Cellobionic Acid Research Articles

Efficient Bio-Oxidation of Cellobiose with Engineered Gluconobacter oxydans to Provide Highly Concentrated Cellobionic Acid

Cellobionic acid (CBA) can be obtained through the oxidation of cellobiose, the monomer of cellulose. CBA serves as a plant-based alternative to its stereoisomer lactobionic acid, which is used in the pharmaceutical, cosmetic, and food industries. Gluconobacter oxydans is a well-established whole-cell biocatalyst with membrane-bound dehydrogenases (mDH) for regio-specific oxidations. As G. oxydans wildtype cells show low cellobiose oxidation activities, the glucose mDH from Pseudomonas taetrolens was overexpressed in G. oxydans BP9, a multi mDH deletion strain. Whole-cell biotransformation studies were performed with resting cells of the engineered G. oxydans in stirred tank bioreactors. Initial biomass specific cellobionate formation rates increased with increasing cellobiose concentrations up to 190 g L−1, and were constant until the solubility limit. The maximal volumetric CBA formation rates and the oxygen uptake rates increased linearly with the concentration of engineered G. oxydans. This enables the estimation of the maximum biocatalyst concentration limited by the maximum oxygen transfer rate of any bioreactor. Thus, 5.2 g L−1 G. oxydans was sufficient to produce 502 g L−1 CBA with >99% yield in a simple aerobic batch process. The highly concentrated CBA will reduce downstream processing costs considerably after cell separation.

Recombinant cellobiose dehydrogenase from Thermothelomyces thermophilus: Its functional characterization and applicability in cellobionic acid production

The fungus Thermothelomyces thermophilus is a thermotolerant microorganism that has been explored as a reservoir for enzymes (hydrolytic enzymes and oxidoreductases). The functional analysis of a recombinant cellobiose dehydrogenase (MtCDHB) from T. thermophilus demonstrated a thermophilic behavior, an optimal pH in alkaline conditions for inter-domain electron transfer, and catalytic activity on cellooligosaccharides with different degree of polymerization. Its applicability was evaluated to the sustainable production of cellobionic acid (CBA), a potential pharmaceutical and cosmetic ingredient rarely commercialized. Dissolving pulp was used as a disaccharide source for MtCDHB. Initially, recombinant exoglucanases (MtCBHI and MtCBHII) from T. thermophilus hydrolyzed the dissolving pulp, resulting in 87% cellobiose yield, which was subsequently converted into CBA by MtCDHB, achieving a 66% CBA yield after 24 h. These findings highlight the potential of MtCDHB as a novel approach to obtaining CBA through the bioconversion of a plant-based source.

Sustainable cellobionic acid biosynthesis from starch via artificial in vitro synthetic enzymatic biosystem

Cellobionic acid (CBA), a kind of aldobionic acid, offers potential applications in the fields of pharmaceutical, cosmetic, food, and chemical industry. To tackle the high cost of the substrate cellobiose in CBA production using quinoprotein glucose dehydrogenase, this study developed a coenzyme-free and phosphate-balanced in vitro synthetic enzymatic biosystem (ivSEBS) to enable the sustainable CBA synthesis from cost-effective starch in one-pot via the CBA-synthesis module and gluconic acid-supply module. The metabolic fluxes of this artificial biosystem were strengthened using design−build−test−analysis strategy, which involved exquisite pathway design, meticulous enzyme selection, module validation and integration, and optimization of the key enzyme dosage. Under the optimized conditions, a remarkable concentration of 6.2 g/L CBA was achieved from initial 10 g/L maltodextrin with a starch-to-CBA molar conversion rate of 60 %. Taking into account that the biosystem simultaneously accumulated 3.6 g/L of gluconic acid, the maltodextrin utilization rate was calculated to be 93.3 %. Furthermore, a straightforward scaling-up process was performed to evaluate the industrial potential of this enzymatic biosystem, resulting in a yield of 21.2 g/L CBA from 50 g/L maltodextrin. This study presents an artificial ivSEBS for sustainable production of CBA from inexpensive starch, demonstrating an alternative paradigm for biomanufacturing of other aldobionic acids.

