A novel esterase from Burkholderia sp. YD106 capable of hydrolysis of methyl (R, S)-N-(2, 6-dimethylphenyl) alaninate, and its mutation for improving enantioselectivity

Methyl ( R, S )-2, 6-dimethylphenylaminopropionate (( R, S )-1), is an intermediate in the production of the agricultural fungicide ( R, S )-metalaxyl. ( R, S )-1 can be hydrolyzed enantioselectively by some hydrolases to produce ( R )-1, which was used for production of ( R )-metalaxyl. In this work, a strain Burkholderia sp. YD106 that could hydrolyze ( R, S )-1 was screened from the activated sludge, but it had almost no enantioselectivity. The intracellular active esterase WZest was successfully heterologous expressed in the recombinant E. coli BL21 (DE3)-pET-28a (+)-GE04845. Using the recombinant strain as the parent strain, the mutants were constructed by site-directed mutation. Among all 33 mutants, 7 had altered enantioselectivity, of which 4 mutants were ( R )-enantioselective, and 3 were ( S )-enantioselective. The mutant WZest-W23T had the highest ( R )-enantioselectivity. When it catalyzed hydrolysis of ( R, S )-1 at 44.6% substrate conversion, e.e. _p reached 94.70% with enantiomeric ratio (E) of 85.0. WZest showed significant amino acid sequence differences from the two reported esterases capable of hydrolyzing ( R, S )-1. It was both active in two kinds of solutions. One was emulsion with the substrate ( R, S )-1 emulsified with Tween80, the other was homogeneous solution with acetone as co-solvent. The activity of WZest in the former was higher than that in the latter.