Reduction Of Xylose Research Articles

Continuous Xylitol Production from Synthetic Xylose Solutions by Candida guilliermondii: Influence of pH and Temperature

AbstractThe effects of pH and temperature on continuous xylitol production by Candida guilliermondii have been studied under semiaerobic conditions. The specific productivites obtained at different pH have been used in the model proposed by Villadsen et al. with the aim to get information on the enzyme likely limiting xylitol production (xylose reductase). The kinetic results obtained at different temperatures have been utilized to estimate the thermodynamic parameters either of the reduction of xylose to xylitol (ΔH# = 62 kJ mol–1; ΔS# = –0.12 kJ mol–1 K–1) or of the biocatalyst thermal inactivation (ΔHD° = 90 kJ mol–1; ΔSD° = 0.30 kJ mol–1 K–1). Finally, the well‐known empirical procedure of Sizer has been compared with the so‐called “thermodynamic approach” from the viewpoint of their ability to explain the physical meaning of the related thermodynamic parameters.

Effect of enhanced xylose reductase activity on xylose consumption and product distribution in xylose-fermenting recombinant Saccharomyces cerevisiae.

Recombinant Saccharomyces cerevisiae TMB3001, harboring the Pichia stipitis genes XYL1 and XYL2 (xylose reductase and xylitol dehydrogenase, respectively) and the endogenous XKS1(xylulokinase), can convert xylose to ethanol. About 30% of the consumed xylose, however, is excreted as xylitol. Enhanced ethanol yield has previously been achieved by disrupting the ZWF1 gene, encoding glucose-6-phosphate dehydrogenase, but at the expense of the xylose consumption. This is probably the result of reduced NADPH-mediated xylose reduction. In the present study, we increased the xylose reductase (XR) activity 4-19 times in both TMB3001 and the ZWF1-disrupted strain TMB3255. The xylose consumption rate increased by 70% in TMB3001 under oxygen-limited conditions. In the ZWF1-disrupted background, the increase in XR activity fully restored the xylose consumption rate. Maximal specific growth rates on glucose were lower in the ZWF1-disrupted strains, and the increased XR activity also negatively affected the growth rate in these strains. Addition of methionine resulted in 70% and 50% enhanced maximal specific growth rates for TMB3255 (zwfl Delta) and TMB3261 (PGK1-XYL1, zwf1 Delta), respectively. Enhanced XR activity did not have any negative effect on the maximal specific growth rate in the control strain. Enhanced glycerol yields were observed in the high-XR-activity strains. These are suggested to result from the observed reductase activity of the purified XR for dihydroxyacetone phosphate.

Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose.

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD(+). In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g(-1)) and the lowest xylitol yield (0.05 g g(-1)) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.

Mutational study of the role of tyrosine‐49 in the Saccharomyces cerevisiae xylose reductase

The xyl1 gene encoding xylose reductase was cloned from Saccharomyces cerevisiae and expressed in Escherichia coli. The purified enzyme readily carried out xylose reduction in vitro. It prefers NADPH as the co-enzyme by about 80-fold over NADH. Compared to the native enzyme purified from S. cerevisiae (Kuhn et al., 1995), the recombinant xylose reductase displayed slightly higher (about two-fold) affinities (Km) for the substrate (xylose) and co-factor (NADPH), as well as a 3.9-fold faster turnover number (Kcat) and 7.4-fold greater catalytic efficiency (Kcat/Km). The reason for the apparent discrepancies in kinetic constants between the recombinant and native S. cerevisiae xylose reductases is not known. Replacement of Tyr49 by Phe in the recombinant enzyme led to greater than 98% loss of activity, suggesting that this residue plays a critical role in catalysis. Intrinsic enzyme fluorescence spectroscopic analysis showed that the wild-type and the Y49F variant both bound the co-enzyme NADPH with similar affinity. This supports the view that Tyr49 is involved in interaction with the substrate and not the co-factor during catalysis. Copyright © 2001 John Wiley & Sons, Ltd.