Electrochemical Cellobiose Valorization: Anodic Oxidation to Cellobionic Acid and Cathodic Reduction to Cellobitol

AbstractThis study elucidates key parameters allowing the electrochemical valorization of cellobiose, by creating value‐added products by both, anodic oxidation, and cathodic reduction. We demonstrate the first electrochemical reduction of cellobiose to cellobitol in a divided cell using Pb as cathode, leading up to 49 % conversion, though with long reaction times (over 26 h) and low coulombic efficiency ( ) of 4 %. The successful synthesis of cellobionic acid was demonstrated by mediated oxidation in an undivided electrochemical cell, using a graphite anode and CaBr2 as electrocatalytic mediator. Cellobiose oxidation was highly selective, allowing for up to 92 % conversion to cellobionic acid at 48 % when using a charge of 2 FE.

The role of lignin in the conversion of wheat straw to cellobionic acid by Neurospora crassa HL10

An engineered Neurospora crassa strain (HL10) naturally produces cellulases, cellobiose dehydrogenase (CDH), and heterologously expresses laccase. It can convert Avicel (cellulose) to cellobionic acid (cellobionate) without any enzyme addition at high yields. However, the addition of a catalytic amount of artificial redox mediators was required to achieve a high yield conversion. When a lignocellulosic substrate like wheat straw was used as the carbon source, it was found that adding exogenous artificial redox mediators did not improve cellobionate yield. We hypothesized that lignin and lignin degradation products were able to serve as redox mediators for the CDH-laccase conversion system. Experiments were designed to prove the hypothesis. When N. crassa HL10 was grown on Avicel, the addition of enzyme hydrolyzed lignin (EHL) can achieve a similar level of cellobionate production as with ABTS addition. The addition of EHL can increase the cellobionate production rate in the CDH-laccase bi-enzyme system using cellobiose as the substrate. The formation of lignin radicals, and the quenching of lignin radicals by reduced CDH, were verified by an electron paramagnetic resonance (EPR) experiment, providing further evidence that lignin radicals can serve as the electron acceptor of reduced CDH. Hence, lignocellulosic biomass is a self-sufficient substrate for the production of cellobionate. The conversion of the lignocellulosic substrate to cellobionate using N. crassa HL10 can be achieved without any exogenous enzyme addition or redox mediator addition.

Fluorescence labeling of C1-oxidized cellulose. Part 1: Method development

A novel method for fluorescence labeling and subsequent profiling of oxidized end groups along the molar mass distribution of cellulose has been developed. The notorious labeling reluctance of this group due to lactone (gluconolactone) formation was overcome by introducing amide-linked rather than ester-linked markers. A coupling method based on carbodiimide chemistry to bind the fluorophore N-(1-naphthyl)ethylenediamine (NED) was used and optimized with the model compound cellobionic acid towards appropriate reaction conditions and complete conversion. The reaction product, NED-labeled cellobionic acid (CBN), was obtained in 98 % yield, isolated by flash chromatography and comprehensively analytically characterized. CBN was used as a standard for HPLC quantification regarding reaction kinetics and stability analysis. The optimized labeling conditions were transferred to the heterogeneous derivatization of cellulose followed by GPC MALLS/IR/fluorescence analysis for molar mass-related profiling of oxidized reduced ends in cellulosic pulps.

Valorization of cellulose in waste paper into value-added cellobionic acid by genetically engineered Pseudomonas taetrolens

To expand the applications of waste paper (WP), we attempted to valorize waste office paper (WOP) into cellobionic acid (CBA) using a two-step biocatalytic method. First, we prepared cellobiose from WOP using commercial cellulases. To increase cellobiose production, we optimized various reaction conditions; the type of cellulases, reaction temperature, the amount of WOP, and the amount of cellulases. Under the optimized reaction conditions, 23 g/L cellobiose was generated from 80 g/L of WOP. CBA was then produced from the prepared cellobiose using recombinant Pseudomonas taetrolens homologously expressing quinoprotein glucose dehydrogenase. From 23 g/L cellobiose derived from WOP, this strain produced 24.1 g/L CBA. We demonstrated for the first time that WOP could be successfully valorized into CBA using cellobiogenic cellulases and a CBA-producing strain. This study sets the groundwork to produce CBA at a low price and is expected to expand the application of CBA in industries.

