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BackgroundTrichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized.ResultsTo address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose.ConclusionThis study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

This study investigated the impact of Candida tropicalis NITCSK13 on sugarcane bagasse (SCB) consolidated bioprocessing (CSB) using various parameters, such as pH, steam explosion (STEX) pretreatment, and temperature (at two different temperatures, cellulose hydrolysis and ethanol fermentation). The backpropagation neural network (BPNN) method simulated the optimal CSB conditions, achieving a maximum ethanol yield of 44 ± 0.32 g/L (0.443 g of ethanol/g of SCB) from STEX pretreated SCB within 48 h at 55 °C for cellulose hydrolysis and 33 °C for ethanol fermentation and pH 3.5. The simulated conditions were experimentally validated and showed an R2 value of 0.998 and absolute average deviation (AAD) of 1.23%. The strain NITCSK13 also exhibited a high ethanol tolerance of 16% (v/v). The interactions between the inhibitors, cellobiose, furfural, and thermocellulase were assessed through molecular docking. The results revealed a maximum inhibitory constant of 3.7 mM for furfural against the endoglucanase (EnG) of Humicola insolens (2ENG) at 50 °C. Acremonium chrysogenum endoglucanase (5M2D) exhibited a maximum of 88.7 µM for cellobiose at 50 °C. The SWISS homology model of EnG from Candida viswanathii exhibited inhibitory effects similar to those of EnG from Thermoascus and Thermotoga, indicating that the moderately thermophilic yeast Candida sp. cellulase may be capable of efficiently tolerating inhibitors and could be a promising candidate for consolidated bioprocessing of cellulosic ethanol. [ABSTRACT FROM AUTHOR]

A novel metal–organic framework functionalized with amino and triazole moieties, namely Zn-TDA, has been synthesized. It represents the first example of a MOF-based sensor for highly selective sensing of cellobiose over other saccharides. Furthermore, Zn-TDA was used as a catalyst for the conversion of cellobiose to 5-hydroxymethylfurfural (5-HMF), achieving an excellent yield of 68.7% within 90 min under mild conditions. [ABSTRACT FROM AUTHOR]

Formic acid is considered a promising hydrogen carrier. Biomass‐derived formic acid can be obtained by oxidative decomposition of sugars. This study explored the production of formic acid from cellobiose, a disaccharide consisting of d‐glucose linked by β‐glycosidic bonds using heterogeneous catalysts under mild reaction conditions. The use of alkaline earth metal oxide solid base catalysts like CaO and MgO in the presence of hydrogen peroxide could afford formic acid from cellobiose at 343 K. While CaO gave 14 % yield of formic acid, the oxide itself was converted to a harmful metal peroxide, CaO2 after the reaction. In contrast, MgO could produce formic acid without the formation of the metal peroxide. The difficulty in selectively synthesizing formic acid from cellobiose using these solid base catalysts was due to the poor conversion of cellobiose to glucose. Using a combination of solid acid and base catalysts, a high formic acid yield of 33 % was obtained under mild reaction conditions due to the quantitative hydrolysis of cellobiose to glucose by a solid acid followed by the selective decomposition of glucose to formic acid by a solid base. [ABSTRACT FROM AUTHOR]

The interdomain electron transfer (IET) between the catalytic flavodehydrogenase domain and the electron-transferring cytochrome domain of cellobiose dehydrogenase (CDH) plays an essential role in biocatalysis, biosensors and biofuel cells, as well as in its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domains of CDH, which is hypothesised to limit IET in solution by small angle X-ray scattering (SAXS). CDH from Myriococcum thermophilum (syn. Crassicarpon hotsonii, syn. Thermothelomyces myriococcoides) was probed by SAXS to study the CDH mobility at different pH and in the presence of divalent cations. By comparison of the experimental SAXS data, using pair-distance distribution functions and Kratky plots, we show an increase in CDH mobility at higher pH, indicating alterations of domain mobility. To further visualise CDH movement in solution, we performed SAXS-based multistate modelling. Glycan structures present on CDH partially masked the resulting SAXS shapes, we diminished these effects by deglycosylation and studied the effect of glycoforms by modelling. The modelling shows that with increasing pH, the cytochrome domain adopts a more flexible state with significant separation from the dehydrogenase domain. On the contrary, the presence of calcium ions decreases the mobility of the cytochrome domain. Experimental SAXS data, multistate modelling and previously reported kinetic data show how pH and divalent ions impact the closed state necessary for the IET governed by the movement of the CDH cytochrome domain.

