Influence of different parameters on reverse micelle extraction combined with acetone precipitation to purify sn-1,3 extracellular lipase from Aspergillus niger GZUF36
Influence of different parameters on reverse micelle extraction combined with acetone precipitation to purify sn-1,3 extracellular lipase from Aspergillus niger GZUF36
复制标题
不同参数对反胶束提取联合丙酮沉淀纯化黑曲霉GZUF36 sn-1,3胞外脂肪酶的影响
DOI：
10.1007/s13197-019-03743-4
复制
发表时间：
2019-06-01
影响因子：
3.1
通讯作者：
He, Laping
中科院分区：
文献类型：
--
作者：
Chen, Cuicui;Tian, Hua;He, Laping
采用反胶束萃取和丙酮沉淀相结合的方法获得电泳酶的可行性报道较少。我们的目标是通过这种组合从一株新的黑曲霉GZUF36中纯化出一种sn-1，3胞外脂肪酶。通过控制粗酶液与丙酮的体积比(1：2.5)，对该脂肪酶进行了初步纯化。然后，研究了不同参数对反胶束萃取的影响。正向萃取的适宜表面活性剂、pH值、盐和助溶剂及萃取时间分别为125mM十六烷基三甲基溴化铵，9.0，0.075 mM氯化钠，10%正己醇，30min。在此条件下，正向萃取率可达90.3% ± 3.2%。反萃的适宜食盐、pH、提取时间和短链乙醇依次为1.5M KCl6.5、60min和10%乙醇。添加10%乙醇对提高提取效率具有明显的优势。在此优化条件下，脂肪酶的总萃取率和纯化倍数分别达到76.8%和10.14。SDS-PAGE分析表明，该脂肪酶纯蛋白的相对分子质量为42.7 kDa，TLC显示该脂肪酶具有sn-1，3选择性。LC-MS/MS分析表明，该脂肪酶含有297个氨基酸残基，可能是糖基化的。通过对不同参数的研究，证明了以CTAB为表面活性剂、正己醇为助溶剂的反胶束萃取和乙醇反萃取丙酮沉淀法是一种新的、简单的方法，可以获得几乎纯净的sn-1，3脂肪酶。
There are few reports on the feasibility of combined reverse micelle extraction and acetone precipitation to obtain electrophoretic pure enzymes. We aimed to purify a sn-1,3 extracellular lipase from a novel Aspergillus niger GZUF36 through this combination in this work. This lipase preliminarily purified by controlling the volume ratio (1:2.5) of crude enzyme solution and acetone. Then, we studied effects of different parameters on reverse micelle extraction. The suitable surfactant, pH, salt and cosolvent and extraction time for forward extraction were 125 mM cetyl trimethylammonium bromide (CTAB), 9.0, 0.075 M NaCl, 10% n-hexanol and 30 min, respectively. Under these conditions, the forward extraction rate reached 90.3% ± 3.2%. The suitable salt, pH, extraction time and short chain alcohol for backward extraction were consecutively 1.5 M KCl, 6.5, 60 min and 10% ethanol. Adding 10% ethanol shows a significant advantage of improvement the extraction rate. Under these optimal conditions, the total extraction rate and purification factor of lipase reached 76.8% and 10.14, respectively. SDS-PAGE showed that molecular weight of the pure protein was 42.7 kDa and TLC exhibited sn-1,3 selectivity of this lipase. LC–MS/MS analysis revealed that the lipase had 297 amino acid residues and was likely to glycosylate. Through the study of different parameters, it demonstrated that the new and simple combination of reverse micelle extraction using CTAB as surfactant and n-hexanol as cosolvent for forward extraction and adding ethanol for backward extraction and acetone precipitation is a promising method to get almost an electrophoretically pure sn-1,3 lipase.