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The L-rhamnose isomerase gene (rhi) of Mesorhizobium lotiwas 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.

The xylitol dehydrogenase gene (xdh) of Bacillus palliduswas cloned and overexpressed in Escherichia coliusing 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.

D-Psicose, a new alternative sweetener, was produced from allitol by microbial oxidation of the newly isolated strain Enterobacter aerogenesIK7. 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.

The L-arabinose metabolic gene cluster, araA, araB, araD, araG, araHand araR, encoding L-arabinose isomerase (L-AI) and its accessory proteins was cloned from Mycobacterium smegmatisSMDU and sequenced. The deduced amino acid sequence of araAdisplayed highest identity with that of Bacillus subtilis(52%). These six genes comprised the L-arabinose operon, and its genetic arrangement was similar to that of B. subtilis. The L-AI gene (araA), encoding a 501 amino acid protein with a calculated molecular mass of 54,888 Da, was expressed in Escherichia coli. The productivity and overall enzymatic properties of the recombinant L-AI were almost same as the authentic L-AI from M. smegmatis. Although the recombinant L-AI showed high substrate specificity, as did L-AI from other organisms, this enzyme catalyzed not only isomerization of L-arabinose-L-ribulose and D-galactose-D-tagatose but also isomerization of L-altrose-L-psicose and L-erythrulose-L-threose. In combination with L-AI from M. smegmatis, L-threose and L-altrose can be produced from cheap and abundant erythritol and D-fructose respectively, indicating that this enzyme has great potential for biological application in rare sugar production. Transcription analysis using various sugars revealed that this enzyme was significantly induced not only by L-arabinose and D-galactose but also by L-ribose, galactitol, L-ribulose, and L-talitol. This different result of transcription mediated by sugars from that of E. colisuggests that the transcriptional regulation of araAfrom M. smegmatisagainst sugar is loose compared with that from E. coli, and that it depends on the hydroxyl configuration at C2, C3 and C4 positions of sugars.

The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzaeKBN616, used for making shoyu koji.The structural part of xynF3was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3was interrupted by ten short introns and encoded 323 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids. The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi. The xynF3gene was successfully overexpressed in A. oryzaeand the XynF3 was purified. The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000. This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence. The purified XynF3 showed an optimum activity at pH 5.0 and 58°C. It had a Km of 6.5 mg/ml and a Vmaxof 435 μmol•min−1•mg−1when birch wood xylan was used as a substrate. Expression of the xynF3gene was analyzed using an Eschelichia coliβ-glucuronidase gene as a reporter. The result indicated that xynF3is expressed in the medium containing wheat bran as a carbon source.

The gene encoding xylanase G2 (xynG2) was isolated from a genomic library of Aspergillus oryzaeKBN616, used for making shoyu koji. The structural part of xynG2was found to be 767 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynG2was interrupted by a single intron which was 71 bp in size and encoded 232 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynG2 had a signal peptide of 44 amino acids. The predicted amino acid sequence of XynG2 has strong similarity to other family 11 xylanases from fungi. The xynG2gene was successfully overexpressed in A. oryzaeand the overpexpressed XynG2 was purified. The molecular weight of XynG2 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 21,000. This was almost the same as the molecular weight of 20,047 calculated from the deduced amino acid sequence. The purified XynG2 showed an optimum activity at pH 6.0 and 58°C. It had a Km of 5.1 mg/ml and a Vmax of 123 μmol/min/mg when birch wood xylan was used as a substrate.