Mutational study of the role of tyrosine‐49 in the Saccharomyces cerevisiae xylose reductase

The xyl1 gene encoding xylose reductase was cloned from Saccharomyces cerevisiae and expressed in Escherichia coli. The purified enzyme readily carried out xylose reduction in vitro. It prefers NADPH as the co-enzyme by about 80-fold over NADH. Compared to the native enzyme purified from S. cerevisiae (Kuhn et al., 1995), the recombinant xylose reductase displayed slightly higher (about two-fold) affinities (K(m)) for the substrate (xylose) and co-factor (NADPH), as well as a 3.9-fold faster turnover number (K(cat)) and 7.4-fold greater catalytic efficiency (K(cat)/K(m)). The reason for the apparent discrepancies in kinetic constants between the recombinant and native S. cerevisiae xylose reductases is not known. Replacement of Tyr49 by Phe in the recombinant enzyme led to greater than 98% loss of activity, suggesting that this residue plays a critical role in catalysis. Intrinsic enzyme fluorescence spectroscopic analysis showed that the wild-type and the Y49F variant both bound the co-enzyme NADPH with similar affinity. This supports the view that Tyr49 is involved in interaction with the substrate and not the co-factor during catalysis.

Xylitol Production by a Culture ofCandida guilliermondii2581

The yeast strain Candida guilliermondii2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose. The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed. The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm. It was shown that the growth under conditions of limited aeration favors the reduction of xylose.

Continuous enzymatic production of xylitol with simultaneous coenzyme regeneration in a charged membrane reactor

We have developed a new process for the production of xylitol from D-xylose by enzyme technology. An NADH-dependent xylose reductase (XR) from Candida tenuis catalyzes the reduction of xylose, which is coupled to enzymatic oxidations of D-glucose or D-xylose by glucose dehydrogenase from Bacillus cereus to make achievable an up to 10,000-fold regeneration of NADH per cycle of discontinuous conversion. Using a simple kinetic model as a tool for process optimization, suitable conditions with regard to performance and stability of the multi-component reaction system were established, and 300 g/L of substrate could be converted in yields above 96% in one single batch reaction. Due to selective and over 98% complete retention of the native coenzyme by negatively charged nanofiltration membranes used in a continuously operated enzyme reactor, a specific productivity of 80 g xylitol per liter, day, and kilounit of XR was maintained over the 150-h reaction time with only a single dosage of NADH. (c) 1996 John Wiley & Sons, Inc.

Xylose utilisation: cloning and characterisation of the Xylose reductase from Candida tenuis.

Xylose reductases catalyse the initial reaction in the xylose utilisation pathway, the NAD(P)H+H+ dependent reduction of xylose to xylitol. In this work, the xylose reductase gene from Candida tenuis CBS 4435 was cloned and successfully expressed in E. coli. From the purified and partially sequenced protein primers were deduced for PCR. The fragment obtained was used for Southern blot analysis and screening of a subgenomic library. The clone containing the open reading frame was sequenced; the gene consisted of 969 nucleotides coding for a 322 amino acids protein with a molecular mass of 36 kDa. Putative regulatory signals were identified with the help of a Saccharomyces cerevisiae regulatory sequence database. In order to express the xylose reductase in E. coli, the gene was placed under positive and negative control. At low temperatures, the xylose reductase was expressed in soluble and active form up to about 10% of the soluble protein; with rising temperatures formation of visible inclusion bodies occurred. In refolding experiments we were able to recover the major portion of xylose reductase activity from the pellet fraction.

A preliminary information about continuous fermentation using cell recycling for improving microbial xylitol production rates(scientific note).

