Study On The Preparation Of L-tryptophan By Biocatalysis

The stereoselective amidase has become an important tool for the preparation of chiral compounds.With the characteristic of wide substrate spectrum and strict stereospecificity,amidase are playing more and more important role in the preparation for production of optically pure pharmaceuticals,especially of optically pure amino acids.L-Tryptophan is one of the essential amino acids in human body,which has been involved in various metabolic activities in human life.Preparation of L-tryptophan by R-enantioselective amidase catalyzed proceeds under mild conditions with excellent enantioselectivity and has great potential for industrialization.This paper was focused on the process,and investigated in terms of detection,characteristic,preparation and application of this biocatalyst.Firstly,a HPLC analysis of tryptophan and tryptophanamide has been established.Then stain ZJB-09211,which with high stereselectivity and activity toward tryptophanamide was isolated from soil samples.Based on morphology,physiological tests,ATB system and the 16S rRNA sequence,ZJB-09211 was identified as Flavobacterium aquatile.The intracellular amidase exhibited not pretty excellent thermostability with half-life(t1/2)of 32.30 and 27.17 h at 30 and 40?,respectively.To improve the enzyme production of ZJB-09211,the medium components and cultivation conditions of ZJB-09211 were optimized.The results showed that the optimal medium components as follows(g/1):sucrose 4.3,gelatin 5.0,beef extract 8.0,NaNO3 0.17,caprolactam 1.0,K2HPO4 1.0,KH2PO4 1.0,and NaCl 1.0.The optimal cultivation conditions were initial pH 8.0,loading capacity 60ml/500ml,inoculum size 4%.Under this condition,the specific growth rate was 0.251 h-1,and after 27 h the enzyme activity of amidase reached 586.45 U/l,which which was 7.2 times higher than that obtained under initial conditions.Further,the biomass and enzyme activity in the fermentation have been studied,and cell growth kinetic model and fermentation kinetic model have been established.Cell growth fermentation kinetics equations are Cx=0.0604e0.251t/0.9452+0.0548e0.251t and Ce=35.9378e0.251t/0.9452+0.0548e0.251t,respectively.Simultaneously,the asymmetric resolution of tryptophanamide by resting cell was optimized.Results indicated that optimal reaction system was two-phase aqueous system of Tris-HCl buffer solution(pH 7.5)and ethyl acetate(70:30,v:v).The maximum initial reaction rate was got at 45?,0.2g wet cells in 10ml reaction system.After 60min reacting with 80mM tryptophanamide under the optimal conditions,the yield and e.e.of L-Tryptophan reached 40.05mM and 99%,respectively.When the product concentration reached 40 mM,the conversion rate was only 10.67%of the control.Metal ions had no significant impact on enzyme activity and enantioselectivity of the amidase.The study of enzyme kinetic parameters found that Km and Vm and Ks values of the cells were 53.81 mmol/1,1.24mmol/l·min and 14.93mmol/l respectively.