Keiko Uechi

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%.

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.

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 2.7u2005Å 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.

Enzymatic production of three 6-deoxy-aldohexoses from l-rhamnose

6-Deoxy-l-glucose, 6-deoxy-l-altrose, and 6-deoxy-l-allose were produced from l-rhamnose with an immobilized enzyme that was partially purified (IE) and an immobilized Escherichia coli recombinant treated with toluene (TT). 6-Deoxy-l-psicose was produced from l-rhamnose by a combination of l-rhamnose isomerase (TT-PsLRhI) and d-tagatose 3-epimerase (TT-PcDTE). The purified 6-deoxy-l-psicose was isomerized to 6-deoxy-l-altrose and 6-deoxy-l-allose with l-arabinose isomerase (TT-EaLAI) and l-ribose isomerase (TT-AcLRI), respectively, and then was epimerized to l-rhamnulose with immobilized d-tagatose 3-epimerase (IE-PcDTE). Following purification, l-rhamnulose was converted to 6-deoxy-l-glucose with d-arabinose isomerase (TT-BpDAI). The equilibrium ratios of 6-deoxy-l-psicose:6-deoxy-l-altrose, 6-deoxy-l-psicose:6-deoxy-l-allose, and l-rhamnulose:6-deoxy-l-glucose were 60:40, 40:60, and 27:73, respectively. The production yields of 6-deoxy-l-glucose, 6-deoxy-l-altrose, and 6-deoxy-l-allose from l-rhamnose were 5.4, 14.6, and 25.1%, respectively. These results indicate that the aldose isomerases used in this study acted on 6-deoxy aldohexoses.

Novel process for producing 6-deoxy monosaccharides from l-fucose by coupling and sequential enzymatic method.

We biosynthesized 6-deoxy-L-talose, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose, which rarely exist in nature, from L-fucose by coupling and sequential enzymatic reactions. The first product, 6-deoxy-L-talose, was directly produced from L-fucose by the coupling reactions of immobilized D-arabinose isomerase and immobilized L-rhamnose isomerase. In one-pot reactions, the equilibrium ratio of L-fucose, L-fuculose, and 6-deoxy-L-talose was 80:9:11. In contrast, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose were produced from L-fucose by sequential enzymatic reactions. D-Arabinose isomerase converted L-fucose into L-fuculose with a ratio of 88:12. Purified L-fuculose was further epimerized into 6-deoxy-L-sorbose by D-allulose 3-epimerase with a ratio of 40:60. Finally, purified 6-deoxy-L-sorbose was isomerized into both 6-deoxy-L-gulose with an equilibrium ratio of 40:60 by L-ribose isomerase, and 6-deoxy-L-idose with an equilibrium ratio of 73:27 by D-glucose isomerase. Based on the amount of L-fucose used, the production yields of 6-deoxy-L-talose, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose were 7.1%, 14%, 2%, and 2.4%, respectively.

Production of L-allose and D-talose from L-psicose and D-tagatose by L-ribose isomerase.

l-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert l-psicose and d-tagatose to l-allose and d-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce l-allose and d-talose. Conversion reaction was performed with the reaction mixture containing 10% l-psicose or d-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of l-allose and d-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert l-psicose to l-allose without remarkable decrease in the enzyme activity over 7 times use and d-tagatose to d-talose over 37 times use. After separation and concentration, the mixture solution of l-allose and d-talose was concentrated up to 70% and crystallized by keeping at 4 °C. l-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% l-allose and 7.30% d-talose that were obtained from l-psicose and d-tagatose, respectively. Graphical Abstract We described the production of l-allose and d-talose from l-psicose and d-tagatose using immobilized enzyme of recombinant L-RI.

Essentiality of tetramer formation of Cellulomonas parahominis L-ribose isomerase involved in novel L-ribose metabolic pathway.

L-Ribose isomerase from Cellulomonas parahominis MB426 (CpL-RI) can catalyze the isomerization between L-ribose and L-ribulose, which are non-abundant in nature and called rare sugars. CpL-RI has a broad substrate specificity and can catalyze the isomerization between D-lyxose and D-xylulose, D-talose and D-tagatose, L-allose and L-psicose, L-gulose and L-sorbose, and D-mannose and D-fructose. To elucidate the molecular basis underlying the substrate recognition mechanism of CpL-RI, the crystal structures of CpL-RI alone and in complexes with L-ribose, L-allose, and L-psicose were determined. The structure of CpL-RI was very similar to that of L-ribose isomerase from Acinetobacter sp. strain DL-28, previously determined by us. CpL-RI had a cupin-type β-barrel structure, and the catalytic site was detected between two large β-sheets with a bound metal ion. The bound substrates coordinated to the metal ion, and Glu113 and Glu204 were shown to act as acid/base catalysts in the catalytic reaction via a cis-enediol intermediate. Glu211 and Arg243 were found to be responsible for the recognition of substrates with various configurations at 4- and 5-positions of sugar. CpL-RI formed a homo-tetramer in crystals, and the catalytic site independently consisted of residues within a subunit, suggesting that the catalytic site acted independently. Crystal structure and site-direct mutagenesis analyses showed that the tetramer structure is essential for the enzyme activity and that each subunit of CpL-RI could be structurally stabilized by intermolecular contacts with other subunits. The results of growth complementation assays suggest that CpL-RI is involved in a novel metabolic pathway using L-ribose as a carbon source.