Efficient production of cellobionic acid using whole-cell biocatalyst of genetically modified Pseudomonas taetrolens.

Pseudomonas taetrolens has previously been shown to convert cellobiose to cellobionic acid (CBA), which can potentially be used in cosmetics, food, and pharmaceutical industries. The cellobiose-oxidizing activity of the P. taetrolens strain, which expressed the homologous quinoprotein glucose dehydrogenase (GDH), was increased by approximately 50.8% compared to the original strain. Whole-cell biocatalyst (WCB) of the genetically modified P. taetrolens strain [pDSK-GDH] was prepared simply by fermentation and washing processes. Reaction conditions for the proper use of WCB, such as reaction temperature, cell density to be added, and cell harvest time for preparing WCB, were investigated. The highest CBA productivity (18.2g/L/h) was achieved when WCB prepared in the late-exponential phase of cell culture was used at 35°C with cell density of 10 at OD600nm. Under these conditions, 200g/L of cellobiose was all converted to CBA in 11h, and the WCB of P. taetrolens [pDSK-GDH] maintained the maximum catalytic activity during at least six cycles without a significant decline in the productivity. Our results suggest that the manufacture of WCB based on genetically engineered P. taetrolens and its optimized use could be further developed as an economically viable option for the large-scale production of CBA.

Efficient production of cellobionic acid from cellobiose by genetically modified Pseudomonas taetrolens

Pseudomonas taetrolens is one of the most efficient lactobionic acid (LBA)-producing microorganisms. We previously found that it also produced cellobionic acid (CBA) and that quinoprotein glucose dehydrogenase (GDH) from P. taetrolens could convert lactose into LBA. Here, we discovered that GDH could also convert cellobiose into CBA when it was heterologously expressed in Escherichia coli. To enhance the CBA-producing ability of P. taetrolens, we homologously expressed GDH in P. taetrolens, and demonstrated that the intracellular cellobiose-oxidizing activity (1.75 U/μg) and CBA titer (21.4 g/L) of the recombinant P. taetrolens were approximately 54% and 60% higher than those (1.14 U/μg and 13.4 g/L, respectively) of the wild-type P. taetrolens in 12 h flask cultures. We optimized culture conditions of this recombinant strain, such as cellobiose concentration, growth temperature, and cell density of seed culture. Thereafter, we scaled up the CBA production using a 5-L bioreactor under the optimized culture condition determined in flask culture experiments. The CBA production titer, yield, and productivity of the recombinant P. taetrolens strain after batch fermentation were 200 g/L, 95.6%, and 9.52 g/L/h, respectively, which far exceeded those obtained in previous studies. Our study provides important clues to achieve economically feasible production of CBA.

Homoethanol production from cellobionate and glycerol using recombinant Klebsiella oxytoca strains

Cellobionic acid, an oxidized cellulose degradation product, can be produced from cellulosic biomass. Engineered strains of Klebsiella oxytoca BW21 and WT26 can effectively use cellobionate for ethanol production. However, because cellobionate is a more oxidized substrate than cellobiose, oxidized products such as acetate must be produced as by-products to maintain the overall redox balance, and homoethanol production is not possible. One of the strategies to avoid the redox imbalance problem and achieve homoethanol production is co-fermentation of cellobionate with a more reduced substrate glycerol. When cellobionate and glycerol were used as the carbon source by K. oxytoca BW 21, ethanol yield was about 62%–67%. Co-utilization of cellobionate and glycerol in batch fermentation increased the yield of ethanol to about 77% and substantially decreased the accumulation of by-products (such as acetate and 1,3-propanediol). However, glycerol imposes carbon catabolite repression on cellobionate utilization. The utilization rate mismatch between these two substrates limits the benefits of redox recycling between the two substrates’ utilization pathways. When recombinant strain WT26 (which is disabled in glycerol utilization when fermented alone) was used to co-ferment cellobionate and glycerol, the two substrates’ utilization rates exactly matched, which led to an ethanol yield improvement to 96%.

