Purification, characterization of novel 2-keto-D-galactonic acid reductase (KGAR) from P. fluorescens.


Ritsuko Tinimura1, Rie Nomoto2, Harumi Hosaka3, Takanori Matsuura3, Atsushi Nakagawa3, Kenji Ikehara2,Ryoko Iwamoto2

1Graduate School of Human Culture, Nara Womens University
2Department of Chemistry, Faculty of Scinece, Nara Womens University
3Insttitute for Protein Research, Osaka University

The 77th Annual Meeting of the Japanese Biochemical Society (Yokohama Japan, 2004.10.13-16)

2-keto-D-galactonic acid was formed from D-galactose (Gal) in the cell culture of P. fluorescens. It was guessed that during the reaction, Gal dehydrogenase and KGAR catalyzed the conversion of sugars. In this work, KGAR was purified, characterized, and its complete amino acid sequence was determined. Enzymatic activity was measured the change of A340 of NADPH in the presence of substrate and enzyme at 25 °C. KGAR was purified from supernatant of sonic treated P. fluorescens cells followed by ammonium sulfate precipitation, and three column chromatographies.

[Results]

KGAR was purified to homogeneity about 200 times and yield was 8%. The enzyme composed of two identical subunit with MW 37,000. Some properties of the enzyme were shown in table. N-terminal 20 amino acid sequence was determined by the peptide sequencer and complete DNA sequence was find ORD of P. fluorescens pf-5 DNA gene as 1215 DNA. Thus the resulting 404 amino acid sequence was determined. Similarity research of Blast showed that KGAR belongs to 2-keto-D-aldonic acid reductase family. And KGAR is a novel and different enzyme from 2-keto-gluconate reductase already reported.

Sub Vmax Vmax for NADPH Optimal pH Optimal Temp.
2-ketoGlcA 152 U/mg 12.6 mM 14.9 µM 8.5 35 °C
2-ketoGalA 585 U/mg 12.7 mM 29.7 µM 7-8.5 40 °C


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