10, and remained 45% of its activity after incubation at a pH of 10 for 1 h. However, the lipase produced by C737-11 was stable at a pH range of 6-8, and the activity dropped sharply after incubation at pH>9.0.
菌株C737-11粗酶的最适作用温度为37℃,最适pH值为8.0,初步优化了C737-11的产脂肪酶条件,获得高达10.5 U mL-1的脂肪酶活性。
应用单因子试验和正交实验对铜绿假单胞菌C7828-5产脂肪酶发酵培养基进行了快速优化。首先应用单因子试验确定铜绿假单胞菌C7828-5产脂肪酶最适碳源和最适氮源分别是花生油和蔗糖组合及牛肉膏。在此基础上通过L9(34)正交试验,考察了花生油、蔗糖、牛肉膏、硫酸铵等四个因素对产酶的影响,获得优化的培养基组成。最后通过单因子试验确定最适发酵温度、最适起始pH、最适接种量及最适摇床转速等。试验获得的优化培养条件为：蔗糖5gL-1、牛肉膏20 g L-1、(NH4)2SO4 1 g L-1、MgSO4·7H2O 0.5 g/L、CaCl20.5gL-1,聚乙烯醇花生油乳化液120 mLL1,起始pH7.0,培养温度28℃,转速为200 r min-1,培养72h后,获得高达8.08 U mL-1的脂肪酶表达量。
对菌株的发酵粗酶液进行了酶学性质探讨,结果表明菌株C737-11和C7828-5脂肪酶的最适作用温度和最适作用pH分别都是37℃和8.0,都属于中温弱碱性酶。菌株C737-11脂肪酶热稳定性良好,50℃处理60 min后还有82%的酶活力；60℃处理40 min后还有55%的酶活力；70℃处理20 min后还有63%的酶活力；该脂肪酶在pH6.0-8.0范围内具有较好的稳定性,不过,在强碱性条件下(pH9-10),该酶稳定性较差。菌株C7828-5脂肪酶60℃处理1 h后酶活力几乎丧失,在pH 5.0-10.0范围内具有较好的稳定性,而且在中性条件下(pH 7.0),该酶酶活性仍保持不变,pHH10.0温浴1 h后仍有45%的酶活性。
Ten oil-contaminated soil samples were collected from Haikou city.145 lipolytic bacterial strains were isolated from soil samples rich in oil. The screening method is based on the formation of clearance zones on turbid solid media supplemented with emulsified peanut oil. Two strains with high ability of lipases production were obtained by second flask assay. These strains were characterized according to their conventional morphplogical, physiological, biochenmistrica characteristics and also with phylogenetic methods. The strains C737-11 and C7828-5 were identified as B. cepacia and P. aeruginosa, respectively.
The optimal reaction conditions of the lipase produced by C737-11 were 37℃, at a pH of 8.0. The optional medium for the induction or supporting of lipase from C737-11 was preliminarily determined, and the lipase activity reached the maximal level of 10.5 U mL-1.
Lipase-induction conditions of P. aeruginosa C7828-5 were optimized using monofactorial experiment and orthogonal test. The optimum carbon source and nitrogen source for P. aeruginosa C7828-5 were peanut oil, sucrose and beaf extract. Fours factors, peanut oil, sugar, beaf extract and (NH4)2SO4 were investigated by orthogonal test to obtain optimal medium composition. The effect of pH、temperature、inoculum and speed of shaker on the production of the lipase was also examined with the optimized medium by monofactorial experiment. The optimized condition was:sugar 5 g L-1, beaf extract 20 g L"1, (NH4)2SO4 1 g L-1, MgSO4·7H2O 0.5 g L-1, CaCl2 0.5 g L-1, emulsified peanut oil 120 mL L-1; the initial pH of fermentation was pH 7.0, and the temperature was 28℃; After 72 h, the alkaline lipase activity reached the maximal level of 8.08 U mL"1.
The enzymatic properties of the lipase produced by C737-11 and C7828-5 in the fermentation medium was studied. Result showed that they had the same optimal reaction pH and temperature, which were 8.0 and 37℃, respectively, suggesting that they were warm-adapted alkaline lipases. The lipase produced by C737-11 retained 97%,87.7%, and 62.9% of its activity after incubation at 50,60, and 70℃for 20 min, respectively; and 85.9%,55.3%, and 22.6% of its activity after incubation at 50,60, and 70℃for 40 min, respectively, similar to the lipase produced by C7828-5, which retained 97.8%,97%, and 54.3 % of its activity after incubation at 50,60 and 70℃for 20 min, respectively, and 87.2%, 61.5%, and 6.3% of its activity after incubation at 50,60, and 70℃for 40 min, respectively. The lipase produced by C7828-5 was stable between pH 5 and 10, and remained 45% of its activity after incubation at a pH of 10 for 1 h. However, the lipase produced by C737-11 was stable at a pH range of 6-8, and the activity dropped sharply after incubation at pH>9.0.
引文
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