Cloning, sequencing, overexpression and characterization of L-rhamnose isomerase from Bacillus pallidus Y25 for rare sugar production.
The L-rhamnose isomerase gene (L-rhi) encoding for L-rhamnose isomerase (L-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6xHis sequence at a C-terminal of the protein. The open reading frame of L-rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced L-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant L-RhI exhibited maximum activity at 65 degrees C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60 degrees C for 60 min. The apparent affinity (K(m)) and catalytic efficiency (k(cat)/K(m)) for L-rhamnose (at 65 degrees C) were 4.89 mM and 8.36 x 10(5) M(-1) min(-1), respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50 degrees C, for D: -allose, L-mannose, D-ribulose, and L-talose from D-psicose, L-fructose, D-ribose and L-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant L-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.
Poonperm
Wayoon
W
Rare Sugar Research Center and Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki, Kagawa Prefecture 761-0795, Japan.
Takata
Goro
G
Okada
Hiromi
H
Morimoto
Kenji
K
Granström
Tom Birger
TB
Izumori
Ken
K
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins; D001483:Base Sequence; D002241:Carbohydrates / Q000096:biosynthesis*; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002241:Carbohydrates; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D019747:Aldose-Ketose Isomerases; C062878:L-rhamnose isomerase