The interaction of poly(trimethylene carbonate) (PTMC) with hydroxyl group compounds was investigated as a model for polymer blending with polysaccharides. While 1-butanol, 2-butanol, ethylene glycol, and 1,2-cyclohexanediol showed almost no detectable interaction with PTMC in both solution states with the 1H NMR and solid states with the FT-IR, glucose and cellobiose suggested a slight change in the spectral pattern in FT-IR analysis. The thermal properties of the blended samples of PTMC and these hydroxyl groups were also investigated. Although the blends of PTMC with 1-butanol and 2-butanol did not influence thermal degradation behaviors due to their low boiling points, the PTMC blend with a higher number of hydroxyl groups, especially glucose and cellobiose, tended to increase thermal resistance and glass transition temperature, hence showing the existence of an interaction through hydrogen bonding.

The lyotropic properties of alkyl thioglycosides with varying sugar headgroup (lactose, cellobiose, maltose, galactose, or glucose) and alkyl chain length (octyl, decyl, or dodecyl chains) are investigated by surface tensiometry, visual observation, and fluorescence spectroscopy. The results substantiate that the glycosidic S‐linkage confers considerably different solution aggregation behavior on these surfactants relative to their O‐linked counterparts, where the properties of the latter are known. The materials properties of the aggregated structures from the alkyl thioglycosides vary considerably. Micelles are formed by octyl thiocellobioside and all alkyl thiomaltosides. Turbid aggregate solutions are formed by the alkyl thioglucosides and octyl thiogalactoside, whereas the longer chain alkyl thiogalactosides are minimally soluble. Fluorescence spectroscopy of these systems confirms their aggregation in lamellar‐like structures. The alkyl thiocellobiosides and alkyl thiolactosides form hydrogels from these low‐molecular weight materials at concentrations almost an order of magnitude lower than gels using other low‐molecular weight materials. Here, hydrogels form at concentrations <0.3 wt% with some forming hydrogels at concentrations as low as 0.03 wt% from alkyl thiocellobiosides and thiolactosides, with hydrogel properties differing significantly with this slight change in the sugar headgroup. Alkyl thiocellobiosides form a nanofiber network and alkyl thiolactosides form globular hydrogels. Overall, these results clearly document materials properties that can readily be controlled and designed depending on molecular structure. [ABSTRACT FROM AUTHOR]

[Objectives] This study aimed to clarify the effect of a cellobiose-utilization gene cluster on the growth and virulence of Streptococcus pasteurianus. [Methods] A similarity search was performed based on the cellobiose-utilization gene cluster of S.pneumoniae to analyze the homologous gene cluster in S.pasteurianus WUSP067. RT-qPCR, deletion mutant construction, RT-PCR, growth curve determination under different conditions, and mice infection experiments were performed to evaluate the effect of the cellobiose-utilization gene cluster on the growth and virulence of S.pasteurianus. [Results] Two potential cellobiose-utilization gene clusters(CL)were found in the WUSP067, named CL1 and CL2, respectively. Both of them included the phosphotransferase system and β-glucosidase. RT-qPCR results showed no significant difference in the expression of BglP1wp and BglAwp genes in CL1 in DMEM medium with 10 g·L-1 cellobiose. In contrast, the expression of celAwp and celBwp genes in CL2 were significantly up-regulated (P<0.000 1) in DMEM medium with 10 g·L-1cellobiose, with 49-fold and 33-fold, respectively, suggesting that they were involved in utilizing cellobiose. CL2 was chosed for further study, and RT-PCR results showed that it comprised two operons. A deletion mutant strain of the gene celAwp encoding β-glucosidase was generated. Growth curves analysis showed no significant difference in the growth of wild-type strain and ΔcelAwp in THB medium or DMEM medium supplemented with 10 g·L-1 glucose. However, in the presence of 10 g·L-1 cellobiose as the sole carbon source, compared with the wild-type strain, the growth of ΔcelAwp was severely impaired. Its D600 value of 12 h growth was lower than 0.5(P<0.000 1). The virulence of the wild-type strain and ΔcelAwp was compared using a weaned mouse infection model. Mice infected with the wild-type strain with the dose of 3×108 CFU per mouse died in three days, while the survival rate of mice infected with the same dose of ΔcelAwp was 20% on the 14th day post-infection(P<0.05). [Conclusions] S.pasteurianus was able to grow in the cellobiose as a single carbon source, and CL2 was related to cellobiose utilization, among which celAwp gene not only helped to promote the utilization of cellobiose but also contributed to S.pasteurianus virulence. [ABSTRACT FROM AUTHOR]

