Goro Takata

Izumoring: A novel and complete strategy for bioproduction of rare sugars

Starch, whey or hemicellulosic waste can be used as a raw material for the industrial production of rare sugars. D-glucose from starch, whey and hemicellulose, D-galactose from whey, and D-xylose from hemicellulose are the main starting monosaccharides for production of rare sugars. We can produce all monosaccharides; tetroses, pentoses and hexoses, from these raw materials. This is achieved by using D-tagatose 3-epimerase, aldose isomerase, aldose reductase, and oxidoreductase enzymes or whole cells as biocatalysts. Bioproduction strategies for all rare sugars are illustrated using ring form structures given the name Izumoring.

Cloning and characterization of a novel gene encoding L-ribose isomerase from Acinetobacter sp. strain DL-28 in Escherichia coli.

The gene encoding a novel L-ribose isomerase (L-RI) from Acinetobacter sp. was cloned into Escherichia coli and nucleotide sequence was determined. The gene corresponded to an open reading frame of 747 bp that codes for a deduced protein of 249 amino acids, which showed no amino acid sequence similarity with any other sugar isomerases. After expression of the gene in E. coli using pUC118 the recombinant L-RI was purified to homogeneity using different chromatographic methods. The overall enzymatic properties of the purified recombinant L-RI were the same as those of the authentic L-RI. To our knowledge, this is the first time report concerning the L-RI gene.

Gene Cloning and Characterization of L-Ribulose 3-epimerase from Mesorhizobium loti and Its Application to Rare Sugar Production

A gene encoding L-ribulose 3-epimerase (L-RE) from Mesorhizobium loti, an important enzyme for rare sugar production by the Izumoring strategy, was cloned and overexpressed. The enzyme showed highest activity toward L-ribulose (230 U/mg) among keto-pentoses and keto-hexoses. This is the first report on a ketose 3-epimerase showing highest activity toward keto-pentose. The optimum enzyme reaction conditions for L-RE were determined to be sodium phosphate buffer (pH 8.0) at 60 °C. The enzyme showed of higher maximum reaction a rate (416 U/mg) and catalytic efficiency (43 M(-1) min(-1)) for L-ribulose than other known ketose 3-epimerases. It was able to produce L-xylulose efficiently from ribitol in two-step reactions. In the end, 7.2 g of L-xylulose was obtained from 20 g of ribitol via L-ribulose at a yield of 36%.

Direct Production of l-Tagatose from l-Psicose by Enterobacter aerogenes 230S

L-tagatose was produced directly from L-psicose by subjecting the same biomass suspension to microbial reduction followed by oxidation using a newly isolated bacteria Enterobacter aerogenes 230S. After various optimizations, it was observed that cells grown on xylitol have the best conversion potential. Moreover, E. aerogenes 230S converted L-psicose to L-tagatose at a faster rate in the presence of polyols such as glycerol, D-sorbitol, ribitol, L-arabitol, D-mannitol and xylitol. At 5% substrate concentration, the conversion ratio of L-psicose to L-tagatose was above 60% in the presence of glycerol. Identity of crystalline L-tagatose was confirmed by HPLC analysis, (13)C-NMR spectra, and optical rotation.

Characterization of Mesorhizobium loti L-Rhamnose Isomerase and Its Application to L-Talose Production

The L-rhamnose isomerase gene (rhi) of Mesorhizobium loti was cloned and expressed in Escherichia coli, and then characterized. The enzyme exhibited activity with respect to various aldoses, including D-allose and L-talose. Application of it in L-talose production from galactitol was achieved by a two-step reaction, indicating that it can be utilized in the large-scale production of L-talose.

Polyol Conversion Specificity of Bacillus pallidus

The conversion specificity of Bacillus pallidus Y25 for polyols, including elusive rare sugar alcohols, was investigated. B. pallidus cells showed transformation potential for several rare polyols, including allitol, L-mannitol, D/L-talitol, and D-iditol, and converted them to their corresponding ketoses. This indicates that the bacterium had two polyol dehydrogenases specific for polyols that have D-erythro and D-threo configurations. By combination with intrinsic isomerases, polyols were converted directly to various aldoses, including L-xylose, L-talose, D-altrose, and L-glucose.

