Screening Of Strains For Enantioselective Hydrolyzing HMPC Acetate

Allethrin which belongs to the family of synthetic pyrethroids has excellent insecticidal activity and low toxicity to mammals. It has fast knock-down and lethal effects against household insect pests. 4-Hydroxy-3-methyl-2-(2-propynyl) -cyclopent-2-enone(HMPC) is the alcohol moiety of allethrin. Studies on the relationship between the stereochemistry and insecticidal activity have revealed that the (S)-HMPC is the active stereoisomer in allethrin and possesses four times higher activity than that of (R)-HMPC. Therefore, it is valuable to produce optically active (S)-HMPC.Two hundreds and fifty strains were isolated from soil samples. Among them, a strain designated as C61 exhibited high enantioselectivity to (R)-HMPC acetate through multiple screening steps.The strain was identified as Bacillus Horil based on its physiological and biochemical properties.The fermentation conditions and reaction conditions were optimized.The optimum medium consisted of 17.8g/l of soybean powder, 10.2g/l of olive oil, 2g/l of (NH₄)₂SO₄ 1.78g/l of K₂HPO₄, 1g/l of NaCl, and 0.2g/l of MgSO₄. with the initial pH of 7. The fermentation conditions including seed age, inoculum size, temperature, initial pH, liquid volume, cultivating time were investigated and the optimum value was 18h,10%,30℃.8.0,5% and 24h. respectively. The production of hydrolase was increased from 94U/l to 585U/l,,nearly 500% enhancement. The optimal reaction was conducted at temperature 30℃, pH 8.5 and rotation rate 240rpm. The E-value increased from 44 to 70.The hydrolase reactions of different substrate concentrations were investigated. It was found that the (R)-HMPC could be get with the addition of 45mmol/l substrate concentration, while the conversion was 37%, and ee_p reached to 96%;it could give the (S)- HMPC acetate yield of 47% when substrate concentration was 180mmol/l, while the conversion was 53%, and ee_s reached to 95%.