Enantioselective Hydrolysis Of HMPC Acetate By Microbial Method

Allethrin is the first industrialized product among the family of synthetic pyrethroides. Allethrin is well known for its excellent insecticidal activity and low toxicity to mammals. 4-Hydroxy-3-methyl-3-methyl-2-(2-propynyl)-cyclopent-2-enone(HMPC) is the alcohol moiety of allethtin. Studies on the relationship between the stereochemistry and insecticidal activity have revealed that the(S)-HMPC is the active isomer in allethrin and possesses four times higher activity than that of (R)-HMPC. Therefore, it is valuable to produce optically active (S)-HMPC.About 200 strains were isolated from soil, using rac-HMPC acetate as the sole carbon source. Among them, a strain numbered 7, exhibited higher activity and enantioselectivity to (R)-HMPC acetate. The strain was identified as Pseudomonas Aeruginosa based on its 16sRNA gene sequence and physiological and biochemical properties.Two ways of mutagenesis treatment, including UV-irradiation and DES, were carried on to increase the hydrolase production of strain 7. Finally, the high yield mutant named L8-3b19 was selected and produced hydrolase activity 222% more than that of parent strain. The fermentation potency of the mutant was 248.3U/1.The fermentation conditions and reaction conditions were optimized for mutant L8-3b19. The optimum medium (per liter) consisted of 10g Yeast Extract, 10g Tween-80, (NH₄)₂SO₄2g, K₂HPO₄2g, NaCl 1g, MgSO₄ 0.2g, pH 7.0. The optimum fermentation conditions were determined as follows: seed age of 18h, inoculum amount of 10%, medium volume of 30ml for 250ml shake flask, temperature of 30℃, rotation rate of 200rpm, initial pH7.0, and cultivation time of 18h. After optimization, hydrolase production in shake flasks was enhanced from 240U/l to 456U/l, nearly 100%. Considering both the reaction rate and selectivity of enantioselective hydrolysis of racemic HMPC acetate, we optimized the reaction conditions as followes: temperature of 50℃, pH 8.0, and rotation rate of 200rpm,substrate of 105.4mmol/l HMPC acetate .After optimization ,when the reaction was carried out for about 24h, the degree of hydrolysis and ees were 50.81% and 96.59% .The E-value increased from 98 to 120.