Bioconversion Of Xylose Research Articles

Research progress in the biosynthesis of xylitol: feedstock evolution from xylose to glucose.

Xylitol, as an important food additive and fine chemical, has a wide range of applications, including food, medicine, chemical, and feed. This review paper focuses on the research progress of xylitol biosynthesis, from overcoming the limitations of traditional chemical hydrogenation and xylose bioconversion, to the full biosynthesis of xylitol production using green and non-polluting glucose as substrate. In the review, the molecular strategies of wild strains to increase xylitol yield, as well as the optimization strategies and metabolic reconfiguration during xylitol biosynthesis are discussed. Subsequently, on the basis of existing studies, the paper further discusses the current status of research and future perspectives of xylitol production using glucose as a single substrate. The evolution of raw materials from xylose-based five-carbon sugars to glucose is not only cost-saving, but also safe and environmentally friendly, which brings new opportunities for the green industrial chain of xylitol.

Yeast as a cell factory for fermentative production of ethanol from xylose

BackgroundXylose is the most plentiful pentose sugar found in the lignocellulosic biomass in hemicellulose fractions. This pentose sugar has high potential to use as raw material for bioethanol production, as hemicellulose fractions account for 20–30 % in the lignocellulose biomass. However, bioconversion of xylose into ethanol is challenging for industrial prospective a sonly few microbes encompass the pentose fermentation pathway. MethodsBioconversion of xylose into bioethanol using yeasts is preferred since these organisms are more resistant and have higher ethanol tolerance limit compared to bacteria. During fermentation process xylose is not readily consumed by the microbes like glucose. Metabolic engineering has so far been unsuccessful to furnish adequate outcomes; and one of the reasons of this is the lack of consideration of a range of environmental factors which play potential role fermentative ethanol production form xylose. Significant FindingsThis review focuses on the bioethanol production from xylose highlighting the metabolic engineering segment, when microbes enter into fermenting phase. On this basis, the best possible technology can be evolved for subsequent commercialization of the product.

Sortase E-mediated site-specific immobilization of green fluorescent protein and xylose dehydrogenase on gold nanoparticles

Sortase, a bacterial transpeptidase enzyme, is an attractive tool for protein engineering due to its ability to break a peptide bond at a specific site and then reform a new bond with an incoming nucleophile. Here, we present the immobilization of two recombinant proteins, enhanced green fluorescent protein (eGFP) and xylose dehydrogenase (XylB) over triglycine functionalized PEGylated gold nanoparticles (AuNPs) using C. glutamicum sortase E. For the first time, we used a new class of sortase from a non-pathogenic organism for sortagging. The site-specific conjugation of proteins with LAHTG-tagged sequences on AuNPs via covalent cross-linking was successfully detected by surface-enhanced Raman scattering (SERS) and UV–vis spectral analysis. The sortagging was initially validated by an eGFP model protein and later with the xylose dehydrogenase enzyme. The catalytic activity, stability, and reusability of the immobilized XylB were studied with the bioconversion of xylose to xylonic acid. When compared to the free enzyme, the immobilized XylB was able to retain 80% of its initial activity after four sequential cycles and exhibited no significant variations in instability after each cycle for about 72 h. These findings suggest that C. glutamicum sortase could be useful for immobilizing site-specific proteins/enzymes in biotransformation applications for value-added chemical production.

Big data mining, rational modification, and ancestral sequence reconstruction inferred multiple xylose isomerases for biorefinery.

The isomerization of xylose to xylulose is considered the most promising approach to initiate xylose bioconversion. Here, phylogeny-guided big data mining, rational modification, and ancestral sequence reconstruction strategies were implemented to explore new active xylose isomerases (XIs) for Saccharomyces cerevisiae. Significantly, 13 new active XIs for S. cerevisiae were mined or artificially created. Moreover, the importance of the amino-terminal fragment for maintaining basic XI activity was demonstrated. With the mined XIs, four efficient xylose-utilizing S. cerevisiae were constructed and evolved, among which the strain S. cerevisiae CRD5HS contributed to ethanol titers as high as 85.95 and 94.76 g/liter from pretreated corn stover and corn cob, respectively, without detoxifying or washing pretreated biomass. Potential genetic targets obtained from adaptive laboratory evolution were further analyzed by sequencing the high-performance strains. The combined XI mining methods described here provide practical references for mining other scarce and valuable enzymes.