Levulinic acid (LA) is one of the top bio‐based platform molecules that can be converted into many valuable chemicals. Herein, we report the sustainable synthesis of LA acid from various sugars using heteropolyacid catalysts. By using a Box–Behnken design of experiment, both LA yield (up to 69 mol%) and complete suppression of parasitic humin formation could be achieved within a 5 h reaction time at 140°C using fructose as a substrate. The effects of various reaction parameters like temperature, sugar concentration, addition of organic co‐solvent and reaction time on LA yield and humin formation were examined in a three‐dimensional space. Moreover, the results could be successfully transferred to other sugars like glucose or cellobiose, paving the way for an atom‐efficient and sustainable LA synthesis process. [ABSTRACT FROM AUTHOR]

Consolidated bioprocessing candidate, Clostridium thermocellum, is a cellulose hydrolysis specialist, with the ability to ferment the released sugars to produce bioethanol. C. thermocellum is generally studied with model substrates Avicel and cellobiose to understand the metabolic pathway leading to ethanol. In the present study, adaptive laboratory evolution, allowing C. thermocellum DSM 1237 to adapt to growth on glucose, fructose, and sorbitol, with the prospect that some strains will adapt their metabolism to yield more ethanol. Adaptive growth on glucose and sorbitol resulted in an approximately 1 mM and 2 mM increase in ethanol yield per millimolar glucose equivalent, respectively, accompanied by a shift in the production of the other expected fermentation end products. The increase in ethanol yield observed for sorbitol adapted cells was due to the carbon source being more reduced compared to cellobiose. Glucose and cellobiose have similar oxidation states thus the increase in ethanol yield is due to the rerouting of electrons from other reduced metabolic products excluding H2 which did not decrease in yield. There was no increase in ethanol yield observed for fructose adapted cells, but there was an unanticipated elimination of formate production, also observed in sorbitol adapted cells suggesting that fructose has regulatory implications on formate production either at the transcription or protein level. [ABSTRACT FROM AUTHOR]

The interaction of poly(trimethylene carbonate) (PTMC) with hydroxyl group compounds was investigated as a model for polymer blending with polysaccharides. While 1-butanol, 2-butanol, ethylene glycol, and 1,2-cyclohexanediol showed almost no detectable interaction with PTMC in both solution states with the 1H NMR and solid states with the FT-IR, glucose and cellobiose suggested a slight change in the spectral pattern in FT-IR analysis. The thermal properties of the blended samples of PTMC and these hydroxyl groups were also investigated. Although the blends of PTMC with 1-butanol and 2-butanol did not influence thermal degradation behaviors due to their low boiling points, the PTMC blend with a higher number of hydroxyl groups, especially glucose and cellobiose, tended to increase thermal resistance and glass transition temperature, hence showing the existence of an interaction through hydrogen bonding. [ABSTRACT FROM AUTHOR]

Developing recombinant Saccharomyces cerevisiae strains capable of transporting and fermenting cellobiose directly is a promising strategy for second-generation ethanol production from lignocellulosic biomass. In this study, we cloned and expressed in the S. cerevisiae CEN.PK2-1C strain an intracellular β-glucosidase (SpBGL7) from Spathaspora passalidarum and co-expressed the cellobiose transporter SiHXT2.4 from Scheffersomyces illinoinensis, and two putative transporters, one from Candida tropicalis (CtCBT1 gene), and one from Meyerozyma guilliermondii (MgCBT2 gene). While all three transporters allowed cell growth on cellobiose, only the MgCBT2 permease allowed cellobiose fermentation, although cellobiose consumption was incomplete. The analysis of the β-glucosidase and transport activities revealed that the cells stopped consuming cellobiose due to a drop in the transport activity. Since ubiquitinylation of lysine residues at the N- or C-terminal domains of the permease are involved in the endocytosis and degradation of sugar transporters, we constructed truncated versions of the permease lacking lysine residues at the C-terminal domain (MgCBT2ΔC), and at both the C- and N-terminal domain (MgCBT2ΔNΔC) and co-expressed these permeases with the SpBGL7 β-glucosidase in an industrial strain. While the strain harboring the MgCBT2ΔC transporter continued to produce incomplete cellobiose fermentations as the wild-type MgCBT2 permease, the strain with the MgCBT2ΔNΔC permease was able to consume and ferment all the cellobiose present in the medium. Thus, our results highlight the importance of expressing cellobiose transporters lacking lysine at the N- and C-terminal domains for efficient cellobiose fermentation by recombinant S. cerevisiae. [ABSTRACT FROM AUTHOR]

