Screening For Enantioselective Amidase: Kinetic Resolution Of Racemate To (S)-(+)-2, 2-Dimethylcyclopropane Carboxamide

Chiral biocatalysis has already been a classical method in preparationenantiomerically pure compounds. Nitrile-converting enzymes, especially amidase,with wide substrate spectrum and strict stereospecificity, are playing more and moreimportant roles in industrial biotransformations for production of optically purepharmaceuticals and agrochemicals. S-（+）-2,2-dimethylcyclopropane carboxamideis a key intermediate in the synthesis of cilastatin, a renal dehydropeptidase inhibitor.Preparation of S-（+）-2,2-dimethylcyclopropane carboxamide by R-enantioselectiveamidase catalyzed kinetic resolution proceeds under mild conditions with excellentenantioselectivity and has great potential for industrialization.A novel enantioselective amidase screening system was developed and proved tobe efficient and accurate. This screening system employed acyl transfer activity ofamidase in the presence of hydroxylamine, leading to the formation of hydroxamicacids, followed by spectrophotometric quantification of hydroxamic acid/iron （Ⅲ）complex. To prove the accuracy of the screening system, the difference betweenenantioselectivity of acyl transfer reaction and that of hydrolysis reaction wasevaluated. With this method, we obtained eight microorganism strains withenantioselective amidase from 523 isolates, two of which showed R-stereospecificactivity for （R,S）-2,2-dimethylcyclopropane carboxamide.In order to monitor and control the bioconversion process, enantioseparation of2,2-dimethylcyclopropanecarboxamide and corresponding acid was performed on acommercial chiral column BGB-175. Based on chiral balance in kinetic resolutionprogress, a novel method, employed enantiomeric excess of both substrate andproduct, was developed for determination of concentration of enantiomers in bioconversion broth. Since only relative quantity （ee） was required in the proposedmethod, calibration and cumbersome quantitative sample handling can be avoided andanalytical accuracy can be greatly improved.Strain ZJB-05174, capable of R-enantioselective degradation of2,2-dimethylcyclopropane carboxamide, was isolated through the screening system.Based on morphology, physiological tests, ATB system and the 16S rRNA sequence,this strain was identified as Delftia tsuruhatensis. This is the first report on strains inthis species with R-amidase activity. D. tsuruhatensis ZJB-05174 catalyzed hydrolysisof 2,2-dimethylcyclopropane carboxamide with an enantiomeric ratio （E value） of 27at 30℃. The intracellular amidase exhibited excellent thermostability with half-life(t_1/2) of 78.6 and 46.2 h at 30 and 40℃, respectively. Urea, regular inhibitor ofamidase, was not effective to amidase from D. tsuruhatensis ZJB-05174, whichsuggested that this amidase might have a different active site structure with otherreported ones.The effects of medium composition and culture conditions on the amidaseactivity of D. tsuruhatensis ZJB-05174 were evaluated experimentally. The acyltransfer activity catalyzed by amidase with acetamide as substrate was firstly provedto be in accordance with correspondence hydrolysis activity. The optimized mediumcomposition was as follows （g/l）: glucose 8.4, acetamide 3.56, yeast extraction 6.3,peptone 0.7, KH₂PO₄1, K₂HPO₄ 1, NaCl 1. The satisfactory fermentation conditionsfor cell growth and formation of amidase were as follows: initial pH value, 7.0; 30℃;inoculum volume, 4% （v/v）; medium volumetric ratio, 30% （v/v）. Under theseconditions, D. tsuruhatensis ZJB-05174 multiplied with growth rate （μ） of 0.33 h^-1.When D. tsuruhatensis ZJB-05174 was cultivated for 20h, the enzyme activity,expressed as acyl transfer activity, reached 1.51 U/ml of culture broth, which was 1.65times higher than before optimization.Influences of reaction conditions on amidase activity and enantioselectivity werealso investigated. Results indicated that optimal working pH of the amidase rangedfrom 7.6 to 8.8. Moreover, the amidase exhibited stricter stereospecificity underpartial acid （pH 5.4） or partial alkali （pH 9.4） conditions than it under neutral conditions. The amidase showed highest activity at 41℃, but its enantiomeric ratiodecreased with increase of temperature, until reversal of stereospecificity wasobserved. Two thermodynamic parameters of the reaction, activation enthalpy andactivation entropy, changed dramatically after cell suspensions pre-incubated at 56℃,which was the reason why the reversal of stereospeccificity was irreversible. Additionof cosolvent, ethanol and acetonitrile, had significant effect not only on enzymeactivity but also on enantioselectivity. The enzyme activity was 2.7 and 2.2 timeshigher, and E-value increased from 32 to 91 and 140, respectively. After optimizationof the reaction conditions, amidase activity increased from 14.3μmol min^-1 g^-1 to 91.8μmol min^-1 g^-1. S-（+）-2,2-dimethylcyclopropane carboxamide was isolated andpurified from reaction mixture with total yield of 43.6%. The sample wascharacterized by polarimeter, IR and ¹H NMR analysis, and the results demonstratedthat chemical and optical purity of the sample were both above 99%.Further, enantioselective acyl transfer reaction catalyzed by D. tsuruhatensisZJB-05174 was investigated. Results showed that addition of cosolvent acceleratedinitial reaction rate of amide hydrolysis, but had no effect on that of acyl transferreaction. However, acyl transfer reaction then slowed down because substrateconcentration dropped sharply due to accelerated side reaction. On the contrary,increasing hydroxylamine concentration effectively inhibited hydrolysis reaction.When concentration ratio of hydroxylamine and substrate reached 10:1, reaction rateof acyl transfer and hydrolysis was almost the same. Moreover, kinetic studies of theamidase catalyzed acyl transfer reaction demonstrated that the reaction occurredaccording to a Ping Pong Bi Bi mechanism. The kinetic constant were as follows:V_m=129.9μmol min^-1 g^-1, K_NH2OH=150mM, K_amide=10mM.