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Xylitol is the first rare sugar that has global markets. It has beneficial health properties and represents an alternative to current conventional sweeteners. Industrially, xylitol is produced by chemical hydrogenation of d-xylose into xylitol. The biotechnological method of producing xylitol by metabolically engineered yeasts, Saccharomyces cerevisiae or Candida, has been studied as an alternative to the chemical method. Due to the industrial scale of production, xylitol serves as an inexpensive starting material for the production of other rare sugars. The second part of this mini-review on xylitol will look more closely at the biotechnological production and future applications of the rare sugar, xylitol. [ABSTRACT FROM AUTHOR]

The rare sugar xylitol is a five-carbon polyol (pentitol) that has beneficial health effects. Xylitol has global markets and, therefore, it represents an alternative to current dominant sweeteners. The research on microbial reduction of d-xylose to xylitol has been focused on metabolically engineered Saccharomycess cerevisiae and Candida strains. The Candida strains have an advantage over the metabolically engineered S. cerevisiae in terms of d-xylose uptake and maintenance of the intracellular redox balance. Due to the current industrial scale production of xylitol, it has become an inexpensive starting material for the production of other rare sugar. The first part of this mini-review concentrates on the biochemistry of xylitol biosynthesis and the problems related to intracellular redox balance. [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]

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. [Copyright &y& Elsevier]

We have studied the metabolism of xylose by Candida tropicalis in oxygen-limited chemostat. In vitro enzyme assays indicated that glycolytic and gluconeogenetic enzymes are expressed simultaneously facilitating substrate cycling. Enhancing the redox imbalance by cofeeding of formate increased xylose and oxygen consumption rates and ethanol, xylitol, glycerol and CO2 production rates at steady state. Metabolic flux analysis (MFA) indicated that fructose 6-phosphate is replenished from the pentose phosphate pathway in sufficient amounts without contribution of the gluconeogenetic pathway. Substrate cycling between pyruvate kinase, pyruvate carboxylase and phospho-enol-pyruvate kinase increased ATP turnover. Cofeeding of formate increased the ATP yield. The ATP yields of xylose and xylose–formate cultivation were 6.9 and 8.7 mol ATP/C-mol CDW, respectively, as calculated from the MFA. [Copyright &y& Elsevier]

Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol dehydrogenase with oxidized carbohydrates was also confirmed by fluorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase. [ABSTRACT FROM AUTHOR]

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]

The study was focused on developing a continuous method to produce an alcohol mixture suitable to be used as a gasoline supplement. The immobilized column reactor with wood pulp fibers was successfully used for the continuous production of butanol and isopropanol using Clostridium beijerinckii DSM 6423. A sugar mixture (glucose, mannose, galactose, arabinose and xylose) representing lignocellulose hydrolysate was used as a substrate for the production of solvents. The effect of dilution rate on solvent production was studied during continuous operation. The maximum total solvent concentration of 11.99 g/l was obtained at a dilution rate of 0.16 h. The maximum solvent productivity (5.58 g/l h) was obtained at a dilution rate of 1.5 h. The maximum solvent yield of 0.45 g/g from sugar mixture was observed at 0.25 h. The system will be further used for the solvent production using wood hydrolysate as a substrate. [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 biomass obtained after the extraction of forskolin from the roots of Coleus forskohlii was evaluated as a substrate for the production of acetone-butanol-ethanol (ABE). The spent biomass constituting more than 90% of the raw material showed 50–70% carbohydrates with starch and cellulose being the major constituents. This study was undertaken to optimize enzymatic hydrolysis of C. forskohlii roots for maximum release of fermentable sugars and subsequent fermentation to ABE. The root biomass was hydrolyzed using the Stargen ® 002 and Accellerase ® 1500. Cocktail of both enzymes (16U Stargen ® 002 and 60 FPU Accellerase ® 1500) could produce 41.2 g/l of total reducing sugars (glucose equivalent to 32.33 g/l). The production of ABE was optimized in a batch fermentation using Clostridium acetobutylicum NCIM 2877. The maximum ABE production using the root hydrolysates was 0.55 g/l. Pretreatment with lime and Amberlite XAD-4 increased the production of total solvent to 5.33 g/l. [ABSTRACT FROM AUTHOR]