A smart self-balancing biosystem with reversible competitive adsorption of in-situ anion exchange resin for whole-cell catalysis preparation of lignocellulosic xylonic acid

Xylonic acid (XA) bioproduction via whole-cell catalysis of Gluconobacter oxydans is a promising strategy for xylose bioconversion, which is hindered by inhibitor formation during lignocellulosic hydrolysates. Therefore, it is important to develop a catalytic system that can directly utilize hydrolysate and efficiently produce XA. Determination of the dynamic adsorption characteristics of 335 anion exchange resin resulted in a unique and interesting reversible competitive adsorption between acetic acid-like bioinhibitor, fermentable sugar and XA. Xylose in crude lignocellulosic hydrolysates was completely oxidized to 52.52 g/L XA in unprecedented self-balancing biological system through reversible competition. The obtained results showed that in-situ resin adsorption significantly affected the direct utilization of crude lignocellulosic hydrolysate for XA bioproduction (p ≤ 0.05). In addition, the resin adsorbed ca. 90 % of XA during bioconversion. The study achieved a multiple functions and integrated system, “detoxification, neutralization and product separation” for one-pot bioreaction of lignocellulosic hydrolysate.

Xylose Dehydrogenase Immobilized on Ferromagnetic Nanoparticles for Bioconversion of Xylose to Xylonic Acid.

d-Xylonic acid (XA), derived from pentose sugar xylose, is a multifunctional high-value chemical with a wide range of applications in the fields of medicines, food, agriculture and is a valuable chemical reagent for the synthesis of other useful commodity chemicals. In the bacterial system, xylose dehydrogenase (XDH) catalyzes the oxidation of d-xylose into d-xylonolactone, consuming NAD+ or NADP+ as a cofactor. The d-xylonolactone then undergoes auto-oxidation into d-xylonic acid. Herein, the XDH enzyme overexpressed in Escherichia coli is purified and immobilized on ferromagnetic nanoparticles, effectively converting xylose into xylonic acid. Parameters deciding the bioconversion were statistically optimized and obtained a maximum of 91% conversion rate. Kinetic parameters of immobilized xylose dehydrogenase showed a 2-fold increase in the maximum velocity of the reaction and catalytic efficiency compared to free enzyme. The operational stability test for the enzyme-nanoparticle conjugate retained 93% relative activity after 10 successive experiments, exhibiting the good recyclability of the biocatalyst for XA production.

Enzymatic membrane reactor in xylose bioconversion with simultaneous cofactor regeneration

In this study, we present the concept of co-immobilization of xylose dehydrogenase and alcohol dehydrogenase from Saccharomyces cerevisiae on an XN45 nanofiltration membrane for application in the process of xylose bioconversion to xylonic acid with simultaneous cofactor regeneration and membrane separation of reaction products. During the research, the effectiveness of the co-immobilization of enzymes was confirmed, and changes in the properties of the membrane after the processes were determined. Using the obtained biocatalytic system it was possible to obtain 99% xylonic acid production efficiency under optimal conditions, which were 5 mM xylose, 5 mM formaldehyde, ratio of NAD+:NADH 1:1, and 60 min of reaction. Additionally, the co-immobilization of enzymes made it possible to improve stability of the co-immobilized enzymes and to carry out xylose conversion in six consecutive cycles and after 7 days of storage at 4 °C with over 90% efficiency. The presented data confirm the effectiveness of the co-immobilization, improvement of the stability and reusability of the biocatalysts, and show that the obtained enzymatic system is promising for use in xylose bioconversion and simultaneous regeneration of nicotinamide cofactor.

Effects of agitation rate and dissolved oxygen on xylose reductase activity during xylitol production at bioreactor scale

The study of xylose reductase (XR) - one of the key enzymes in the production of xylitol - is important in the fermentation process to have maximum efficiency in the bioconversion of xylose to xylitol in lignocellulosic hydrolysate. The aim was to evaluate the effect of agitation rate and dissolved oxygen at 7 L bioreactor scale on the production of xylose reductase (XR) from Candida tropicalis during the bioconversion of xylose into xylitol in the non-detoxified oil palm empty fruit bunch (OPEFB) hydrolysate. The highest xylose consumption (95.5%) and the maximum xylitol production (5.46 g.L-1) were presented under 30% dissolved oxygen and 50 rpm. The maximum XR activity (0.646 U mg-1 protein) was obtained after 144 h of fermentation and at the same conditions of dissolved oxygen and agitation rate mentioned above. The oxygen availability influences the XR activity of C. tropicalis and the xylitol production, observing a xylitol yield factor (YP/S) of 0.27 g.g-1 and volumetric productivity (QP) of 0.33 g.L-1 h-1. At lower dissolved oxygen regardless of the agitation conditions evaluated, an increase in xylitol production was evidenced.