Cloning and characterization of the l-ribose isomerase gene from Cellulomonas parahominis MB426

A newly isolated bacterium, Cellulomonas parahominis MB426, produced l-ribose isomerase (CeLRI) on a medium containing l-ribose as a sole carbon source. A 32 kDa protein isomerizing l-ribose to l-ribulose was purified to homogeneity from this bacterium. A set of degenerated primers were synthesized based on amino acid sequences of the purified CeLRI, and a 747 bp gene encoding CeLRI was cloned, sequenced and overexpressed in Escherichia coli. This gene encoded a 249 amino acid protein with a calculated molecular mass of 27,435. The deduced amino acid sequence of this gene showed the highest identity with l-ribose isomerase from Acinetobacter calcoaceticus DL-28 (71%). The recombinant l-ribose isomerase (rCeLRI) was optimally active at pH 9.0 and 40°C, and was stable up to 40°C for 1 h and not dependent for metallic ions for its activity. The rCeLRI showed widely substrate specificity for the rare sugar which involved l-erythro form such as l-ribose, d-lyxose, d-talose, d-mannose, l-gulose, and l-allose.

Structural insight into L-ribulose 3-epimerase from Mesorhizobium loti.

L-Ribulose 3-epimerase (L-RE) from Mesorhizobium loti has been identified as the first ketose 3-epimerase that shows the highest observed activity towards ketopentoses. In the present study, the crystal structure of the enzyme was determined to 2.7 Å resolution. The asymmetric unit contained two homotetramers with the monomer folded into an (α/β)8-barrel carrying four additional short α-helices. The overall structure of M. loti L-RE showed significant similarity to the structures of ketose 3-epimerases from Pseudomonas cichorii, Agrobacterium tumefaciens and Clostridium cellulolyticum, which use ketohexoses as preferred substrates. However, the size of the C-terminal helix (α8) was much larger in M. loti L-RE than the corresponding helices in the other enzymes. In M. loti L-RE the α8 helix and the following C-terminal tail possessed a unique subunit-subunit interface which promoted the formation of additional intermolecular interactions and strengthened the enzyme stability. Structural comparisons revealed that the relatively small hydrophobic pocket of the enzyme around the substrate was likely to be the main factor responsible for the marked specificity for ketopentoses shown by M. loti L-RE.

Cloning and Overexpression of the Xylitol Dehydrogenase Gene from Bacillus pallidus and Its Application to L-Xylulose Production

The xylitol dehydrogenase gene (xdh) of Bacillus pallidus was cloned and overexpressed in Escherichia coli using pQE60 vector, for the first time. The open reading frame of 759 bp encoded a 253 amino acid protein with a calculated molecular mass of 27,333 Da. The recombinant xylitol dehydrogenase (XDH) was purified to homogeneity by three-step column chromatography, producing a single SDS–PAGE band of 28 kDa apparent molecular mass. The enzyme exhibited maximal activity at 55 °C in glycine-NaOH buffer pH 11.0, with 66% of initial enzyme activity retained after incubation at 40 °C for 1 h. In further application of the recombinant bacterium to L-xylulose production from xylitol (initial concentration 5%) using a resting cell reaction, 35% L-xylulose was produced within 24 h. This result indicates that this recombinant XDH is applicable in the large-scale production of L-xylulose.

Bioproduction of D-Psicose from Allitol with Enterobacter aerogenes IK7: A New Frontier in Rare Ketose Production

D-Psicose, a new alternative sweetener, was produced from allitol by microbial oxidation of the newly isolated strain Enterobacter aerogenes IK7. Cells grown in tryptic soy broth medium (TSB) supplemented with D-mannitol at 37 °C were found to have the best oxidation potential. The cells, owing to broad substrate specificity, oxidized various polyols (tetritol, pentitol, and hexitol) to corresponding rare ketoses. By a resting cell reaction, 10% of allitol was completely transformed to the product D-psicose, which thus becomes economically feasible for the mass production of D-psicose. Finally, the product was crystallized and confirmed to be D-psicose by analytical methods.