Acetone being a non-fuel solvent produced during the traditional acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum ; reduces the overall fuel alcohol yield. However, the conversion of acetone into isopropanol has been recommended to improve the process economy. The present study aims to develop an engineered C. acetobutylicum DSM 792 strain to convert acetone into isopropanol by introducing the secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B593 using the allele-coupled exchange approach. Batch and continuous fermentation experiments were carried out with a modified strain C. acetobutylicum DSM 792-ADH to improve the isopropanol yield and titer. The growth and production behavior of the modified strain in stationary flask culture and controlled batch culture was studied. Almost 50% of acetone was converted into isopropanol with highest total solvent yield to be 0.39 g g −1 glucose. The modified strain also utilized sugar mixture and SO 2 –ethanol–water spent liquor as a substrate to produce the solvents. [ABSTRACT FROM AUTHOR]

The co-production of acetone during traditional butanol fermentation (acetone–butanol–ethanol) lowers the yield of alcohol biofuels. The present study was aimed to develop an improved Clostridium acetobutylicum strain with enhanced alcohol fuel production capability. The previously developed IBE (isopropanol-butanol-ethanol) producing C. acetobutylicum DSM792-ADH strain was further optimized to enhance the solvent productivity and yield. Immobilized cell column reactor was used to investigate the effect of dilution rate on IBE fermentation. Pure glucose, artificial sugar mixture and SEW spent liquor from spruce chips were used as substrates to study the behavior of the modified microorganism. The highest total solvent concentrations of approximately 10.60 g l −1 , 10 g l −1 and 6 g l −1 were obtained when glucose, sugar mixture and SEW spent liquor were used as substrates respectively. The modified microorganism could effectively utilize the lignocellulosic biomass hydrolyzate (SEW spent liquor) to yield a solvent productivity of 1.67 g l −1 h −1 . [ABSTRACT FROM AUTHOR]

GH10 xylanase from Thermoascus aurantiacus strain SL16W (TasXyn10A) showed high stability and activity up to 70-75 °C. The enzyme's half-lives were 101 h, 65 h, 63 min and 6 min at 60, 70, 75 and 80 °C, respectively. The melting point ( T), as measured by DSC, was 78.5 °C, which is in line with a strong activity decrease at 75-80 °C. The biomass-dissolving ionic liquid 1-ethyl-3-methylimidazolium acetate ([emim]OAc) in 30 % concentration had a small effect on the stability of TasXyn10A; T decreased by only 5 °C. It was also observed that [emim]OAc inhibited much less GH10 xylanase (TasXyn10A) than the studied GH11 xylanases. The K of TasXyn10A increased 3.5-fold in 15 % [emim]OAc with xylan as the substrate, whereas the approximate level of V was not altered. The inhibition of enzyme activity by [emim]OAc was lesser at higher substrate concentrations. Therefore, high solid concentrations in industrial conditions may mitigate the inhibition of enzyme activity by ionic liquids. Molecular docking experiments indicated that the [emim] cation has major binding sites near the catalytic residues but in lower amounts in GH10 than in GH11 xylanases. Therefore, [emim] cation likely competes with the substrate when binding to the active site. The docking results indicated why the effect is lower in GH10. [ABSTRACT FROM AUTHOR]

Highlights: [•] Carbohydrate profiling of Coleus forskohlii root. [•] Utilization of Coleus forskohlii root biomass for bioethanol production. [•] Immobilization of yeast on wood chips. [•] Continuous lignocellulosic fermentation to produce bioethanol. [ABSTRACT FROM AUTHOR]

Highlights: [•] Study suggests the preference of biomass feedstocks appropriate for ABE-fermentation. [•] Effect of varying lignocellulosic sugar concentrations in continuous column reactor. [•] Multi linear regression (MLR) modeling successfully used for continuous production of solvents. [•] Scale up of column reactor is successfully demonstrated. [Copyright &y& Elsevier]

Highlights: [•] Oil palm empty fruit bunches (OPEFB) are fractionated by SO2–ethanol–water mixture. [•] Fractionation of OPEFB feedstock produces cellulosic fibers and spent liquor. [•] Spent liquor contains dissolved hemicellulose sugars (mostly monomers) and lignin. [•] Fractionation and sugar hydrolysis is hindered by the high cation content of OPEFB. [•] Acidic leaching of OPEFB only partly removes cations; more intense deashing needed. [Copyright &y& Elsevier]

Highlights: [•] Advanced and economical production of biobutanol. [•] Continuous two stage fermentation with integrated liquid–liquid recovery technique. [•] Utilization of lignocellulosic sugar mixture (pentoses and hexoses). [•] Long run system operation with high solvent productivity (10.85g/Lh) and yield. [•] Can be integrated to the wood based biorefinery to make it industrially feasible. [ABSTRACT FROM AUTHOR]

