Construction And Expression Of ?-amino Acid Ester Acyltransferase Engineering Bacteria And Its Catalytic Synthesis Of L-alanyl-L-glutamine

L-Alanyl-L-glutamine is much more tolerant to high temperature and soluble than each of the constitutive amino acids.Based on this property,it is applied in intravenous infusions as the carrier of L-glutamine.?-Amino acid ester acyltransferase?Aet?catalyzes the L-alanyl-L-glutamine forming reaction from L-alaine methylester hydrochloride and L-glutamine.In this study,a strain producing?-amino acid ester acyltransferase was constructed,and its expression conditions were optimized.What's more,its enzymatic properties and catalytic applications were studied.In this study,we first optimized the codons of the genes coding of the?-amino acid ester acyltransferases derived from E.brevis ATCC 14234 and siyangensis AJ2458,and then synthesized them by whole genes and introduced them into different plasmid vectors.SAET-QC01?SAET-QC02?EAET-DW01 were constructed,the SAET-QC01 is significantly higher than others,it is indicated that SAET-QC01 is more suitable for?-amino acid ester acyltransferase expression.Next,the fermentation conditions suitable for enzyme production optimized preliminarily were as follows:0.5%glycerol,0.5%tryptone,1%yeast,0.1%MgSO₄,initial pH value 6.3,induction temperature was 20?,the induction stage(OD₆₀₀=2.0-2.5),IPTG concentration was 0.6 mmol/L,induction time was 12 h,the enzyme activity was increased from 221.6 U/m L to 998.0 U/mL,showing 5 fold improvement over that of the initial enzyme activity.The recombinant protein was separated and purified by Ni-NTA affinity chromatography,The enzyme was approximately 68 kDa with an optimum temperature of 27? and pH of 8.5,it was most stable at pH 7.0-8.0 and relatively stable in an acidic environment;Metal ions and compounds have a certain influence on the activity of the enzyme,within a certain concentration range,low concentration of Co²⁺or EDTA could promote the enzyme activity,high concentration of Fe³⁺,Ni²⁺or SDS could inhibite the enzyme activity,and measured Km and Vmax values of the enzyme were 0.30 mol/L and 0.02 mol/min·g.The catalytic applications of the enzyme were explored preliminarily,used recombinant?-Amino acid ester acyltransferase prepared L-alanyl-L-glutamine.Under the condition of enzyme added 50 U/mmol,glutamine and alanine hydrochloride were added at a ratio of 0.8,when the concentration of L-alaine methylester hydrochloride was 100 mmol/L,200 mmol/L,300 mmol/L,400 mmol/L,500 mmol/L and 600mmol/L,the yields of L-alanyl-L-glutamine were 15.1 g/L,29.2 g/L,42.3 g/L,56.2 g/L,69.5 g/L,78.2 g/L,and the conversion rate to glutamine were 86.7%,84.0%,81.1%,80.9%,80.0%,75.0%.Finally,the catalytic products were separated and purified,and their structures were characterized to determine the structural characteristics of the L-alanyl-L-glutamine.?-Amino acid ester acyltransferase has excellent acid-basei resistance,high catalytic efficiency.These characteristics suggest its application prospects in the industrial production.