Valorisation of xylose to renewable fuels and chemicals, an essential step in augmenting the commercial viability of lignocellulosic biorefineries.

Biologists and engineers are making tremendous efforts in contributing to a sustainable and green society. To that end, there is growing interest in waste management and valorisation. Lignocellulosic biomass (LCB) is the most abundant material on the earth and an inevitable waste predominantly originating from agricultural residues, forest biomass and municipal solid waste streams. LCB serves as the renewable feedstock for clean and sustainable processes and products with low carbon emission. Cellulose and hemicellulose constitute the polymeric structure of LCB, which on depolymerisation liberates oligomeric or monomeric glucose and xylose, respectively. The preferential utilization of glucose and/or absence of the xylose metabolic pathway in microbial systems cause xylose valorization to be alienated and abandoned, a major bottleneck in the commercial viability of LCB-based biorefineries. Xylose is the second most abundant sugar in LCB, but a non-conventional industrial substrate unlike glucose. The current review seeks to summarize the recent developments in the biological conversion of xylose into a myriad of sustainable products and associated challenges. The review discusses the microbiology, genetics, and biochemistry of xylose metabolism with hurdles requiring debottlenecking for efficient xylose assimilation. It further describes the product formation by microbial cell factories which can assimilate xylose naturally and rewiring of metabolic networks to ameliorate xylose-based bioproduction in native as well as non-native strains. The review also includes a case study that provides an argument on a suitable pathway for optimal cell growth and succinic acid (SA) production from xylose through elementary flux mode analysis. Finally, a product portfolio from xylose bioconversion has been evaluated along with significant developments made through enzyme, metabolic and process engineering approaches, to maximize the product titers and yield, eventually empowering LCB-based biorefineries. Towards the end, the review is wrapped up with current challenges, concluding remarks, and prospects with an argument for intense future research into xylose-based biorefineries.

Increasing NADPH Availability for Xylitol Production via Pentose-Phosphate-Pathway Gene Overexpression and Embden-Meyerhof-Parnas-Pathway Gene Deletion in Escherichia coli.

Cofactor availability is often a rate-limiting factor in the bioconversion of xylose to xylitol. The overexpression of pentose phosphate pathway genes and the deletion of Embden-Meyerhof-Parnas pathway genes can modulate the glucose metabolic flux and increase the intracellular NADPH supply, enabling Escherichia coli cells to produce xylitol from corncob hydrolysates. The effects of zwf and/or gnd overexpression and pfkA, pfkB, and/or pgi deletion on the intracellular redox environment and xylitol production were examined. The NADPH-enhanced strain 2bpgi produced 162 g/L xylitol from corncob hydrolysates after a 76 h fed-batch fermentation in a 15 L bioreactor, which was 13.3% greater than the 143 g/L xylitol produced by the IS5-d control strain. Additionally, the xylitol productivity and xylitol yield per glucose for 2bpgi were 2.13 g/L/h and 2.50 g/g, respectively. Thus, the genetic modifications in 2bpgi significantly enhanced NADPH regeneration, making 2bpgi a potentially useful strain for the industrial-scale production of xylitol from detoxified corncob hydrolysates.

A cost-practical cell-recycling process for xylonic acid bioproduction from acidic lignocellulosic hydrolysate with whole-cell catalysis of Gluconobacter oxydans

Xylonic acid (XA), as a bio-based platform chemical, is of considerable interest for xylose bioconversion. The whole-cell catalysis of Gluconobacter oxydans presents a promising application potential, while the hard works of cell culture still severely hinder XA business from the crude toxics-containing lignocellulosic hydrolysate. Hence, the bacterial cells should be recycled to reduce commercial production cost. The implementation of diatomite detoxification not only absorbs most of the degraded inhibitors in hydrolysate, but also confines the sugar contents loss with 10% and allows the bacterial cells to maintain 90% bioconversion performance during cell-recycling operation. Additionally, a scale-up of XA bioproduction was achieved in a sealed oxygen supply fermenter. Finally, 210 g XA was produced from 1000 g corncob originated hydrolysate within 24 h of whole-cell catalysis. Diatomite treatment provides an efficient and cost-practical approach for the commercial bioproduction of biochemicals like XA from lignocellulosic biomass.

