Study On The Preparation Of Levetiracetam By Biocatalysis

Levetiracetam,a new anti-epileptic drug(AED)with a structure of 2-oxo-l-pyrrolidine,offers lots of advantages over traditional therapy,as highly favourable therapeutic index,wide margin of safety,lower side-effects,no drug/drug interactions,which is highly worthy to be exploited with extensive clinical application.Preparation of(S)-a-ethyl-2-oxo-pyrrolidine acetamide(S-1)by combination of nitrile hydratase(NHase)and R-enantioselective amidase catalyzed kinetic resolution proceeds under mild conditions with excellent enantioselectivity and great potential for industrialization.Firstly,the reaction conditions on NHase activity of Rhodococcus boritolerans FW815 were investigated.It was indicated that the NHase had a broad pH-optimum between 7 and 9,and exhibited maximal activity at 40?,the apparent activation energy(Ea)was 40.38 kJ/mol.It had excellent thermostability with half-life(t1/2)of 10.8h at 30? and good reusability with 60%activity remained in the seventh batch.Addition of metal ions and cosolvents would not enhance NHase activity.There were no substrate and product inhibitive effect,but the conversion ratio would drop by the increase of substrate concentration.Moreover,kinetic studies of the NHase catalyzed reaction showed that the kinetic constants were as follows:Km=24.92 mM,Vm=3.13mmolmin-1g-1.The strain ZJB-09227,capable of R-enantioselective degradation of 2-(2-oxo-pyrrolidine)butanamide,was isolated through a colorimetric screening method.Based on morphology,physiological tests,Biology system and 16S rDNA sequence,the new isolate was identified as Mycobacterium diernhoferi.To our best knowledge,this stain was the first report in the genus Mycobacterium that harbored R-stereospecific amidase.The intracellular amidase of this strain had excellent thermostability with half-life(t1/2)of 161.97,77.25 and 7.94h at 30,40 and 45 ?,respectively.High dose of urea would inhibited its activity.To improve the cell growth and amidase formation of M.diernhoferi ZJB-09227,its fermentation medium composition and culture conditions were optimized by single factors experiment and orthogonal experimental design.The optimized medium composition was as follows(g/L):glucose 11,yeast extract 13,?-caprolactam 1.5,K2HPO4 1,KH2PO4 1,NaCl 1.The satisfactory culture conditions were that growing temperature 30?,initial pH value 6.5,inoculum volume 4%(v/v),medium volumetric 80mL/500mL.Under these conditions,the amidase activity reach maximum(315.5U/liter of culture broth)after cultivated for 45h,which was 1.78 folds of that with initial medium.Further,influences of reaction conditions on amidase activity and enantioselectivity were also evaluated.Results indicated that the amidase.exhibited maximal activity in phosphate buffer(pH 8.0)at 50? with 0.7g wet cell in 10mL reaction system.Its enantioselectivity was little influenced by temperature,the apparent activation energy(Ea)was 74.67kJ/mol.Adding cosolvents would not improve its activity and enantioselectivity.There is no substrate inhibitive effect in reaction,but the product would inhibit amidase activity,which was caused by the product ammonium rather than the product acid(R-2).Reaction dynamics study shows that the Michaelis-Menten constant Km and Vmax were 7.899mM and 1.58?molmin-1g-1 respectively.