Overexpression of the gene for N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli and continuous produciton of optically active methionine by a bioreactor.
A DNA sequence encoding N-acylamino acid racemase (AAR) was inserted downstream from the T7 promoter in pET3c. The recombinant plasmid was introduced into Escherichia coli MM 294 lysogenized with a bacteriophage lambda having a T7 RNA polymerase gene. The amount of AAR produced by the E. coli transformant was 1100-fold more than that produced by Amycolatopsis sp. TS-1-60, the DNA donor strain. The AAR was purified to homogeneity from the crude extract of the E. coli transformant by two steps: heat treatment and Butyl-Toyopearl column chromatography. Bioreactors for the production of optically active amino acids were constructed with DEAE-Toyopearl-immobilized AAR and D- or L-aminoacylase. D- or L-methionine was continuously produced with a high yield from N-acetyl-DL-methionine by the bioreactor.
Tokuyama
S
S
Technology Development Division Takeda Chemical Industries Ltd., Osaka, Japan.
Hatano
K
K
D039903:Actinobacteria / Q000201:enzymology / Q000235:genetics*; D000581:Amidohydrolases; D000591:Amino Acid Isomerases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000837:Anion Exchange Resins; D001483:Base Sequence; D019149:Bioreactors; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008715:Methionine / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D012117:Resins, Synthetic; D013237:Stereoisomerism; D014764:Viral Proteins
D000837:Anion Exchange Resins; D012117:Resins, Synthetic; D014764:Viral Proteins; C075368:Toyopearl DEAE 650 M; D008715:Methionine; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000581:Amidohydrolases; C019672:aminoacylase I; D000591:Amino Acid Isomerases