Optimized Bioconversion of Xylose Derived from Pre-Treated Crop Residues into Xylitol by Using Candida boidinii

Crop residues can serve as low-cost feedstocks for microbial production of xylitol, which offers many advantages over the commonly used chemical process. However, enhancing the efficiency of xylitol fermentation is still a barrier to industrial implementation. In this study, the effects of oxygen transfer rate (OTR) (1.1, 2.1, 3.1 mmol O2/(L × h)) and initial xylose concentration (30, 55, 80 g/L) on xylitol production of Candida boidinii NCAIM Y.01308 on xylose medium were investigated and optimised by response surface methodology, and xylitol fermentations were performed on xylose-rich hydrolysates of wheat bran and rice straw. High values of maximum xylitol yields (58–63%) were achieved at low initial xylose concentration (20–30 g/L) and OTR values (1.1–1.5 mmol O2/(L × h)). The highest value for maximum xylitol productivity (0.96 g/(L × h)) was predicted at 71 g/L initial xylose and 2.7 mmol O2/(L × h) OTR. Maximum xylitol yield and productivity obtained on wheat bran hydrolysate were 60% and 0.58 g/(L × h), respectively. On detoxified and supplemented hydrolysate of rice straw, maximum xylitol yield and productivity of 30% and 0.19 g/(L × h) were achieved. This study revealed the terms affecting the xylitol production by C. boidinii and provided validated models to predict the achievable xylitol yields and productivities under different conditions. Efficient pre-treatments for xylose-rich hydrolysates from rice straw and wheat bran were selected. Fermentation using wheat bran hydrolysate and C. boidinii under optimized condition is proved as a promising method for biotechnological xylitol production.

Enhanced xylitol production using non-detoxified xylose rich pre-hydrolysate from sugarcane bagasse by newly isolated Pichia fermentans

BackgroundIntegrated management of hemicellulosic fraction and its economical transformation to value-added products is the key driver towards sustainable lignocellulosic biorefineries. In this aspect, microbial cell factories are harnessed for the sustainable production of commercially viable biochemicals by valorising C5 and C6 sugars generated from agro-industrial waste. However, in the terrestrial ecosystem, microbial systems can efficiently consume glucose. On the contrary, pentose sugars are less preferred carbon source as most of the microbes lack metabolic pathway for their utilization. The effective utilization of both pentose and hexose sugars is key for economical biorefinery.ResultsBioprospecting the food waste and selective enrichment on xylose-rich medium led to screening and isolation of yeast which was phylogenetically identified as Pichia fermentans. The newly isolated xylose assimilating yeast was explored for xylitol production. The wild type strain robustly grew on xylose and produced xylitol with > 40% conversion yield. Chemical mutagenesis of isolated yeast with ethyl methanesulphonate (EMS) yielded seven mutants. The mutant obtained after 15 min EMS exposure, exhibited best xylose bioconversion efficiency. This mutant under shake flask conditions produced maximum xylitol titer and yield of 34.0 g/L and 0.68 g/g, respectively. However, under the same conditions, the control wild type strain accumulated 27.0 g/L xylitol with a conversion yield of 0.45 g/g. Improved performance of the mutant was attributed to 34.6% activity enhancement in xylose reductase with simultaneous reduction of xylitol dehydrogenase activity by 22.9%. Later, the culture medium was optimized using statistical design and validated at shake flask and bioreactor level. Bioreactor studies affirmed the competence of the mutant for xylitol accumulation. The xylitol titer and yield obtained with pure xylose were 98.9 g/L and 0.67 g/g, respectively. In comparison, xylitol produced using non-detoxified xylose rich pre-hydrolysate from sugarcane bagasse was 79.0 g/L with an overall yield of 0.54 g/g.ConclusionThis study demonstrates the potential of newly isolated P. fermentans in successfully valorising the hemicellulosic fraction for the sustainable xylitol production.