Xylitol is a sugar-alcohol with important technological properties, such as anticariogenicity, low caloric value, and negative dissolution heat. It can be used successfully in food formulations and pharmaceutical industries. Its production is therefore in great demand. Biotechnological xylitol production has several economic advantages in comparison with the conventional process based on the chemical reduction of xylose. The efficiency and the productivity of this fermentation chiefly depends on the microorganism and the process conditions employed. In this article a simple continuous culture with cell recycling was evaluated to enhance the capability of Candida guilliermondii FTI 20037 to produce xylitol. The fermentation was initiated batchwise by directly inoculating the grown seed culture in a 2-L bench-scale fermentor. Continuous feeding was begun at a dilution rate (D) of 0.060/h after the xylose concentration had completely consumed and the cell concentration was about 4.0 g/L. At a dilution rate of 0.060/h the xylitol concentration was about 15 g/L and increased by about 35%, whereas the dilution rate decreased by about 58%. Furthermore, the volumetric productivity, Qp, markedly depended on the dilution rate, diminishing by about 37% as D was changed from 0.060 to 0.025/h. These preliminary results show us that continuous fermentation with cell recycling is a good way to study the xylitol production by xylose-fermenting yeasts.

Mutational analysis of the role of the conserved lysine-270 in the Pichia stipitis xylose reductase.

Xylose reductase catalyzes the NAD(P)H-dependent reduction of xylose to xylitol and is essential for growth on xylose by yeasts. To understand the nature of coenzyme binding to the Pichia stipitis xylose reductase, we investigated the role of the strictly conserved Lys270 in the putative IPKS coenzyme binding motif by site-directed mutagenesis. The Lys270Met variant exhibited lower enzyme activity than the wild-type enzyme. The apparent affinity of the variant for NADPH was decreased 5-16-fold, depending on the substrate used, while the apparent affinity for NADH, measured using glyceraldehyde as the substrate, remained unchanged. This resulted in 4.3-fold higher affinity for NADH over NADPH using glyceraldehyde as the substrate. The variant also showed a 14-fold decrease in Km for xylose, but only small changes were observed in Km values for glyceraldehyde. The wild-type enzyme, but not the Lys270Met variant, was susceptible to modification by the Lys-specific pyridoxal 5'-phosphate. Results of our chemical modification and site-directed mutagenesis study indicated that Lys270 is involved in both NADPH and D-xylose binding in the P. stipitis xylose reductase.

Mutational analysis of the role of the conserved lysine-270 in the Pichia stipitis xylose reductase

Xylose reductase catalyzes the NAD(P)H-dependent reduction of xylose to xylitol and is essential for growth on xylose by yeasts. To understand the nature of coenzyme binding to the Pichia stipitis xylose reductase, we investigated the role of the strictly conserved Lys270 in the putative IPKS coenzyme binding motif by site-directed mutagenesis. The Lys270Met variant exhibited lower enzyme activity than the wild-type enzyme. The apparent affinity of the variant for NADPH was decreased 5–16-fold, depending on the substrate used, while the apparent affinity for NADH, measured using glyceraldehyde as the substrate, remained unchanged. This resulted in 4.3-fold higher affinity for NADH over NADPH using glyceraldehyde as the substrate. The variant also showed a 14-fold decrease in K m for xylose, but only small changes were observed in K m values for glyceraldehyde. The wild-type enzyme, but not the Lys270Met variant, was susceptible to modification by the Lys-specific pyridoxal 5′-phosphate. Results of our chemical modification and site-directed mutagenesis study indicated that Lys270 is involved in both NADPH and d-xylose binding in the P. stipitis xylose reductase.

Production of Xylitol by Electrical Reduction of Xylose

Production of Xylitol by Electrical Reduction of Xylose

Production of Xylitol by Electrical Reduction of Xylose

Production of Xylitol by Electrical Reduction of Xylose

Influence of cosubstrate concentration on xylose conversion by recombinant, XYL1-expressing Saccharomyces cerevisiae: a comparison of different sugars and ethanol as cosubstrates.

Conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae expressing the XYL1 gene, encoding xylose reductase, was investigated by using different cosubstrates as generators of reduced cofactors. The effect of a pulse addition of the cosubstrate on xylose conversion in cosubstrate-limited fed-batch cultivation was studied. Glucose, mannose, and fructose, which are transported with high affinity by the same transport system as is xylose, inhibited xylose conversion by 99, 77, and 78%, respectively, reflecting competitive inhibition of xylose transport. Pulse addition of maltose, which is transported by a specific transport system, did not inhibit xylose conversion. Pulse addition of galactose, which is also transported by a specific transporter, inhibited xylose conversion by 51%, in accordance with noncompetitive inhibition between the galactose and glucose/ xylose transport systems. Pulse addition of ethanol inhibited xylose conversion by 15%, explained by inhibition of xylose transport through interference with the hydrophobic regions of the cell membrane. The xylitol yields on the different cosubstrates varied widely. Galactose gave the highest xylitol yield, 5.6 times higher than that for glucose. The difference in redox metabolism of glucose and galactose was suggested to enhance the availability of reduced cofactors for xylose reduction with galactose. The differences in xylitol yield observed between some of the other sugars may also reflect differences in redox metabolism. With all cosubstrates, the xylitol yield was higher under cosubstrate limitation than with cosubstrate excess.

Site-directed mutagenesis of the cysteine residues in the Pichia stipitis xylose reductase

Xylose reductase catalyzes the reduction of xylose to xylitol and is known to play a pivotal role in pentose metabolism in yeasts. We previously showed that a cysteine residue may be involved in binding of the coenzyme NADPH to the Pichia stipitis xylose reductase through chemical modification studies. The question arose as to which of the three cysteine residues in this enzyme may be involved in coenzyme binding. We cloned the XYL1 gene encoding xylose reductase from P. stipitis into the phagemid pEMBL18(+) suitable for site-directed mutagenesis. Each of the three cysteine residues (Cys 19, Cys 27 and Cys 130) was individually mutated to serine. All three Cys→Ser variants remained functional, but with reduced catalytic activity. Sensitivity of the P. stipitis xylose reductase to thiol-specific reagents was attributed to both Cys 27 and Cys 130 residues as substitution of either residue with Ser resulted in a significant but incomplete loss of sensitivity to PCMBS. The apparent K m values of the Cys variants for NADPH, NADH and xylose did not differ from those of the wild-type enzyme isolated from yeast by more than 4-fold. Our results suggest that none of the Cys residues are directly involved in NADPH binding, although Cys 130 may reside in or near the coenzyme binding region and might play a role in coenzyme specificity.

A heterologous reductase affects the redox balance of recombinant Saccharomyces cerevisiae

Recombinant Saccharomyces cerevisiae harbouring the xylose reductase (XR) gene XYL1 from Pichia stipitis was grown in anoxic chemostat culture at two different dilution rates. At each dilution rate a transient experiment, encompassing a shift in the sugar content of the medium from glucose to glucose plus xylose was performed. The steady states at the beginning and the end of the transients were compared in terms of specific product fluxes from glucose metabolism. At both dilution rates, the specific glycerol flux decreased and the specific acetate and CO2 fluxes increased. The specific ethanol flux was not affected. At the lower dilution rate, the production of biomass decreased during the transient, but at the higher dilution rate it increased. The changes in product pattern can be explained as being due to the redox perturbation caused by the consumption of reduced cofactors in the XR-catalysed reaction. Regeneration of NAD partly through xylose reduction instead of glycerol production decreased the formation of glycerol. Additionally, xylose reduction activated those pathways which produce reduced cofactors, such as acetate formation and the pentose phosphate pathway, indicated by increased acetate and CO2 production. The dual cofactor specificity of XR, with a preference for NADPH over NADH, was evident from the effects of xylose reduction on product fluxes. Comparison of the xylose reduction rates at low and high glucose flux indicated that the supply of reduced cofactors partly controlled the reaction rate. At the higher dilution rate, control by some other factor such as xylose transport or XR activity increased. Calculation of carbon balances at the steady states showed that all substrate carbon was recovered in biomass or products. Based on the specific product fluxes, calculations of quantitative cofactor balances at the steady states was attempted. However, sensitivity calculations showed that analysis errors in the range of 5% caused substantial errors in the cofactor balance, without affecting the carbon balance.

