Production of d-tagatose 3-epimerase of Pseudomonas cichorii ST-24 using recombinant Escherichia coli

• Published in: Journal of fermentation and bioengineering Vol. 84; no. 4; pp. 348 - 350
• Main Authors: Ishida, Yutaka, Kamiya, Takanori, Izumori, Ken
• Format: Journal Article
• Language: English
• Published: Osaka Elsevier B.V 01.01.1997; Society for Fermentation and Bioengineering
• Subjects: Amino acids; Biological and medical sciences; Biotechnology; Cell culture; CETOSAS; Coliform bacteria; Column chromatography; d-tagatose 3-epimerase; Electrophoresis; Enzymes; ESCHERICHIA COLI; EXPRESION GENICA; expression; EXPRESSION DES GENES; Fundamental and applied biological sciences. Psychology; GENE EXPRESSION; Genetic engineering; Genetic technics; ISOMERASAS; ISOMERASE; ISOMERASES; KETOSE; KETOSES; Methods. Procedures. Technologies; Modification of gene expression level; pH effects; Polyethylene glycols; Precipitation (chemical); PSEUDOMONAS CICHORII; Thermodynamic stability; Pseudomonas cichorii; expression; d-tagatose 3-epimerase; Pseudomonadales; Recombinant microorganism; Purification; Enzyme; Escherichia coli; Environmental factor; Gene expression; Characterization; Plasmid; Isomerases; Enzymatic activity; Production; Bacteria; Pseudomonadaceae; Kinetic parameter; Recombinant protein; Hybrid gene; Physicochemical properties; Enterobacteriaceae
• ISSN: 0922-338X
• DOI: 10.1016/S0922-338X(97)89257-4

The d-tagatose 3-epimerase ( d-TE) gene of Pseudomonas cichorii ST-24 was expressed in Escherichia coli under the control of the trc promoter. The d-TE production level was highest in E. coli JM105 as a host strain and in NZC medium as a culture medium. Production of d-TE by E. coli JM105 was about 100-fold higher than that of d-TE by P. cichorii ST-24, and the enzyme constituted ∼5% of the total protein. d-TE from E. coli JM105 was purified by polyethylene glycol precipitation, DEAE-Toyopearl 650M column chromatography, and Sephadex G-150 column chromatography. The overall purification procedure resulted in 16.7-fold purification with 18.2% recovery. The molecular weight of the purified d-TE was estimated to be 33.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and agreed with that of the d-TE from P. cichorii ST-24. d-TE produced by E. coli JM105 had generally the same enzymological properties, i.e., the optimum pH and temperature, pH and thermal stabilities, and K m value, as that from Pseudomonas sp. ST-24, but the V max value of d-TE from E. coli was 6 times higher than that from Pseudomonas sp. ST-24. The N-terminal amino acid sequence of the recombinant d-TE agreed with that of the d-TE from P. cichorii ST-24.