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

(S)-2,2-dimethylcyclopropane carboxamide is a key intermediate in the synthesis of cilastatin,a renal dehydropeptidase inhibitor.Chiral biocatalysis has already been a classical method in preparation enantiomerically pure compounds.Preparation of（S）-2,2-dimethylcyc-lopropane carboxamide（S-1）by R-enantioselective amidase catalyzed kinetic resolution proceeds under mild conditions with excellent enantioselectivity and has great potential for industrialization.Firstly,a novel R-stereospecific amidase-producing strain ZJB-07021 was isolated through a sophisticated colorimetric screening method.Based on morphology,physiological tests,Biolog system（GP2） and 16S rRNA sequence,the new isolate was identified as Brevibacterium epidermidis.It showed strict enantioselectivity for racemic 1 with an E-value of 23 at 35℃.To our knowledge,this was the first report on the species Brevibacterium epidermidis that harbored R-stereospecific amidase.Strain ZJB-07021 could be further improved as a suitable biocatalyst for the stereoselective bioconversion of racemic 1 after optimization of culture and biotransformation process.The fermentation medium of B.epidermidis ZJB-07021 producing R-amidase was optimized by using response surface methodology（RSM）. Firstly,the optimal fermentation conditions were screened out using single factor experiment.Based on this,the effects of 8 factors related to R-amidase producing were evaluated by using a Plackett-Burmam design. The results showed that the concentration of glucose,yeast extract and acetamide had strong effects on the R-amidase enzyme activity.A 2³ central composite design and RSM were applied to determine the optimal concentration of three significant variables:glucose,yeast extract and acetamide.The optimum fermentation medium were glucose 17.00 g 1（-1）; yeast extract 15.74 g 1^-1;acetamide 7.05 g 1^-1,allowed the amidase activity yield to increase from 41.62 U 1^-1to 72.14 U 1^-1in flask-shaking batch fermentation.Influences of reaction conditions on amidase activity and enantioselectivity were also investigated.Results indicated that optimal working pH of the amidase was 7.6（Tris-HCl buffer）.The amidase showed highest activity at 45℃.The enzyme activity was 3.6 times higher as 14℃,but its enantiomeric ratio decreased with increase of temperature.From correlation linear equation of Arrhenius equation and equation In E=△△S⁺/R-△△H⁺/（RT）,different thermodynamic parameters for B.epidermidis ZJB-07021 catalyzed hydrolysis of（R,S）-1 was determined(E_a=32.92 kJ mol^-1△△H⁺=-37.3 kJ mol^-1,△△S⁺=-95.7 J mol^-1).Strain ZJB-07021 also had a good thermal stability and addition of 10%（v/v）Methanol,had significant effect not only on enzyme activity but also on enantioselectivity.Reaction dynamics shows that the Michaelis-Menten constant K_m and V_maxwere 1.06 mM and 48.22μmol min^-1respectively.