Abstract: A process for the fermentative production of propionic acid from whole wheat flour using starch and gluten as nutrients is presented. Hydrolysis of wheat flour starch using amylases was optimized. A batch fermentation of hydrolysate supplemented with various nitrogen sources using Propionibacterium acidipropionici NRRL B 3569 was performed. The maximum production of 48.61, 9.40, and 11.06g of propionic acid, acetic acid and succinic acid, respectively, was found with wheat flour hydrolysate equivalent to 90g/l glucose and supplemented with 15g/l yeast extract. Further, replacement of yeast extract with wheat gluten hydrolysate showed utilization of gluten hydrolysate without compromising the yields and also improving the economics of the process. The process so developed could be useful for production of animal feed from whole wheat with in situ production of preservatives, and also suggest utilization of sprouted or germinated wheat for the production of organic acids. [Copyright &y& Elsevier]

Abstract: The present study proposes the use of market rejected and waste vegetables as a supplement for improved acetone–ethanol–butanol (ABE) solvent production. In the present study, three different perishable vegetables were evaluated as supplements for ABE fermentation. These vegetables were found to stimulate the solvent production at as low as 1% (w/v) concentration. The supplementation showed improved production of solvents using 4-fold diluted SO2–ethanol–water (SEW) spent liquor supplemented with 25g/l glucose as a substrate. The maximum total solvent production obtained with 5% (w/v) supplementation of carrot, cabbage and tomato was 9.96g/l, 10.49g/l and 10.65g/l, respectively as compared to the control value of 7.43g/l. The applicability of vegetables was successfully evaluated as a complex medium for solvent production. [Copyright &y& Elsevier]

Abstract: The objective of this study was to optimize continuous acetone–butanol–ethanol (ABE) fermentation using a two stage chemostat system integrated with liquid–liquid extraction of solvents produced in the first stage. This minimized end product inhibition by butanol and subsequently enhanced glucose utilization and solvent production in continuous cultures of Clostridium acetobutylicum B 5313. During continuous two-stage ABE fermentation, sugarcane bagasse was used as the cell holding material for the both stages and liquid–liquid extraction was performed using an oleyl alcohol and decanol mixture. An overall solvent production of 25.32g/L (acetone 5.93g/L, butanol 16.90g/L and ethanol 2.48g/L) was observed as compared to 15.98g/L in the single stage chemostat with highest solvent productivity and solvent yield of 2.5g/Lh and of 0.35g/g, respectively. Maximum glucose utilization (83.21%) at a dilution rate of 0.051/h was observed as compared to 54.38% in the single stage chemostat. [Copyright &y& Elsevier]

Abstract: SO2–ethanol–water (SEW) spent liquor from spruce chips was successfully used for batch and continuous production of acetone, butanol and ethanol (ABE). Initially, batch experiments were performed using spent liquor to check the suitability for production of ABE. Maximum concentration of total ABE was found to be 8.79g/l using 4-fold diluted SEW liquor supplemented with 35g/l of glucose. The effect of dilution rate on solvent production, productivity and yield was studied in column reactor consisting of immobilized Clostridium acetobutylicum DSM 792 on wood pulp. Total solvent concentration of 12g/l was obtained at a dilution rate of 0.21h−1. The maximum solvent productivity (4.86g/lh) with yield of 0.27g/g was obtained at dilution rate of 0.64h−1. Further, to increase the solvent yield, the unutilized sugars were subjected to batch fermentation. [Copyright &y& Elsevier]

Clostridium beijerinckii DSM 6423 was studied using different continuous production methods to give maximum and stable production of isopropanol and n-butanol. In a single-stage continuous culture, when wood pulp was added as a cell holding material, we could increase the solvent productivity from 0.47 to 5.52 g L h with the yield of 54% from glucose. The overall solvent concentration of 7.51 g L (39.4% isopropanol and 60.6% n-butanol) with the maximum solvent productivity of 0.84 g L h was obtained with two-stage continuous culture. We were able to run the process for more than 48 overall retention times without losing the ability to produce solvents. [ABSTRACT FROM AUTHOR]

The objective of this study is to develop a process for conditioning spent liquor produced by SO2-ethanol-water (SEW) fractionation of spruce wood chips for fermentation to butanol, ethanol and acetone/isopropanol, i.e., by means of the so called acetone-butanol-ethanol (ABE) process using Clostridia bacteria. This study serves as part of an overall project aiming at the development of economic processes for producing chemicals and biofuels from mixed forest biomass via SEW fractionation and ABE fermentation technologies. [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: 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]

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°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 13C-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. [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]

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. [Copyright &y& Elsevier]