Amylolytic Enzymes Acquired from L-Lactic Acid Producing Enterococcus faecium K-1 and Improvement of Direct Lactic Acid Production from Cassava Starch

An amylolytic lactic acid bacterium isolate K-1 was isolated from the wastewater of a cassava starch manufacturing factory and identified as Entercoccus faecium based on 16S rRNA gene sequence analysis. An extracellular α-amylase was purified to homogeneity and the molecular weight of the purified enzyme was approximately 112xa0kDa with optimal pH value and temperature measured of 7.0 and 40xa0°C, respectively. It was stable at a pH range of 6.0–7.0, but was markedly sensitive to high temperatures and low pH conditions, even at a pH value of 5. Ba2+, Al3+, and Co2+ activated enzyme activity. This bacterium was capable of producing 99.2% high optically pure L-lactic acid of 4.3 and 8.2xa0g/L under uncontrolled and controlled pH at 6.5 conditions, respectively, in the MRS broth containing 10xa0g/L cassava starch as the sole carbon source when cultivated at 37xa0°C for 48xa0h. A control pH condition of 6.5 improved and stabilized the yield of L-lactic acid production directly from starch even at a high concentration of starch at up to 150xa0g/L. This paper is the first report describing the properties of purified α-amylase from E. faecium. Additionally, pullulanase and cyclodextrinase activities were also firstly recorded from E. faecium K-1.

Structure of D-tagatose 3-epimerase-like protein from Methanocaldococcus jannaschii

The crystal structure of a D-tagatose 3-epimerase-like protein (MJ1311p) encoded by a hypothetical open reading frame, MJ1311, in the genome of the hyperthermophilic archaeon Methanocaldococcus jannaschii was determined at a resolution of 2.64u2005Å. The asymmetric unit contained two homologous subunits, and the dimer was generated by twofold symmetry. The overall fold of the subunit proved to be similar to those of the D-tagatose 3-epimerase from Pseudomonas cichorii and the D-psicose 3-epimerases from Agrobacterium tumefaciens and Clostridium cellulolyticum. However, the situation at the subunit-subunit interface differed substantially from that in D-tagatose 3-epimerase family enzymes. In MJ1311p, Glu125, Leu126 and Trp127 from one subunit were found to be located over the metal-ion-binding site of the other subunit and appeared to contribute to the active site, narrowing the substrate-binding cleft. Moreover, the nine residues comprising a trinuclear zinc centre in endonuclease IV were found to be strictly conserved in MJ1311p, although a distinct groove involved in DNA binding was not present. These findings indicate that the active-site architecture of MJ1311p is quite unique and is substantially different from those of D-tagatose 3-epimerase family enzymes and endonuclease IV.

Crystal structure of L-ribose isomerase from Cellulomonas parahominis MB426

Monosaccharides and their derivatives which hardly exist in nature are so-called “rare sugars”. Rare sugars have significance not only in food industries but also pharmaceutical industries. We discovered a novel L-ribose isomerase from Cellulomonas parahominis (CpLRbI, 249 amino acids), which catalyzes the reversible isomerization between L-ribose and L-ribulose, L-allose and L-psicose, and Dtalose and D-tagatose. Since CpL-RbI has a broad substrate specificity, it is useful for the production of various rare sugars. To elucidate the molecular basis of unique enzymatic properties of CpL-RbI, we determined its crystal structure. The N-terminal Histagged CpL-RbI overexpressed in Escherichia coli was purified using a nickel affinity column. Crystals of CpL-RbI were obtained from a reservoir solution of 0.1 M sodium acetate trihydrate (pH 4.6) with 3.9 M ammonium acetate, by a hanging-drop vapor-diffusion method at 293 K (Space group C2221, a = 76.8, b = 88.6, c = 152.3 Å). X-ray diffraction data were collected up to 2.10 Å resolution using a Rigaku R-AXIS VII on a RA-Micro7HF rotating anode generator (40 kV, 30 mA) at 100 K. The structure was solved by a molecular replacement method with a structure of Acinetobacter sp L-ribose isomerase (4NS7) as a search model, and refined to Rfactor of 0.227. CpL-RbI had a cupin-type beta-barrel structure, and the catalytic site was found between two large beta-sheets with a bound metal ion (Fig. 1). There were two protein molecules in an asymmetric unit, forming a homo-dimer with a non-crystallographic 2-fold symmetry (Fig.1). Furthermore, the PISA server showed that two dimers in crystal were associated to form a stable tetramer. Complex structures with substrates, L-ribose, L-allose and L-psicose, were also successfully determined. We will discuss a broad substrate specificity and catalytic reaction mechanism of CpL-RbI based on its three-dimensional structure.