Isao Abe

Enzymatic Production of L -Alanyl- L -glutamine by Recombinant E. coli Expressing α-Amino Acid Ester Acyltransferase from Sphingobacterium siyangensis

An enzymatic production method for synthesizing L-alanyl-L-glutamine (Ala-Gln) from L-alanine methyl ester hydrochloride (AlaOMe) and L-glutamine (Gln) was developed in this study. The cultivation conditions for an Escherichia coli strain overexpressing α-amino acid ester acyltransferase from Sphingobacterium siyangensis AJ 2458 (SAET) and reaction conditions for Ala-Gln production were optimized. A high cell density culture broth prepared by fed-batch cultivation showed 440 units/mL of Ala-Gln-producing activity. In addition, an Ala-Gln-producing reaction using intact E. coli cells overexpressing SAET under optimum conditions was conducted. A total Ala-Gln yield of 69.7 g/L was produced in 40 min. The molar yield was 67% against both AlaOMe and Gln.

Gene cloning and characterization of α-amino acid ester acyl transferase in Empedobacter brevis ATCC14234 and Sphingobacterium siyangensis AJ2458.

The gene encoding α-amino acid ester acyl transferase (AET), the enzyme that catalyzes the peptide-forming reaction from amino acid methyl esters and amino acids, was cloned from Empedobacter brevis ATCC14234 and Sphingobacterium siyangensis AJ2458 and expressed in Escherichia coli. This is the first report on the aet gene. It encodes a polypeptide composed of 616 (ATCC14234) and 619 (AJ2458) amino acids residues. The V max values of these recombinant enzymes during the catalysis of L-alanyl-L-glutamine formation from L-alanine methylester and L-glutamine were 1,010 U/mg (ATCC14234) and 1,154 U/mg (AJ2458). An amino acid sequence similarity search revealed 35% (ATCC14234) and 36% (AJ2458) identity with an α-amino acid ester hydrolase from Acetobacter pasteurianus, which contains an active-site serine in the consensus serine enzyme motif, GxSYxG. In the deduced amino acid sequences of AET from both bacteria, the GxSYxG motif was conserved, suggesting that AET is a serine enzyme.