Biocatalysis Synthesis Of Trans-4-(Aminomethyl) Cyclohexane Carboxylic Acid

Trans-4-(aminomethyl)cyclohexane carboxylic acid(trans-AMCHA,TA),tranexamic acid was reported to have a potent antiplasmin activity,which is important haemostatic agent being extensively clinically used acute chronic bounded or general bleeding caused by primary fibrinolysis hyperfunction.Currently,it is synthesized by chemical hydrogenation of an aromatic precursor,then the resulting products further undergoes high temperature,high pressure and strong alkali to convert cis-monomer to trans-monomer.In recent years,biocatalysis has been reported to become one of the most promising production methods of chemicals,due to its mild reaction conditions,highly stereoselectivity and strong specificity and so on.Up to date,reports on biocatalysis of trans-4-(aminomethyl)cyclohexane carboxylic acid are almost unavailable.Therefore,it is of great significance to develop production technology on biocatalysis of trans-4-(aminomethyl)cyclohexane carboxylic acid.In this paper,biocatalysis of trans-4-(aminomethyl)cyclohexane carboxylic acid was researched.In this thesis,the fast detection method of 4-(aminomethyl)cyclohexane carboxylic acid trans and cis monomer was first explored.The effect of chromatography conditions on the separation of trans and cis monomer were investigated and optimized,and a rapid detection method based on thin-layer chromatography was established.Then the separation of the cis-trans isomers in silica gel G plate was achieved.Using the method establised,strain ZJB-12033,a bacterium being capable of stereoselectively hydrolyzing the trans-AMCHA methyl ester to trans-AMCHA was isolated from over 200 soil samples.Based on the morphology,biochemical characteristics and 16S rDNA sequence analysis,strain ZJB-12033 was identified as Bacillus cereus.The medium components and cultivation conditions of B.cereus ZJB-12033 were optimized by single factor method.The optimum medium composition for strain growth and specific activity was as follows(g/L):glucose 10,peptone 6,yeast extract 4,urea 6,KH2PO4 5,FeSO4·7H20 0.01.The optimal conditions for strain growth and producing ester hydrolase were as follows:medium loading 18%(v/v),inoculum size 6%(v/v),initial pH 6.0,30?,150 rpm for 18 h.Under the optimum conditions,the specific activity of the enzyme increased from 2.03 U/g to 12.46 U/g.Finally,various biocatlytic parameters were also investigated and optimized for B.cereus ZJB-12033 resting cells in terms of selectivity hydrolysis of trans-4-(aminomethyl)cyclohexane carboxylic acid methyl ester.The data demonstrated that the optimal bioconversion system were as follow:the optimum buffer solution was Na2HP04-NaH2PO4(0.20 M),the optimum temperature and pH for the biocatalysis were 45 ? and 6.5,respectively.The optimum substrate concentration was 120 mM.The optimal cell concentration was 0.4 g(wet cell weight)/15 mL.Under the optimum conditions,the yield of TA was 15.37%after 3 h.