Chiral resolution of N-methyl-d,l-aspartic acid using a predicted N-demethylase from Chloroflexi bacterium 54-19

N-methyl-d-aspartic acid(NMDA),an amino acid existing in human and animal central nervous system,exerts agonist action on one of the glutamate receptor subtypes and is clinically used for treatment of diabetes,Parkinson’s and Alzheimer’s syndrome.In this study,an enzymatic protocol for the chiral resolution of N-methyl-d,l-aspartic acid was built with a predicted N-demethylase(GenBank ID:OJV90073.1)from the genome of Chloroflexi bacterium 54-19.Through sequence alignment,the enzyme shares an identity of 32.19%to 2UZZ(PDB ID)with a conserved catalytic center.Recombinantly expressed in Bacillus subtilis WB600,the N-demethylase was characterized with optimal temperature and pH at 55℃and 7.5,and adaptive temperature and pH were 40-60℃ and 6-8.The effects of organic solvents and metal ions were investigated as well.Compared with other previously reported sarcosine oxidases,the enzyme showed a specific N-demethylation activity against N-methyl-l-aspartic acid according to the analysis by chiral liquid chromatography,LC-MS/MS and a detection by polarimeter.The results demonstrated 76%of 4.5 mM N-methyl-d,l-aspartic acid could be chirally separated by 7 mg·L^(−1) enzyme after reaction of 80 min.This work provided a foundation for mild synthesis of NMDA in industry.