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Non-covalent interaction of alcohol dehydrogenase with polysaccharides was studied using three neutral and three anionic polysaccharides. The process of interaction of alcohol dehydrogenase with gum Arabic was optimized with respect to the ratio of enzyme to gum Arabic, pH, and molarity of buffer. Alcohol dehydrogenase-gum Arabic complex formed under optimized conditions showed 93 % retention of original activity with enhanced thermal and pH stability. Lower inactivation rate constant of alcohol dehydrogenase-gum Arabic complex within the temperature range of 45 to 60 °C implied its better stability. Half-life of alcohol dehydrogenase-gum Arabic complex was higher than that of free alcohol dehydrogenase. A slight increment was observed in kinetic constants ( K and V) of gum Arabic-complexed alcohol dehydrogenase which may be due to interference by gum Arabic for the binding of substrate to the enzyme. Helix to turn conversion was observed in complexed alcohol dehydrogenase as compared to free alcohol dehydrogenase which may be responsible for observed stability enhancement. [ABSTRACT FROM AUTHOR]

Continuous production of acetone, n-butanol, and ethanol (ABE) was carried out using immobilized cells of Clostridium acetobutylicum DSM 792 using glucose and sugar mixture as a substrate. Among various lignocellulosic materials screened as a support matrix, coconut fibers and wood pulp fibers were found to be promising in batch experiments. With a motive of promoting wood-based bio-refinery concept, wood pulp was used as a cell holding material. Glucose and sugar mixture (glucose, mannose, galactose, arabinose, and xylose) comparable to lignocellulose hydrolysate was used as a substrate for continuous production of ABE. We report the best solvent productivity among wild-type strains using column reactor. The maximum total solvent concentration of 14.32 g L was obtained at a dilution rate of 0.22 h with glucose as a substrate compared to 12.64 g L at 0.5 h dilution rate with sugar mixture. The maximum solvent productivity (13.66 g L h) was obtained at a dilution rate of 1.9 h with glucose as a substrate whereas solvent productivity (12.14 g L h) was obtained at a dilution rate of 1.5 h with sugar mixture. The immobilized column reactor with wood pulp can become an efficient technology to be integrated with existing pulp mills to convert them into wood-based bio-refineries. [ABSTRACT FROM AUTHOR]

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 6×His 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°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60°C for 60 min. The apparent affinity ( K m) and catalytic efficiency ( k cat/ K m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 105 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°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. [ABSTRACT FROM AUTHOR]

Abstract: Xylitol was used as a raw material for production of l-xylose and l-lyxose using Alcaligenes 701B strain and immobilized l-rhamnose isomerase enzyme. Alcaligenes 701B converted xylitol to l-xylulose with a yield of 34% in the bioreactor. l-Xylulose was converted to l-xylose and l-lyxose using immobilized l-rhamnose isomerase enzyme. The final equilibrium between l-xylulose, l-xylose and l-lyxose was 53:26:21. The enzyme assays indicated that Alcaligenes 701B strain has an NAD-dependent xylitol dehydrogenase enzyme responsible for l-xylulose production. Furthermore, NAD(P)H-dependent l-xylulose reductase enzyme was active during conversion of xylitol to l-xylulose. The highest l-xylulose production rate corresponded with the highest growth rate. The Alcaligenes 701B strain used d-xylose for biomass growth, but xylitol was used only for l-xylulose production during conversion phase. [Copyright &y& Elsevier]

Candida guilliermondii grew on the d- and l-isomers of arabinose, xylose, ribose and lyxose. Xylose reductase was extracted from xylose-grown cells and the enzyme was purified to homogeneity. The purified enzyme used NADPH as a cosubstrate, had a molecular weight of 36 000 Da and pI of 4.5. It reduced the d- and l-forms of all the above pentoses. [ABSTRACT FROM AUTHOR]

Abstract: A novel facultative thermophilic bacterium capable of producing rare ketoses from polyols was isolated from soil and identified as Bacillus pallidus Y25. We have employed this isolated strain for the production of l-xylulose, a rare ketopentose, from xylitol by a resting cell reaction at 50°C. After optimization of culture conditions and reaction states, l-xylulose was produced at up to 85% yield when 2% xylitol was utilized. The key enzyme responsible for the interconversion of xylitol and l-xylulose was purified to homogeneity and identified as NAD-dependent xylitol dehydrogenase (XDH-Y25), with a molecular mass of 28kDa as determined by SDS-PAGE. XDH-Y25 is most active in glycine–NaOH buffer (pH 10) at 40°C and its substrate specificity is restricted to d-threitol, xylitol and d-iditol. This suggested that the enzyme is specific for polyols that have a hydroxyl group at the C-2 and C-3 positions in the l- and d-sides, respectively, in the Fischer projection. N-terminal amino acid sequencing of XDH-Y25 revealed low homology to any known dehydrogenases. To our knowledge, this is the first report on the characterization of xylitol dehydrogenase from a Bacillus species. [Copyright &y& Elsevier]

Abstract: l-Rhamnose isomerase (l-RhI) from Pseudomonas stutzeri LL172 can convert d-psicose to d-allose. Partially purified recombinant l-RhI from Escherichia coli was immobilized on BCW-2510 Chitopearl beads and utilized to produce d-allose. Total 20,000 units of immobilized enzyme converted d-psicose to d-allose without remarkable decrease in the enzyme activity over 17 days. When 50% d-psicose (w/w) was applied to a column with a flow rate of 0.8ml/min at 42°C, approximately 30% d-psicose was isomerized to d-allose for 17 days. However, by reducing the flow rate to 0.4ml/min after 17 days, d-allose was transformed at the same rate for 13 days. The total of 27l reaction mixture was separated by Simulated-Moving-Bed Chromatograph system. Approximately 2.2l/d of 50% (w/w) reaction mixture was separated continuously. After separation, d-allose and d-psicose fractions were 3l of approximately 10% (w/w) with 95% purity and 10l of approximately 8% (w/w) with 95% purity per day, respectively. The separated d-allose solution was concentrated up to about 50% and crystallized gradually by being kept at room temperature. Crystals of d-allose were separated from the syrup by filtration and 1.65kg crystals of 100% purity were obtained. The d-allose crystal yield from the d-psicose substrate was approximately 10%. [Copyright &y& Elsevier]