Assessing the enzymatic effects of cellulases and LPMO in improving mechanical fibrillation of cotton linters

BackgroundThe increasing interest in replacing petroleum-based products by more sustainable materials in the packaging sector gives relevance to cellulose as a biodegradable natural resource. Moreover, its properties can be modified physically, chemically or biotechnologically in order to obtain new bioproducts. Refined cotton linters with high cellulose content were treated with hydrolytic (cellulases) and oxidative (LPMO and Laccase_Tempo) enzymes to evaluate their effect on fibre properties and in improving mechanical fibrillation.ResultsCellulases released cellooligosaccharides, reducing fibre length and partially degrading cellulose. They also improved mechanical fibrillation yielding up to 18% of nanofibrillated cellulose (NFC). LPMO introduced a slight amount of COOH groups in cellulose fibres, releasing cellobionic acid to the effluents. The action of cellulases was improved after LPMO treatment; however, the COOH groups created disappeared from fibres. After mechanical fibrillation of LPMO–cellulase-treated cotton linters a 23% yield of NFC was obtained. Laccase_Tempo treatment also introduced COOH groups in cellulose fibres from cotton, yielding 10% of NFC. Degree of polymerization was reduced by Laccase_Tempo, while LPMO treatment did not significantly affect it but produced a higher reduction in fibre length. The combined treatment with LPMO and cellulase provided films with higher transparency (86%), crystallinity (92%), smoothness and improved barrier properties to air and water than films casted from non-treated linters and from commercial NFC.ConclusionsThe combined enzymatic treatment with LPMO and cellulases boosted mechanical fibrillation of cotton linters, improving the NFC production and providing bioproducts with high transparency and high barrier properties.

Cellobionic acid inhibition of cellobiohydrolase I and cellobiose dehydrogenase

End-product inhibition by cellobiose and glucose is a rate-limiting factor in cellulose hydrolysis by cellulases. While cellobiose and glucose inhibition have been extensively investigated, cellobionate inhibition has been minimally studied despite the discovery that accessory proteins such as cellobiose dehydrogenase (CDH) work synergistically with cellulases to form aldonic acids. The fraction of equilibrated cellobionate in the cellobiono-δ-lactone form was determined by NMR over the range of pH 4–8. To better improve the understanding of cellobiono-δ-lactone and cellobionate inhibition of cellulases, we investigate the inhibition of Trichoderma reesei cellobiohydrolase I (CBHI), one of the most extensively studied CBHs, and Neurospora crassa CDH by equilibrated cellobionate. The well-characterized inhibitor cellobiose is evaluated in parallel for comparison. The mechanisms of inhibition are determined for both inhibitors and the kinetic parameters compared using a Michaelis–Menten inhibition model. Cellobiose and cellobionate inhibition of CBHI is extended to Avicel hydrolysis, along with an investigation of those inhibitors on Accelerase 1500, a commercial cellulase preparation.

Lactobionic and cellobionic acid production profiles of the resting cells of acetic acid bacteria.

Lactobionic acid was produced by acetic acid bacteria to oxidize lactose. Gluconobacter spp. and Gluconacetobacter spp. showed higher lactose-oxidizing activities than Acetobacter spp. Gluconobacter frateurii NBRC3285 produced the highest amount of lactobionic acid per cell, among the strains tested. This bacterium assimilated neither lactose nor lactobionic acid. At high lactose concentration (30%), resting cells of the bacterium showed sufficient oxidizing activity for efficient production of lactobionic acid. These properties may contribute to industrial production of lactobionic acid by the bacterium. The bacterium showed higher oxidizing activity on cellobiose than that on lactose and produced cellobionic acid.

