碱性脂肪酶高产诱变菌株产酶条件优化及其碱性脂肪酶的高效分离纯化
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摘要
脂肪酶(EC3.1.1.3,甘油酯水解酶)是一类能催化长链脂肪酸甘油酯水解为甘油和长链脂肪酸(或者是此反应的逆反应)的生物催化剂.它可以在油水界面上催化甘油三酯水解生成脂肪酸和甘油,以及中间产物甘油一酯和甘油二酯.广泛应用于食品、医药、洗涤、皮革、造纸等行业领域.
本文以实验室保存的碱性脂肪酶高产诱变菌株Lp-11为出发菌株,通过菌种复壮.产酶条件优化,从而提高菌株产酶量.并对其碱性脂肪酶进行高效分离纯化,以期降低纯化成本.提高碱性脂肪酶的活性回收率.
利用平板划线法和平板涂布法对菌株Lp-11进行菌种复壮.得到一株产脂肪酶活性相对最高且传代稳定的菌株:Lp-11-④。
利用逐因子试验及正交设计等方法对菌株Lp-11-④进行产酶条件的优化。首先运用逐因子试验确定Lp-11-④产碱性脂肪酶的最适碳源和氮源分别为:葡萄糖和蛋白胨.然后在此基础上利用正交试验对其发酵培养基成分进行优化,最后得到其最佳培养条件为:葡萄糖0.5%;蛋白胨5.0%;Na_2HPO_40.7%;KH_2PO_40.5%;Tween80 1.0%:橄榄油2.0%;培养基pH8.0:接种量3.0%(v/v):种龄12h:转速250r/min;温度30℃,在此条件下培养(30h)发酵液酶活达到366.996U/ml,比优化前提高84.541%.
对菌株Lp-11-④所产的碱性脂肪酶进行高效分离纯化.菌体发酵液经双水相萃取(ATPS)、Phenyl-Sepharose疏水作用层析处理.超滤浓缩后得到了纯化后的碱性脂肪酶,用SDS聚丙烯酰胺凝胶电泳和等电聚焦检测其纯度为电泳纯.该酶的纯化倍数为26.81倍.活性的回收率为65.15%.比活力达到9901.02U/mg.
理化性质研究表明,脂肪酶的分子量为265.6KD,由四个分子量为64.8KD的相同亚基组成,该酶的等电点pI为5.43。
Lipase(EC3.1.1.3,triacylglycerol acylhydrolase)is a kind of biocatalysts to hydrolysis long-chain triglycerides to glycerol and lang-chain fatty acids.It can hydrolysis Triglyceride to fatty acid and glycerol in the interface of oil-water.Lipase is widely used in food,medicine,scour,leather and paper industry.
In this research work,rejuvenation and enzyme production optimization were used to improve the lipase production of Lp-11-④.Alkalin lipase was purified high efficiently to reduce purification costs.
We used plate streaking and coating methods to rejuvenate Lp-11 which was conserved by our laboratory.Finally,the high yield Lp-11-④strain was selected.Stability test demonstrated that the lipase activity remained the same level after five generations.
Lipase production condition of Lp-11-④was optimized using seriatim-factorial experiment and orthogonal experiment.The optimum carbon source and the optimum nitrogen source for Lp-11-④screened through seriatim-factorial experiment were glucose and peptone,respectively.By orthogonai experiment we obtained result of the optimum culture medium was:glucose0.5%; peptone5.0%;Na_2HPO_40.7%;KH_2PO_40.5%;Tween80 1.0%;olive oil2.0%;pH8.0;inoculation quantity 3.0%(V/V);bacteria age 12h;revolution 250r/min;temperature 30℃.After 30h incubation with the optimum condition,a maximum lipase yield 366.996U/mL was obtained,which was 84.541%higher than that under beginning culture mediums.
The alkaline lipase of the supernatant was purified to homogeneity through aqueous two-phase extraction and hydrophobic interaction chromatography on Phenyl-Sepharose Fast Flow column, and the pure alkaline lipase was characterized by SDS-PAGE and IEF electrophoresis.65.15%of the lipase activity was recovered,and purification fold was 26.81.The specific activity was 9901.02 unit/mg.