Bioproduction of succinic acid from xylose by engineered Yarrowia lipolytica without pH control

BackgroundXylose is the most prevalent sugar available in hemicellulose fraction of lignocellulosic biomass (LCB) and of great interest for the green economy. Unfortunately, most of the cell factories cannot inherently metabolize xylose as sole carbon source. Yarrowia lipolytica is a non-conventional yeast that produces industrially important metabolites. The yeast is able to metabolize a large variety of substrates including both hydrophilic and hydrophobic carbon sources. However, Y. lipolytica lacks effective metabolic pathway for xylose uptake and only scarce information is available on utilization of xylose. For the economica feasibility of LCB-based biorefineries, effective utilization of both pentose and hexose sugars is obligatory.ResultsIn the present study, succinic acid (SA) production from xylose by Y. lipolytica was examined. To this end, Y. lipolytica PSA02004 strain was engineered by overexpressing pentose pathway cassette comprising xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulose kinase (XK) gene. The recombinant strain exhibited a robust growth on xylose as sole carbon source and produced substantial amount of SA. The inhibition of cell growth and SA formation was observed above 60 g/L xylose concentration. The batch cultivation of the recombinant strain in a bioreactor resulted in a maximum biomass concentration of 7.3 g/L and SA titer of 11.2 g/L with the yield of 0.19 g/g. Similar results in terms of cell growth and SA production were obtained with xylose-rich hydrolysate derived from sugarcane bagasse. The fed-batch fermentation yielded biomass concentration of 11.8 g/L (OD600: 56.1) and SA titer of 22.3 g/L with a gradual decrease in pH below 4.0. Acetic acid was obtained as a main by-product in all the fermentations.ConclusionThe recombinant strain displayed potential for bioconversion of xylose to SA. Further, this study provided a new insight on conversion of lignocellulosic biomass into value-added products. To the best of our knowledge, this is the first study on SA production by Y. lipolytica using xylose as a sole carbon source.

Heterologous expression of genes for bioconversion of xylose to xylonic acid in Corynebacterium glutamicum and optimization of the bioprocess

In bacterial system, direct conversion of xylose to xylonic acid is mediated through NAD-dependent xylose dehydrogenase (xylB) and xylonolactonase (xylC) genes. Heterologous expression of these genes from Caulobacter crescentus into recombinant Corynebacterium glutamicum ATCC 13032 and C. glutamicum ATCC 31831 (with an innate pentose transporter, araE) resulted in an efficient bioconversion process to produce xylonic acid from xylose. Process parameters including the design of production medium was optimized using a statistical tool, Response Surface Methodology (RSM). Maximum xylonic acid of 56.32 g/L from 60 g/L xylose, i.e. about 76.67% of the maximum theoretical yield was obtained after 120 h fermentation from pure xylose with recombinant C. glutamicum ATCC 31831 containing the plasmid pVWEx1 xylB. Under the same condition, the production with recombinant C. glutamicum ATCC 13032 (with pVWEx1 xylB) was 50.66 g/L, i.e. 69% of the theoretical yield. There was no significant improvement in production with the simultaneous expression of xylB and xylC genes together indicating xylose dehydrogenase activity as one of the rate limiting factor in the bioconversion. Finally, proof of concept experiment in utilizing biomass derived pentose sugar, xylose, for xylonic acid production was also carried out and obtained 42.94 g/L xylonic acid from 60 g/L xylose. These results promise a significant value addition for the future bio refinery programs.

Xylitol production from lignocellulosic pentosans using Kluyveromyces marxianus: kinetic modelling of yeast growth and fermentation

ABSTRACTKinetics of xylitol production from sugarcane bagasse hydrolysate by Kluyveromyces marxianus IIPE453t was modelled. Yeast growth and xylitol production were represented with logistic and Michaelis–Menten equations. Batch fermentation produced xylitol with varying yields at different xylose concentrations. The maximum xylitol titre of 25.6 g L−1 was obtained with a volumetric productivity of 0.24 g L−1 h−1. Sensitivity analysis demonstrated that xylose bioconversion linked to XRKS activity was the principal factor governing xylitol production kinetics.

Bioconversion of Xylose to Ethylene Glycol and Glycolate in Engineered Corynebacterium glutamicum.