Cellobionic acid utilization: from Neurospora crassa to Saccharomyces cerevisiae.

BackgroundEconomical production of fuels and chemicals from plant biomass requires the efficient use of sugars derived from the plant cell wall. Neurospora crassa, a model lignocellulosic degrading fungus, is capable of breaking down the complex structure of the plant cell wall. In addition to cellulases and hemicellulases, N. crassa secretes lytic polysaccharide monooxygenases (LPMOs), which cleave cellulose by generating oxidized sugars—particularly aldonic acids. However, the strategies N. crassa employs to utilize these sugars are unknown.ResultsWe identified an aldonic acid utilization pathway in N. crassa, comprised of an extracellular hydrolase (NCU08755), cellobionic acid transporter (CBT-1, NCU05853) and cellobionic acid phosphorylase (CAP, NCU09425). Extracellular cellobionic acid could be imported directly by CBT-1 or cleaved to gluconic acid and glucose by a β-glucosidase (NCU08755) outside the cells. Intracellular cellobionic acid was further cleaved to glucose 1-phosphate and gluconic acid by CAP. However, it remains unclear how N. crassa utilizes extracellular gluconic acid. The aldonic acid pathway was successfully implemented in Saccharomyces cerevisiae when N. crassa gluconokinase was co-expressed, resulting in cellobionic acid consumption in both aerobic and anaerobic conditions.ConclusionsWe successfully identified a branched aldonic acid utilization pathway in N. crassa and transferred its essential components into S. cerevisiae, a robust industrial microorganism.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0303-2) contains supplementary material, which is available to authorized users.

Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase, Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes

The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes.

Isobutanol production from cellobionic acid in Escherichia coli.

BackgroundLiquid fuels needed for the global transportation industry can be produced from sugars derived from plant-based lignocellulosics. Lignocellulosics contain a range of sugars, only some of which (such as cellulose) have been shown to be utilizable by microorganisms capable of producing biofuels. Cellobionic acid makes up a small but significant portion of lignocellulosic degradation products, and had not previously been investigated as an utilizable substrate. However, aldonic acids such as cellobionic acid are the primary products of a promising new group of lignocellulosic-degrading enzymes, which makes this compound group worthy of study. Cellobionic acid doesn’t inhibit cellulose degradation enzymes and so its inclusion would increase lignocellulosic degradation efficiency. Also, its use would increase overall product yield from lignocellulose substrate. For these reasons, cellobionic acid has gained increased attention for cellulosic biofuel production.ResultsThis study describes the discovery that Escherichia coli are naturally able to utilize cellobionic acid as a sole carbon source with efficiency comparable to that of glucose and the construction of an E. coli strain able to produce the drop-in biofuel candidate isobutanol from cellobionic acid. The gene primarily responsible for growth of E. coli on cellobionic acid is ascB, a gene previously thought to be cryptic (expressed only after incurring specific mutations in nearby regulatory genes). In addition to AscB, the ascB knockout strain can be complemented by the cellobionic acid phosphorylase from the fungus Neurospora crassa. An E. coli strain engineered to express the isobutanol production pathway was successfully able to convert cellobionic acid into isobutanol. Furthermore, to demonstrate potential application of this strain in a sequential two-step bioprocessing system, E. coli was grown on hydrolysate (that was degraded by a fungus) and was successfully able to produce isobutanol.ConclusionsThese results demonstrate that cellobionic acid is a viable carbon source for biofuel production. This work suggests that with further optimization, a bacteria-fungus co-culture could be used in decreased-cost biomass-based biofuel production systems.

Production of cellobionate from cellulose using an engineered Neurospora crassa strain with laccase and redox mediator addition.

