Preparation Of Chiral Amino Acids And Their Derivatives By Microbial Enzyme

Chiral is one of the basic properties of nature.Most molecules of constituting living organism are asymmetric chiral molecules,which play important physiological functions in vivo.Amino acid is a kind of chiral compound,and was widely used in several industries related to human life and health such as medical,food,health products,feed and so on.D-amino acid,the synthetic intermediates,are widely used in the preparation of antibiotics,peptide hormone,pesticides and artificial sweeteners.Generally,amino acids can be prepared by fermentation,chemosynthesis or enzymatic synthesis.Compared with chemical synthesis,enzymatic synthesis has many advantages such as high efficiency,environment-friendly and safty.With increased environmental awareness,people have increasingly paid attention to comprehensive utilization of resources,clean production,low-carbon and energy consumption,which have become a global trend of development.Sustainable development,namely with minimal environmental impact,minimal resource consumpation and the optima management model to achieve the most reasonable economic growth,has become consensus on ecomomic and social development fields.In this study,a series of genetically engineered strains were constructed.The way of the preparation of natural or non-natural chiral amino acids was studied.Chiral amino acids were synthesized by single or double enzymatic way in the presence of genetically engineered strains,which had high-activity,good selectivity and salt tolerance.At the same time,the reaction conditions of the enzyme were optimized.Specifically the following:1.Recombinant expression of Peptidase B.The enzyme has broad substrate specificity,and can optical resolution DL-amino amides to obtained L-amino acids.In the second chapter of the thesis,DL-3-methoxy-alanine amide was synthesized from acrylamide as raw materials,which was optical resolutioned by Peptidase B.The reaction conditions of the enzyme were optimized.Optimum conditions:pH 9.0,reaction temperature 40 ?,substrate concentration 400 mmol/L,and the enzyme was significantly activated by low concentration metal ions such as Co2+,Ca2+,Mn2+ and Mg2+.Under optimal conditions,after 14h the molar conversion rate of L-3-methoxy-alanine amide was 96%.The L-3-methoxy alanine and D-3-methoxy-alanine amide were hydrolyzed by the hydrobromide to obtained D-serine and L-serine.Also the author prepared L-2-amino fatty acids from 2-bromo fatty acid amides by chemical and biological.The specific rotation of L-2-amino butyric acid,L-norvaline and L-norleucine were consistent with international standard.2.Recombinant expression of D-stereospecific amino-acid amidase.The enzyme has broad substrate specificity,and can optical resolution DL-amino amides to obtained D-amino acids.DL-Phenylalaninamide was optical resolutioned by Daa A.The reaction conditions of the enzyme were optimized.Optimum conditions:pH 9.0,reaction temperature 45?,substrate concentration 500 mmol/L.Under optimal conditions,after 8h the molar conversion rate of D-Phenylalaninamide was 96%.Also the author prepared D-2-amino fatty acids from 2-bromo fatty acid amides by chemical and biological.The specific rotation of D-2-amino butyric acid,L-norvaline and L-norleucine were consistent with international standard.3.Recombinant expression of a-amino-s-caprolactam racemase.The enzyme has broad substrate specificity,racemic amino amides could be obtained by use of ACLR from L or D amino amides.L-Alaninamide was transformed into DL-Alaninamide in the presence of ACLR.The reaction conditions of the enzyme were optimized.Optimum conditions:pH 7.5,reaction temperature 45?,substrate concentration 800 mmol/L.Under optimal conditions,after 4h the molar racemation rate of L-Alaninamide was 98%.The combination of PepB with ACLR or DaaA with ACLR could be used to resolution DL-amino amides.The dual-enzyme system had good resolution results,on the one hand,D or L amino acids could be obtained,on the other hand,raw material waste was avoided,which had importance to industrial production of amino amides.DL-Methioninamide was optical resolutioned by PepB-ACLR or DaaA-ACLR.The reaction conditions of the dual-enzyme systems were optimized.Optimum conditions of PepB-ACLR:pH 8.5,reaction temperature 40?,substrate concentration 300 mmol/L,ACLR:PepB is 1:3,time 10 h.Optimum conditions of DaaA-ACLR:pH 8.0,reaction temperature 45?,substrate concentration 500 mmol/L,ACLR:DaaA is 1:3,time 18 h.4.An efficient chemoenzymatic synthesis of lacosamide with high enantiopurity was performed in the resolution process catalyzed by immobilized Escherichia coli cells with aminoacylase(EC3.5.1.14)activity as a key step.D,L-3-Methoxy-alanine was synthesized by previously described procedures from low-cost methyl acrylate.This product was then converted to N-acetyl-D,L-3-methoxy-alanine by acetylation,which was used in the resolution process catalyzed by immobilized Escherichia coli cells with aminoacylase(EC3.5.1.14)activity,and N-acetyl-D-3-methoxy-alanine and L-3-methoxy-alanine were obtained from the resolution system.Lacosamide was synthesized by the amidation of compound N-acetyl-D-3-methoxy-alanine with benzylamine.The paper based on the chemical and biological synthesis of chiral amino acids and their derivates,combined with genetic engineering recombinant enzyme,increased the efficiency of enzymatic conversion,prepared a variety of chiral amino acids compounds,has important theoretical significance and potential applications.