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.

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, expression, and transcription analysis of L-arabinose isomerase gene from Mycobacterium smegmatis SMDU.

The L-arabinose metabolic gene cluster, araA, araB, araD, araG, araH and araR, encoding L-arabinose isomerase (L-AI) and its accessory proteins was cloned from Mycobacterium smegmatis SMDU and sequenced. The deduced amino acid sequence of araA displayed 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. coli suggests that the transcriptional regulation of araA from M. smegmatis against 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.

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 degrees 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.

Efficient conversion of allitol to D-psicose by Bacillus pallidus Y25.

An efficient method for conversion of allitol to D-psicose was achieved by a resting cell reaction of Bacillus pallidus Y25 for the first time. Notably, it was possible to produce D-allose and D-altrose from allitol directly via D-psicose by prolonging the reaction time. This method was applied for the preparation of D-psicose using the extract of Itea virginica as a starting material in this study. D-Psicose which is the absolutely key precursor for the production of other six carbon sugars could be obtained as the sole product at high yield.

Novel substrate specificity of D-arabinose isomerase from Klebsiella pneumoniae and its application to production of D-altrose from D-psicose.

d-Arabinose isomerase from Klebsiella pneumoniae 40bXX was purified 12-fold with a 62.5% yield indicated by its electrophoretic homogeneity. The purified enzyme showed the highest activities toward d-arabinose and l-fucose as substrates at optimum conditions (50 mM glycine-NaOH, pH 9.0, 40 degrees C). The enzyme had a broad range of substrate specificities toward various d/l-aldoses, i.e., d-arabinose, l-fucose, d/l-xylose, d-mannose, d/l-lyxose, l-glucose, d-altrose and d/l-galactose. The equilibrium ratios between d-arabinose and d-ribulose, l-fucose and l-fuculose, d-altrose and d-psicose, and l-galactose and l-tagatose were 90:10, 90:10, 13:87 and 25:75, respectively. Using a combination of the immobilized d-tagatose 3-epimerase and d-arabinose isomerase, we achieved the production of d-altrose from d-fructose in a batch reactor. We successfully produced approximately 12 g of d-altrose from 200 g of d-fructose in a reaction series with an overall yield of 6%. The product obtained was confirmed to be d-altrose by HPLC and (13)C-NMR. To the best of our knowledge, this is the first report on the production of d-altrose from a cheap sugar, d-fructose, using an enzymatic method.

Efficient biosynthesis of D-allose from D-psicose by cross-linked recombinant L-rhamnose isomerase: separation of product by ethanol crystallization.

Mass production of a rare aldohexose D-allose from D-psicose was achieved in a batch reaction by crude recombinant L-rhamnose isomerase (L-RhI) cross-linked with glutaraldehyde. The D-psicose substrate was, in turn, mass produced from a naturally abundant ketohexose D-fructose by immobilized recombinant D-tagatose 3-epimerase (D-TE). At an equilibrium state, 25% of D-psicose was isomerized to D-allose, that is, 25 g of D-allose was obtained from 100 g of D-psicose. The D-allose product was easily separated and crystallized from the reaction mixture that contains 25%D-allose, 8%D-altrose and 67%D-psicose using ethanol. Empirically, approximately 338 g, that is, 90% of a theoretical overall yield for the purification of pure D-allose crystals was produced from 1.5 kg of D-psicose within 30 d using a constructed bioreactor. The cross-linked enzyme had an operative half-life of two months after repeated usages.

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.