The biological production of two-carbon compounds (ethylene glycol (EG) and glycolate) has been studied for the sustainable supply of the compounds to the polymer, cosmetic, textile, and medical industries. Here, we demonstrated the bioconversion of xylose to either ethylene glycol (EG) or glycolate using engineered Corynebacterium glutamicum, a well-known industrial amino acid producer. A synthetic ribulose 1-phosphate (Ru1P) pathway involving heterologous d-tagatose 3-epimerase and l-fuculose kinase/aldolase reactions was introduced in C. glutamicum. Subsequently, heterologous expression of Escherichia coli YqhD reductase with the synthetic Ru1P pathway led to ethylene glycol production from xylose. Additional pathway engineering in C. glutamicum by mutating ald, which encodes an aldehyde dehydrogenase, abolished the by-product formation of glycolate during xylose conversion to EG at a yield of 0.75 mol per mol. In addition, the bioconversion of xylose to glycolate was achieved, and the almost maximum molar yield was 0.99 mol per mol xylose in C. glutamicum via the Ru1P pathway. Thus, the synthetic Ru1P pathway in C. glutamicum led bioconversion of xylose to either ethylene glycol or glycolate with high molar yields.

Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae.

Bioconversion of xylose-the second most abundant sugar in nature-into high-value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long-term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially-targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed-batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value-added products.

Efficient production of xylitol by the integration of multiple copies of xylose reductase gene and the deletion of Embden-Meyerhof-Parnas pathway-associated genes to enhance NADPH regeneration in Escherichia coli.

Cofactor supply is a rate-limiting step in the bioconversion of xylose to xylitol. Strain WZ04 was first constructed by a novel simultaneous deletion-insertion strategy, replacing ptsG, xylAB and ptsF in wild-type Escherichia coli W3110 with three mutated xylose reductase genes (xr) from Neurospora crassa. Then, the pfkA, pfkB, pgi and/or sthA genes were deleted and replaced by xr to investigate the influence of carbon flux toward the pentose phosphate pathway and/or transhydrogenase activity on NADPH generation. The deletion of pfkA/pfkB significantly improved NADPH supply, but minimally influenced cell growth. The effects of insertion position and copy number of xr were examined by a quantitative real-time PCR and a shake-flask fermentation experiment. In a fed-batch fermentation experiment with a 15-L bioreactor, strain WZ51 produced 131.6gL-1 xylitol from hemicellulosic hydrolysate (xylitol productivity: 2.09gL-1h-1). This study provided a potential approach for industrial-scale production of xylitol from hemicellulosic hydrolysate.

In-Vitro and In-Silico Characterization of Xylose Reductase from Emericella nidulans

Background: Lignocellulosic residues generated by various anthropogenic activities can be a potential raw material for many commercial products such as biofuels, organic acids and nutraceuticals including xylitol. Xylitol is a low-calorie nutritive sweetener for diabetic patients. Microbial production of xylitol can be helpful in overcoming the drawbacks of traditional chemical production process and lowring cost of production. Objective: Designing efficient production process needs the characterization of required enzyme/s. Hence current work was focused on in-vitro and in-silico characterization of xylose reductase from Emericella nidulans. Methods: Xylose reductase from one of the hyper-producer isolates, Emericella nidulans Xlt-11 was used for in-vitro characterization. For in-silico characterization, XR sequence (Accession No: Q5BGA7) was used. Results: Xylose reductase from various microorganisms has been studied but the quest for better enzymes, their stability at higher temperature and pH still continues. Xylose reductase from Emericella nidulans Xlt-11 was found NADH dependent and utilizes xylose as its sole substrate for xylitol production. In comparison to whole cells, enzyme exhibited higher enzyme activity at lower cofactor concentration and could tolerate higher substrate concentration. Thermal deactivation profile showed that whole cell catalysts were more stable than enzyme at higher temperature. In-silico analysis of XR sequence from Emericella nidulans (Accession No: Q5BGA7) suggested that the structure was dominated by random coiling. Enzyme sequences have conserved active site with net negative charge and PI value in acidic pH range. Conclusion: Current investigation supported the enzyme’s specific application i.e. bioconversion of xylose to xylitol due to its higher selectivity. In-silico analysis may provide significant structural and physiological information for modifications and improved stability.