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[目的]从土壤中筛选出能选择性水解2-[(2-甲基-6-乙基)苯基氨基]丙甲酯(NEMPAME)的菌株,并对该菌株进行鉴定及优化。[方法]以NEMPAME为碳源,结合传统的罗丹明B平板筛选方法,通过摇瓶复筛,筛选1株具有最高立体选择性水解NEMPAME的菌株。在摇瓶培养下,采用单因素和正交试验,对其培养条件进行优化。[结果]从土壤中筛选出1株催化拆分NEMPAME的菌株,经形态观察和16Sr RNA鉴定,初步认为是铜绿假单胞菌(Pseudomonas)。经培养条件优化后酶活提高了308.98%。[结论]该菌株拆分NEMPAME研究中发现,活性蛋白属于胞内酶,手性选择性为R-型。优化后:葡萄糖10 g/L,酵母20 g/L,Mg SO40.5 g/L,Zn Cl20.5 g/L,橄榄油10 g/L,初始p H值7.5,培养温度37℃,发酵时间48 h。
[Objective] The research aimed to screen the strains which could selectively hydrolyze 2 - [(2-methyl-6-ethyl) phenylamino] propylmethyl ester (NEMPAME) from soil and identify and optimize the strain. [Method] With NEMPAME as carbon source and traditional rhodamine B screening method, one strain with the highest stereoselective hydrolysis of NEMPAME was screened by shake flask screening. Under shaking flask culture, single factor and orthogonal experiment were used to optimize the culture conditions. [Result] A strain of NEMPAME catalyzed by soil was screened from soil. The morphological observation and 16S rRNA identification suggested that it was initially identified as Pseudomonas. After optimization of culture conditions enzyme activity increased by 308.98%. [Conclusion] It was found from the NEMPAME study that the active protein belongs to intracellular enzyme and the chirality is R-type. After optimization: glucose 10 g / L, yeast 20 g / L, Mg SO40.5 g / L, Zn Cl20.5 g / L, olive oil 10 g / L, initial pH 7.5, incubation temperature 37 ℃, Time 48 h.