The molecular weight of the lipase is 265.6KD and submits are 64.8KD determined by gel filtration chromatography on Superdex-200 and SDS-PAGE method.Isoelectric point of the pure lipase was 5.43 showed by IEF-PAGE.
本文以实验室保存的碱性脂肪酶高产诱变菌株Lp-11为出发菌株,通过菌种复壮.产酶条件优化,从而提高菌株产酶量.并对其碱性脂肪酶进行高效分离纯化,以期降低纯化成本.提高碱性脂肪酶的活性回收率.
利用平板划线法和平板涂布法对菌株Lp-11进行菌种复壮.得到一株产脂肪酶活性相对最高且传代稳定的菌株:Lp-11-④。
利用逐因子试验及正交设计等方法对菌株Lp-11-④进行产酶条件的优化。首先运用逐因子试验确定Lp-11-④产碱性脂肪酶的最适碳源和氮源分别为:葡萄糖和蛋白胨.然后在此基础上利用正交试验对其发酵培养基成分进行优化,最后得到其最佳培养条件为:葡萄糖0.5%;蛋白胨5.0%;Na_2HPO_40.7%;KH_2PO_40.5%;Tween80 1.0%:橄榄油2.0%;培养基pH8.0:接种量3.0%(v/v):种龄12h:转速250r/min;温度30℃,在此条件下培养(30h)发酵液酶活达到366.996U/ml,比优化前提高84.541%.
对菌株Lp-11-④所产的碱性脂肪酶进行高效分离纯化.菌体发酵液经双水相萃取(ATPS)、Phenyl-Sepharose疏水作用层析处理.超滤浓缩后得到了纯化后的碱性脂肪酶,用SDS聚丙烯酰胺凝胶电泳和等电聚焦检测其纯度为电泳纯.该酶的纯化倍数为26.81倍.活性的回收率为65.15%.比活力达到9901.02U/mg.
理化性质研究表明,脂肪酶的分子量为265.6KD,由四个分子量为64.8KD的相同亚基组成,该酶的等电点pI为5.43。
Lipase(EC3.1.1.3,triacylglycerol acylhydrolase)is a kind of biocatalysts to hydrolysis long-chain triglycerides to glycerol and lang-chain fatty acids.It can hydrolysis Triglyceride to fatty acid and glycerol in the interface of oil-water.Lipase is widely used in food,medicine,scour,leather and paper industry.
In this research work,rejuvenation and enzyme production optimization were used to improve the lipase production of Lp-11-④.Alkalin lipase was purified high efficiently to reduce purification costs.
We used plate streaking and coating methods to rejuvenate Lp-11 which was conserved by our laboratory.Finally,the high yield Lp-11-④strain was selected.Stability test demonstrated that the lipase activity remained the same level after five generations.
Lipase production condition of Lp-11-④was optimized using seriatim-factorial experiment and orthogonal experiment.The optimum carbon source and the optimum nitrogen source for Lp-11-④screened through seriatim-factorial experiment were glucose and peptone,respectively.By orthogonai experiment we obtained result of the optimum culture medium was:glucose0.5%; peptone5.0%;Na_2HPO_40.7%;KH_2PO_40.5%;Tween80 1.0%;olive oil2.0%;pH8.0;inoculation quantity 3.0%(V/V);bacteria age 12h;revolution 250r/min;temperature 30℃.After 30h incubation with the optimum condition,a maximum lipase yield 366.996U/mL was obtained,which was 84.541%higher than that under beginning culture mediums.
The alkaline lipase of the supernatant was purified to homogeneity through aqueous two-phase extraction and hydrophobic interaction chromatography on Phenyl-Sepharose Fast Flow column, and the pure alkaline lipase was characterized by SDS-PAGE and IEF electrophoresis.65.15%of the lipase activity was recovered,and purification fold was 26.81.The specific activity was 9901.02 unit/mg.
The molecular weight of the lipase is 265.6KD and submits are 64.8KD determined by gel filtration chromatography on Superdex-200 and SDS-PAGE method.Isoelectric point of the pure lipase was 5.43 showed by IEF-PAGE.
引文
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