BACKGROUND: The industrial‐scale application of enzymes faces obstacles due to elevated costs and difficulties in stability and reuse. In this study, magnetic spent coffee grounds, an ecotoxic waste, have been utilized successfully for the first time to immobilize β‐glucosidase to overcome these challenges. RESULTS: The spent coffee grounds were magnetized and amine‐functionalized, followed by characterization using various techniques. Under optimized conditions, forming an imine bond between the functionalized support and β‐glucosidase resulted in a 62% immobilization yield (92.81 mg g−1 enzyme loading) and 12.5 U mg−1 activity after immobilization. A relatively small kinetic change was observed in the Km value (902 to 946 μmol L−1) after immobilization, suggesting minimal hindrance by AMSCG3 on substrate access or product release. Moreover, Glu@AMSCG3 showed exceptional stability (>90% residual activity) within a pH range of 3 to 6 after 2 h of incubation at 25 °C. A residual activity of 87.94% was maintained even at 80 °C and pH 5 after 2 h of incubation compared to the free β‐glucosidase, which showed only 6.5% residual activity at the same temperature. When cellobiose was hydrolyzed using Glu@AMSCG3 under optimum conditions, 91.33% cellobiose conversion was achieved initially, and over 79% conversion was maintained for 10 reusability cycles. CONCLUSION: The improved stability of β‐glucosidase after covalent immobilization on amine‐modified magnetically separable spent coffee grounds indicates their potential as a support matrix for application in enzyme immobilization. © 2024 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

AbstractGlucose-1-phosphate, a crucial pharmaceutical intermediate, can be generated through the synergistic transformation of lignocellulose using cellulase and cellobiose phosphorylase (CBP), thus holding promise for a profitable biorefinery process. However, the reported optimal pH range of CBPs (6.0–7.5) does not align with that of cellulase (4.8), resulting in suboptimal efficiency in the one-pot conversion of cellulose to glucose-1-phosphate. In this study, we identified a novel cellobiose phosphorylase, CBP1942, comprising 811 amino acid residues (92.5 kDa), in tailing pond macrogenomes, exhibiting identity of 60% to 75% with reported CBPs. CBP1942 was efficiently expressed in E. coli BL21 and purified with His tagging. It maintains over 90% enzyme activity across pH 4–9. Compared to the benchmark CtCBP (GenBank ID: AAL67138.1), CBP1942 displays a 1.2-fold increase in specific enzyme activity at the optimal cellulase temperature (50 °C), indicating its superior suitability for cellulase compounding. CBP1942 exhibits Km and kcat values of 6.69 mM and 8.83 s−1, respectively, with a kcat/Km ratio 6.6 times higher than CtCBP, suggesting superior catalytic efficiency. Notably, CBP1942 shows remarkable thermal stability, retaining 94.67% enzyme activity after 3 h at 50 °C, while CtCBP retains only 78.33%. Additionally, CBP1942 complexed with cellulase enhances the conversion of corn stover, resulting in a 6.2-fold increase in glucose-1-phosphate yield compared to CtCBP. These findings indicate CBP1942’s potential as an industrial enzyme for corn stover conversion to glucose-1-phosphate. [ABSTRACT FROM AUTHOR]

Cellobiose has received increasing attention in various industrial sectors, ranging from food and feed to cosmetics. The development of large-scale cellobiose applications requires a cost-effective production technology as currently used methods based on cellulose hydrolysis are costly. Here, a one-pot synthesis of cellobiose from sucrose was conducted using a recombinant Pichia pastoris strain as a reusable whole-cell biocatalyst. Thermophilic sucrose phosphorylase from Bifidobacterium longum (BlSP) and cellobiose phosphorylase from Clostridium stercorarium (CsCBP) were co-displayed on the cell surface of P. pastoris via a glycosylphosphatidylinositol-anchoring system. Cells of the BlSP and CsCBP co-displaying P. pastoris strain were used as whole-cell biocatalysts to convert sucrose to cellobiose with commercial thermophilic xylose isomerase. Cellobiose productivity significantly improved with yeast cells grown on glycerol compared to glucose-grown cells. In one-pot bioconversion using glycerol-grown yeast cells, approximately 81.2 g/L of cellobiose was produced from 100 g/L of sucrose, corresponding to 81.2% of the theoretical maximum yield, within 24 h at 60 °C. Moreover, recombinant yeast cells maintained a cellobiose titer > 80 g/L, even after three consecutive cell-recycling one-pot bioconversion cycles. These results indicated that one-pot bioconversion using yeast cells displaying two phosphorylases as whole-cell catalysts is a promising approach for cost-effective cellobiose production. [ABSTRACT FROM AUTHOR]