The reduction of xylose to xylitol by Candida guilliermondii and Candida parapsilosis: Incidence of oxygen and pH

The ability of C. guilliermondii and C. parapsilosis to ferment xylose to xylitol was evaluated under different oxygen transfer rates in order to enhance the xylitol yield. In C. guilliermondii, a maximal xylitol yield of 0.66 g/g was obtained when oxygen transfer rate was 2.2 mmol/l.h. Optimal conditions to produce xylitol by C. parapsilosis (0.75 g/g) arose from cultures at pH 4.75 with 0.4 mmoles of oxygen/l.h. The response of the yeasts to anaerobic conditions has shown that oxygen was required for xylose metabolism.

Galvanostatic electrochemical reduction of pentoses

Electrochemical reduction of ribose and xylose has been studied in sodium sulphate solution and in phosphate buffers on amalgamated lead cathodes under conditions of galvanostatic electrolysis. The products after peracetylation were determined by gas chromatography. Over the catholyte pH range 8–11 high yields of the pentitols were obtained (84 and 75% for ribose and xylose, respectively). Regardless of the pH, 2-deoxypentitols were also formed in low yields (a few percent). Raising the temperature of the reaction to 45°C intensified the reduction 2 to 3 fold and, with xylose, the yield of xylitol was markedly increased. Large differences were observed between pH readings in the bulk solution and at the cathode. In unbuffered solutions these differences enhanced the rate of reduction of the pentoses autocatalytically.

発酵生産プロセスの膜利用技術

Recent research progresses on membrane technologies to fermentation production processes are reviewed. First, a cross flow filtration of suspended yeast cells by a hollow fiber module was carried out to enhance the ethanol productivity in repeated fedbatch culture. The ethanol productivity obtained in this system was 11.3g ethanol/litre/h compared to 3 in a conventional Melle-Boinnot method. Second, in ethanol fermentation, an immobilized yeast cells system capable of forced permeation of the medium was developed in contrast to conventional immobilized yeast cell system where the mass transfer depends on “diffusion”. In this new system, the cell density on the surface of gel (1 mm thickness) became 1.7×1010 cells/ml gel compared to 4×109 in the conventional system. Hence, the ethanol productivity in this system could increase 60g ethanol/ litre gel/h compared to 20 to 30 in conventional packed bed systems. Third, acetic acid fermentaion from ethanol was conducted in a hollow fiber module bioreactor where the bacterial cells absorbed on the fiber surface could consume oxygen running through the inside of hollow fiber and ethanol running through the outside the fiber. Thus, a mimic vineger production of conventional surface cluture could be established with a remarkable increase of the surface area per reactor volume. Finally, a sulfonated polysulfone membrane was used for in situ regeneration and retention of coenzymes NADP (H) using xylose reductase coupled with oxidoreductase system in the reduction of xylose to xylitol with hydrogen gas. The membrane could almost completely reject the permeation of NADP (H) and F420 and the required enzymes, but not reject for permeation of xylitol.

Regeneration and retention of NADP (H) for xylitol production in an ionized membrane reactor

A sulfonated polysulfone membrane reactor was used forin situ regeneration and retention of coenzymes NADP (H) using the xylose reductase ofCandida pelliculosa coupled with oxidoreductase system ofMethanobacterium sp. in the reduction of xylose to xylitol with hydrogen gas. The membrane could almost completely reject the permeation of NADP (H) (92 and 97%), F420 (97%) and the required enzymes (100%), but not reject for the permeation of xylitol (product). After 4-h reaction for the production of xylitol from xylose (93% yield), although 25% NADP (H) initially added was lost its activity due to unavoidable degradation, the membrane could reject the permeation of the remaining NADP (H) and F420 at the level of 90 and 95%, respectively.