Enzymatic Synthesis Of Tryptophan And Non-natural Amino Aacids And Catalytic Mechanism

Amino acids are a kind of bioactive chiral molecules.Amino acids and their derivatives have a very wide range of applications in medicine and food field.Amino acids have a large market demand.Amino acids and their derivatives became the focus in this field for their special and important application.The methods of preparing amino acids and their derivatives include physical,chemical and biological methods.Biological method is the use of microorganism or enzyme direct transformation.Enzymatic synthesis is believed more efficient,economical,surrounding welcome and safety compared to chemosynthesis.It is against this background that the author carried out a series of related work,constructed genetic engineering bacteria,biologically synthesized of a series of chiral amino acid intermediates,enriched biological preparation of chiral drug intermediates.Specifically the following:1.Trp BA and trp A are two key enzyme gene in L-tryptophan synthetic way.Trp BA and trp A were obtained from Escherichia coli K-12.They were constructed into a vector pETDuet-1 in tandem.Then the recombinant vector was introduced into Escherichia coli.The optimal temperature and pH value were 35 ? and 8,respectively.The optimal L-serine concentration was 100 mmol/L.Under the optimal conditions,bioconversion rate of L-serine reached 81%after 6 h by trp BA and trp A co-expression in Escherichia coli.2.Enzymatic synthesis of L-tryptophan from L-serine and indole catalyzed by tryptophan synthase from recombinant Escherichia coli in an aqueous/organic biphasic system was carried out.The influences of organic solvents,volume fraction of ethyl acetate,reaction temperature,pH value of the aqueous phase,molar ratio of L-serine to indole,L-serine concentration,surfactants,and enzyme amount were investigated.Ethyl acetate was found to be the best organic solvent for the reaction.The optimal volume fraction of ethyl acetate was 2.5%.The optimal temperature and pH value were 35 ? and 8,respectively.The optimal molar ratio of L-serine to indole was 1:1.2.The optimal L-serine concentration was 100 mmol/L.The optimal mass fraction of Tween 80 was 0.4%.The optimal enzyme amount was 20 g/L.Under the optimal conditions,bioconversion rate of L-serine reached 95%after 2.5 h in the aqueous/organic biphasic system.Tryptophan synthetase conformational space were investigated.Tryptophan synthetase conformational space was investigated.There is a stable hydrogen bond formed between indole and tryptophan synthase active site Asp 138.Enzymatic synthesis of L-2-methyltryptophan from L-serine and 2-methylindole catalyzed by tryptophan synthase from recombinant Escherichia coli in an aqueous/organic biphasic system was carried out.Toluene was found to be the best organic solvent for the reaction.The optimal volume fraction of ethyl acetate was 2%.The optimal temperature and pH value were 40? and 8,respectively.The optimal molar ratio of L-serine to 2-methylindole was 1:1.2.The optimal L-serine concentration was 150 mmol/L.The optimal mass fraction of Trion X-100was 2%.The optimal Ca2+ concentration was 0.1 mmol/L.Under the optimal conditions,bioconversion rate of L-serine reached 97.5%after 5 h in the aqueous/organic biphasic system.There is a stable hydrogen bond formed between 2-methylindole and tryptophan synthase active site Gly189.3.The effects of polyethylene glycol(PEG)of different molecular(1000,2000),glycerol,ethylene glycol on the catalytic activity of tryptophan synthase were studied.The results indicated that the addition of PEG 2000 increased the enzymatic activity of tryptophan synthase.Reaction conditions were optimized by using 10 g L-1 PEG 2000 at pH 9 and 40?.L-Serine conversion rate reach 89.9%under the optimal conditions.The kinetic parameters indicated the specificity of TSase to substrate was improved.4.Enzymatic synthesis of L-cysteine,S-phenyl-L-cysteine,S-methyl-L-cysteine,and S-carboxymethyl-L-cysteine catalyzed by tryptophan synthase from recombinant Escherichia coli were carried out.The influences of substrate specificity,reaction temperature,pH value,substrate concentration,molar ratio of substrate and time were investigated.The optimal temperature and pH value were 37? and 8,respectively.The optimal molar ratio of L-serine to sulfhydryl compound was 1:1.2.The optimal of substrate concentrations were 400 mmol/L.The response times of L-cysteine,S-methyl-L-cysteine,S-phenyl-L-cysteine and S-carboxymethyl-L-cysteine catalyzed by tryptophan synthase were 8 h,12 h,16 h and 18 h,respectively.Under the optimal conditions,bioconversion rate of L-serine reached 90%.There was a stable hydrogen bond formed between hydrothion and tryptophan synthase active site Ser235.There was a stable hydrogen bond formed between methanthiol and tryptophan synthase active site Gly234.There were stable hydrogen bonds formed between thiopheno and tryptophan synthase active site Ser235and Gly233.There were stable hydrogen bonds formed between mercaptoacetic acid and tryptophan synthase active site Gly232,Gly233,Gly234,Ser235and Asn236.5.Chiral resolutions of N-acetyl-D,L-tryptophan,N-acetyl-D,L-phenylalanine and N-acetyl-D,L-methionine were investigated.The optimal reaction conditions of N-acetylornithine deacetylase for chiral resolution of N-acetyl-DL-amino acids are as follows:with N-acetyl-D,L-amino acids concentration 300 mmol/L,Co2+ 10-4mol/L,Tween 80 0.4%,at pH=7 and 45?.The average conversion rate of N-acetyl-D,L-methionine,N-acetyl-D,L-phenylalanine and N-acetyl-D,L-tryptophan were 96%,83%and 65%,respectively.