Cloning, sequencing, overexpression and characterization of l-rhamnose isomerase from Bacillus pallidus Y25 for rare sugar production

Abstract

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.

Characterization ofMesorhizobium lotiL-Rhamnose Isomerase and Its Application toL-Talose Production

“…The K m and k cat /K m values of M. loti L-RhI as compared with other known L-RhIs, showed affinity and catalytic efficiency similar to D-allose (Table 2), but the kinetic parameters of M. loti L-RhI to D-allose and L-talose, compared with the phospho-isomerases were significantly different (Table 2). 8,10,20,21) These results suggest that the catalytic mechanisms of L-RhI and phospho-isomerase may be different. Recombinant L-RhI from M. loti was applied to produce L-talose from L-tagatose.…”

“…Genomic DNA preparation and recombinant DNA techniques were performed as previously described. 8) The DNA sequences for both strands were determined by the dideoxy nucleotide chain termination method using a DTCS Quick Start Kit (Beckman Coulter, Fullerton, CA), and they were analyzed on a CEQ8000 system (Beckman Coulter). 14) Homology searches were performed with deduced nucleotide and amino acid sequences available from the DNA Data Bank of Japan (DDBJ) using the BLAST program.…”

Characteristics and Kinetic Properties of L-Rhamnose Isomerase from <i>Bacillus Subtilis</i> by Isothermal Titration Calorimetry for the Production of D-Allose

“…1(B)), which was in agreement with the DNA sequence analysis. (Poonperm et al, 2007). The crystal structure of P. stuzeri L-RhI and E. coli L-RhI revealed that they require a divalent metal ion for their activity, one or two metal ions were observed.…”

“…The alignment of L-RhIs from different species revealed that B. subtilis L-RhI shares 76.42% identity with that from B. halodurans (Prabbu et al, 2011), 57.28% identity with L-RhI from E. coli K12, 56.47% identity with L-RhI from B. pallidus Y25 (Poonperm et al, 2007), 55.45% identity with L-RhI from T. saccharolyticum NTOU1 (Lin et al, 2010), but only 18.30% identity with L-RhI from P. stutzeri (Table 3). So far, crystal structures of L-RhIs from E. coli and P. stuzeris have been resolved, of which the structure of E. coli L-RhI and its relationship with xylose isomerase was the first report (Korndörfer et al, 2000).…”

Advances in the enzymatic production of l-hexoses

“…The specific activities of the enzymes isolated from P. stutzeri, Caldicellulosiruptor saccharolyticus (Lin et al 2011), Thermotoga maritime (Park et al 2010), Thermoanaerobacterium saccharolyticum (Hung et al 2014), Bacillus pallidus (Poonperm et al 2007), M. loti (Takata et al 2011), and Bacillus subtilis (Park 2014) toward L-mannose were 81.6, 38, 15, 6, 4.52, 2.27, and 0.92 U mg −1 , respectively. In addition, L-fructose has also been produced from L-mannose with a conversion ratio of approximately 25 % by an enzyme reactor packed with cross-linked Streptomyces rubiginosus D-glucose isomerase crystals (Jokela et al 2002).…”