We report a novel production process for cellobionic acid from cellulose using an engineered fungal strain with the exogenous addition of laccase and a redox mediator. A previously engineered strain of Neurospora crassa (F5∆ace-1∆cre-1∆ndvB) was shown to produce cellobionate directly from cellulose without the addition of exogenous cellulases. Specifically, N. crassa produces cellulases, which hydrolyze cellulose to cellobiose, and cellobiose dehydrogenase (CDH), which oxidizes cellobiose to cellobionate. However, the conversion of cellobiose to cellobionate is limited by the slow re-oxidation of CDH by molecular oxygen. By adding low concentrations of laccase and a redox mediator to the fermentation, CDH can be efficiently oxidized by the redox mediator, with in-situ re-oxidation of the redox mediator by laccase. The conversion of cellulose to cellobionate was optimized by evaluating pH, buffer, and laccase and redox mediator addition time on the yield of cellobionate. Mass and material balances were performed, and the use of the native N. crassa laccase in such a conversion system was evaluated against the exogenous Pleurotus ostreatus laccase. This paper describes a working concept of cellobionate production from cellulose using the CDH-ATBS-laccase system in a fermentation system.

The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ)-based LC–MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium vs sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.

Discovery of cellobionic acid phosphorylase in cellulolytic bacteria and fungi

A novel phosphorylase was characterized as new member of glycoside hydrolase family 94 from the cellulolytic bacterium Xanthomonas campestris and the fungus Neurospora crassa. The enzyme catalyzed reversible phosphorolysis of cellobionic acid. We propose 4-O-β-d-glucopyranosyl-d-gluconic acid: phosphate α-d-glucosyltransferase as the systematic name and cellobionic acid phosphorylase as the short names for the novel enzyme. Several cellulolytic fungi of the phylum Ascomycota also possess homologous proteins. We, therefore, suggest that the enzyme plays a crucial role in cellulose degradation where cellobionic acid as oxidized cellulolytic product is converted into α-d-glucose 1-phosphate and d-gluconic acid to enter glycolysis and the pentose phosphate pathway, respectively.

Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content

BackgroundThe recent discovery of accessory proteins that boost cellulose hydrolysis has increased the economical and technical efficiency of processing cellulose to bioethanol. Oxidative enzymes (e.g. GH61) present in new commercial enzyme preparations have shown to increase cellulose conversion yields. When using pure cellulose substrates it has been determined that both oxidized and unoxidized cellodextrin products are formed. We report the effect of oxidative activity in a commercial enzyme mix (Cellic CTec2) upon overall hydrolysis, formation of oxidized products and impact on β-glucosidase activity. The experiments were done at high solids loadings using a lignocellulosic substrate simulating commercially relevant conditions.ResultsThe Cellic CTec2 contained oxidative enzymes which produce gluconic acid from lignocellulose. Both gluconic and cellobionic acid were produced during hydrolysis of pretreated wheat straw at 30% WIS. Up to 4% of released glucose was oxidized into gluconic acid using Cellic CTec2, whereas no oxidized products were detected when using an earlier cellulase preparation Celluclast/Novozym188. However, the cellulose conversion yield was 25% lower using Celluclast/Novozym188 compared to Cellic CTec2. Despite the advantage of the oxidative enzymes, it was shown that aldonic acids could be problematic to the hydrolytic enzymes. Hydrolysis experiments revealed that cellobionic acid was hydrolyzed by β-glucosidase at a rate almost 10-fold lower than for cellobiose, and the formed gluconic acid was an inhibitor of the β-glucosidase.Interestingly, the level of gluconic acid varied significantly with temperature. At 50°C (SHF conditions) 35% less gluconic acid was produced compared to at 33°C (SSF conditions). We also found that in the presence of lignin, no reducing agent was needed for the function of the oxidative enzymes.ConclusionsThe presence of oxidative enzymes in Cellic CTec2 led to the formation of cellobionic and gluconic acid during hydrolysis of pretreated wheat straw and filter paper. Gluconic acid was a stronger inhibitor of β-glucosidase than glucose. The formation of oxidized products decreased as the hydrolysis temperature was increased from 33° to 50°C. Despite end-product inhibition, the oxidative cleavage of the cellulose chains has a synergistic effect upon the overall hydrolysis of cellulose as the sugar yield increased compared to using an enzyme preparation without oxidative activity.