Escherichia coli strain JM 109 harboring 6× His-tag l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri allowed a 20-fold increase in the volumetric yield of soluble enzyme compared to the value for the intrinsic yield. Detailed studies on the substrate specificity of the purified His-tagged protein revealed that it catalyzed previously unknown common and rare aldo/ketotetrose, aldo/ketopentose, and aldo/ketohexose substrates in both d- and l-forms, for instance, erythrose, threose, xylose, lyxose, ribose, glucose, mannose, galactose, altrose, tagatose, sorbose, psicose, and fructose. Using a high enzyme–substrate ratio in extended reactions, the enzyme-catalyzed interconversion reactions from which two different products from one substrate were formed: l-lyxose, l-glucose, l-tagatose and d-allose were isomerized to l-xylulose and l-xylose, l-fructose and l-mannose, l-galactose and l-talose, and d-psicose and d-altrose, in that order. Kinetic studies, however, showed that l-rhamnose with Km and Vmax values of 11 mM and 240 U/mg, respectively, was the most preferred substrate, followed by l-mannose, l-lyxose, d-ribose, and d-allose. Based on the observed catalytic mode of action, these new findings reflected a hitherto undetected interrelation between l-RhI and d-xylose isomerase (d-XI). [Copyright &y& Elsevier]

Wild-type strain of Pseudomonas cichorii ST-24 was unable to grow on D-psicose and inductively produced D-tagatose 3-epimerase (D-TE) with D-tagatose as an inducer. We have isolated a constitutive mutant, designated strain Ka75, which had acquired a new ability to grow on a mineral salts medium containing D-psicose as a sole carbon source. The D-psicose-metabolizing mutant synthesized a high level of D-TE. When grown on the culture medium supplemented with Mn2+, the mutant strain produced around 250-fold higher activity than did the parent strain. Enzymatic properties of the constitutive enzyme were similar to those of the wild-type. Using the immobilized D-TE and recombinant L-rhamnose isomerase (L-RhI) from Escherichia coli strain JM109, a two-step enzymatic reaction was performed for massproduction of a rare aldo-hexose monosaccharide, L-galactose, from a common one, L-sorbose. In the first step, L-sorbose was epimerized to L-tagatose in a yield of 28%. The L-tagatose obtained was utilized as a starting material for L-galactose preparation by the immobilized L-RhI. At equilibrium, approximately 30% L-tagatose was isomerized to L-galactose. Finally, 7.5 g of L-galactose was obtained from 100 g of L-sorbose, viz an overall yield of 7.5%. The product obtained was purified and identified to be L-galactose by specific optical rotation and high performance liquid chromatography (HPLC) analysis, and was ultimately confirmed by 13C nuclear magnetic resonance (13C NMR) and IR spectra. [Copyright &y& Elsevier]

• ABE solventogenesis with reducing agents and controlled feeding regimen. • L-cysteine and DTT fed-batches achieved solvent yield of 0.38 and 0.37 g/g. • 24.33 and 22.98 g/L of ABE produced by L-cysteine and DTT fed-batches. • Enhanced solventogenesis led to higher ATP levels in DTT and L-cysteine batches. • Histidine secretion is important in Clostridial acid stress. Acetone-butanol-ethanol (ABE) fermentation process is a promising bioenergy option amid rising concerns over the environmental impact of fossil fuel usage. However, the commercialization of the ABE process has been marred by challenges of low product yield and titer, thereby non-competitive process economics. Here, we coupled cost competitive reducing agents with a controlled feeding strategy to improve both product titer and yield. Reducing agents promote cofactor dependent butanol production, while fed-batch operation enhances glucose consumption, final ABE titer, and partly mitigates product toxicity. The effects of ascorbic acid, L-cysteine, and dithiothreitol (DTT) on ABE fed-batch production using Clostridium acetobutylicum was investigated in current study. NADH, ATP, extracellular amino acid secretion, and NADH-dependent butanol dehydrogenase (BDH) assays were performed to study the metabolic modifications triggered by reducing agents. Incidentally, L-cysteine and DTT improved ABE solvent titer by 2-fold, producing 24.33 and 22.98 g/L ABE with solvent yields of 0.38 and 0.37 g/g, respectively. Elevated NADH, BDH, and ATP levels in fermentation broth reflected in enhanced ABE titer and yield. Furthermore, histidine secretion emerged as an important factor in Clostridial acid stress tolerance in this study. The results demonstrate that addition of reducing agents in fed-batch ABE fermentation operation enables efficient utilization of glucose with significant improvement in solvent production. [ABSTRACT FROM AUTHOR]