| The calcium salt of 2-hydroxy-4-?methylthio?butanoic acid?also known as hydroxymethionine,HMTBA?is a component of compound?-keto acid tablets,treating renal failure disease.At the same time,because it can be converted to L-methionine?L-Met?in the body,it is widely used in livestock and poultry feed additives.In this paper,we developed a process for the production of hydroxymethionine from L-methionine by a cascade reaction.A cascade reaction to produce?R/S?-hydroxymethionine from L-methionine was designed and built.Then,we studied the production of ketonemethionine?KMTB?,?R?-hydroxy-methionine,and?S?-hydroxymethionine.Finally,we established the effective transformation system of L-methionine and realized large-scale preparation of the ketonemethionine,?R?-hydroxymethionine and?S?-hydroxymethionine.1.Designing and building of cascade reaction for production of?R/S?-hydroxymethion-ine.The catalysis cascade platform was designed with two main modules.The first module was the basic module?BM?,in which L-Met was transformed to prochiral KMTB.The other module was the extender module?EM?in which KMTB was converted into HMTBA.EM1was used to produce?R?-HMTBA from KMTB;EM2 was used to produce?S?-HMTBA from KMTB.Compared with the enzyme activity,L-amino acid deaminase from Proteus vulgaris was selected to build BM module.Coexpressing D-lactate dehydrogenase from Pediococcus acidilactici and the formate dehydrogenase from Candida boidinii was selected to build EM1module.Coexpressing L-lactate dehydrogenase from Bacillus coagulans and the formate dehydrogenase from Candida boidinii was used to build EM2 module.2.Production of ketonemethionine.The optimum transformation system of the BM module was investigated.The results showed that the optimal transformation condition was:100 g·L-1 L-methionine,20 g·L-1 wet biomass,25?,pH7.5.After 24 h,the product concentration of ketonemethionine was 98.5 g·L-1.Furthermore,the transformation was carried out on the fermentor with a work volume of 1 L.After 14 h,the concentration of ketonemethionine reached 98.7 g·L-11 with a conversion rate of 99%.After the HPLC and mass spectrometry analysis,the intermediate product was conformed to be ketonemethionine.3.Production of?R?-hydroxymethionine.The optimum transformation system of the EM1 module was investigated.The results showed that the optimal transformation condition was:30?,pH7.0,0.4 mMNAD+.Furthermore,the transformation was carried out on the fermentor with a work volume of 1 L.Under 90 g·L-1 ketomethionine and 20 g·L-1R-stereoselective wet biomass,after 8 h,the product concentration of?R?-hydroxymethionine was 89.6 g·L-1 with a conversion rate of 98.2%.The optimal temperature of BM module was25?and the optimum temperature of EM1 module was 30?.Using a two-stage strategy,we tested the one-pot production of?R?-HMTBA from L-Met at 1 L.In the first stage,the BM was performed at a substrate concentration of 100 g L-1 L-Met at 25?C,and a conversion rate of 99.6%was obtained after a reaction time of 14 h.In the second stage,the reaction temperature was elevated to 30?C,the optimal temperature for EM1.After a further 9 h incubation,the product concentration of?R?-HMTBA was 97.6 g·L-1 with a yield of 96.9%from L-Met.The final product was conformed to be HMTBA by HPLC and mass spectrometry analysis.Further,the ee value of HMTBA was analyzed by chiral column.The results showed that the optical purity of?R?-HMTBA?ee>99%?was produced by EM1module.4.Production of?S?-hydroxymethionine.The optimum transformation system of the EM2 module was investigated.The results showed that the optimal transformation condition was:30?,pH7.0,0.4 mMNAD+.Furthermore,the transformation was carried out on the fermentor with a work volume of 1 L.Under 90 g·L-1 ketomethionine and 20 g·L-1S-stereoselective wet biomass,after 8 h,the product concentration of?S?-hydroxymethionine was 88.2 g·L-1 with a conversion rate of 96.7%.The optimal temperature of BM module was25?and the optimum temperature of EM2 module was 30?.Using a two-stage strategy,we tested the one-pot production of?S?-HMTBA from L-Met at 1 L.In the first stage,the BM was performed at a substrate concentration of 100 g L-1 L-Met at 25?C,and a conversion rate of 99.6%was obtained after a reaction time of 14 h.In the second stage,the reaction temperature was elevated to 30?C,the optimal temperature for EM2.After a further 9 h incubation,the product concentration of?S?-HMTBA was 96.4 g·L-1 with a yield of 95.8%from L-Met.The final product was conformed to be HMTBA by HPLC and mass spectrometry analysis.Further,the ee value of HMTBA was analyzed by chiral column.The results showed that the optical purity of?S?-HMTBA?ee>99%?was produced by EM2module. |
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