Screen And Identification Of A New Strain Capable Of Enantioselective Hydrolyzing Methyl (R,S)-n-(2,6-dimethylphenyl) Alaninate,Clone And Expression Of Its Esterase Gene

The research used methyl?R,S?-N-?2,6-dimethylphenyl?alaninate?MAP?,a key intermediate for the synthesis of Metalaxyl,as the single carbon resources in the enrichment cultures for concentrating MAP-hydrolyzing microorganisms from the activated sludge samples,used Rhodamine B and Bromocresol Purple as indicators in the agar plate medium to select the taget microorganisms.There are 27 strains were obtained.The pure cultures of each of them in the shake flasks were carried out for the further screening.Finally,a lipase or esterase-producing strain OD3,which was the best one for enantioselective hydrolysis of methyl?R,S?-N-?2,6-dimethylphenyl?alaninate,was found.The strain OD3 was identified to be Achromobacter denitrificans by physiologic and biochemical index,morphological and 16S rDNA sequencing method.The high enantiomeric excess(ee_p=92%)were achieved with 29.5%of conversion of methyl?R,S?-N-?2,6-dimethylphenyl?alaninate alaninate at 37?hydrolyzed by the cells.The proteins in the crude enzyme extracts from the cells were separated by DEAE Sepharose Fast Flow anion exchange chromatography.The results showed that the active protein was rarely content.The gene library of Achromobacter denitrificans 1104 was first estabilished.From it,the DNA sequence fragment containing the target gene was found.By sequence analysis and PCR amplification,two esterase genes EHest and BXest were obtained.Their full lengths were708 bp and 819 bp respectively.The BXest was 111 bp longer than EHest while the rest parts were identical.The two genes were ligated with plasmids pET28a?+?respectively,then transformed into E.coli BL21Gold?DE3?plysS.The resulting recombinants successfully expressed EHest and BXest capable of catalyzing enatioelective hydrolysis of methyl?R,S?-N-?2,6-dimethylphenyl?alaninate.Their expression activities were 22.62 and 27.07 times higher than the original strain respectively.SDS-PAGE showed their sizes were 27.1 kDa and 31.4 kDa respectively.Catalyzing the hydrolysis of methyl?R,S?-N-?2,6-dimethylphenyl?alaninate?5%m/v?for 1 h at 37?by EHest and BXest,the substrate conversion were 12.26%and 29.48%respectively and ee_p of the product acid?major in R configuration?were 91.02%and 85.14%.The enzymatic properties of BXest was studied.The optimal pH for the recombination BXest was 9.0.The optimal temperature was about50?.The enzyme was stable at below 50?,it can keep more than 90%of the initial activity after 2 h incubation at these temperatures.It was the most stable at pH 5.0-9.0.After incubation for 2 h?37??at pH 5.0-9.0,it can remain more than 80%of the initial activity.1 mM of Cu²⁺?Fe³⁺can inhibited the activity of the recombinant BXest in different degrees.10%DMSO also plays a certain role in promoting enantioselectivity and catalytic rate of the recombination Bxest.Its activity of hydrolysis of methyl?R,S?-N-?2,6-dimethylphenyl?alaninate was 333 times higher than the activity of hydrolysis of olive oil.Its hydrolysis methyl methyl?R,S?-N-?2,6-dimethylphenyl?alaninate of michaelis-menten kinetics parameters Vm,Km is 0.733 g/l·?min?and 7.49 g/L respectively.