The economical and efficient commercial production of second-generation bioethanol requires fermentation microorganisms capable of entirely and rapidly utilizing all sugars in lignocellulosic hydrolysates. In this study, we developed a recombinant Saccharomyces cerevisiae strain, BLH510, through protoplast fusion and metabolic engineering to enhance its ability to co-ferment glucose, xylose, cellobiose, and xylooligosaccharides while tolerating various inhibitors commonly found in lignocellulosic hydrolysates. The parental strains, LF1 and BLN26, were selected for their superior glucose/xylose co-fermentation capabilities and inhibitor tolerance, respectively. The fusion strain BLH510 demonstrated efficient utilization of mixed sugars and high ethanol yield under oxygen-limited conditions. Under low inoculum conditions, strain BLH510 could completely consume all four kinds of sugars in the medium within 84 h. The fermentation produced 33.96 g/L ethanol, achieving 84.3% of the theoretical ethanol yield. Despite the challenging presence of mixed inhibitors, BLH510 successfully metabolized all four sugars above after 120 h of fermentation, producing approximately 30 g/L ethanol and reaching 83% of the theoretical yield. Also, strain BLH510 exhibited increased intracellular trehalose content, particularly under conditions with mixed inhibitors, where the intracellular trehalose reached 239.3 mg/g yeast biomass. This elevated trehalose content contributes to the enhanced stress tolerance of BLH510. The study also optimized conditions for protoplast preparation and fusion, balancing high preparation efficiency and satisfactory regeneration efficiency. The results indicate that BLH510 is a promising candidate for industrial second-generation bioethanol production from lignocellulosic biomass, offering improved performance under challenging fermentation conditions. Our work demonstrates the potential of combining protoplast fusion and metabolic engineering to develop superior S. cerevisiae strains for lignocellulosic bioethanol production. This approach can also be extended to develop robust microbial platforms for producing a wide array of lignocellulosic biomass-based biochemicals. [ABSTRACT FROM AUTHOR]

Ru/N-doped activated carbon/NbOPO4 material bearing unique surface chemistry (Brønsted acidic NbOPO4, Lewis basic N-groups and oxygen functional groups) is shown to exhibit exceptional catalytic activity upon hydrolytic hydrogenation of cellobiose into sorbitol under mild conditions. The multifunctional catalyst was synthesized mechanically grinding NbOPO4 with Ru/N-doped activated carbon under ambient conditions and characterized by standard techniques. The multifunctional catalyst outperformed the benchmark Ru catalysts viz. Ru/carbon, Ru/N-doped activated carbon, Ru/SBA-15, Ru/NbOPO4, Ru/Nb2O5, and Ru/NbOPO4-N-doped activated carbon (obtained by sequential deposition of NbOPO4 and Ru on N-doped carbon) with respect to desired product yield as well as carbon balance closure. Under optimized conditions (140 °C, 4 h, and 20 bar H2), sorbitol yields up to ~ 90% could be obtained with the Ru/N-doped carbon/NbOPO4 catalyst; analysis of reaction products in the temperature range of 130–150 °C revealed cellobitol as the main intermediate. Most importantly, the catalytic material demonstrated good operational stability and demonstrated excellent stability in a fixed-bed reactor under continuous flow conditions, maintaining constant activity, and sorbitol selectivity during ~ 110 h operation. The exceptional performance and stability of the multifunctional catalyst were attributed to the surface chemistry of carbon support that stabilized the Ru nanoparticles, and the acidic NbOPO4 phase promoting efficient and selective hydrolysis of cellobiose and cellobitol. [ABSTRACT FROM AUTHOR]

Due to the continuous rise in global incidence and severity of invasive fungal infections (IFIs), particularly among immunocompromised and immunodeficient patients, there is an urgent demand for swift and accurate fungal pathogen diagnosis. Therefore, the need for fungal‐specific positron emission tomography (PET) imaging agents that can detect the infection in the early stages is increasing. Cellobiose, a disaccharide, is readily metabolized by fungal pathogens such as Aspergillus species. Recently, our group reported fluorine‐18 labeled cellobiose, 2‐deoxy‐2‐[18F]fluorocellobiose ([18F]FCB), for specific imaging of Aspergillus infection. The positive imaging findings with very low background signal on delayed imaging make this ligand a promising fungal‐specific imaging ligand. Inspired by this result, the decision was made to automate the radiolabeling procedure for better reproducibility and to facilitate clinical translation. A Trasis AllInOne (Trasis AIO) automated module was used for this purpose. The reagent vials contain commercially available 2‐deoxy‐2‐[18F]fluoroglucose ([18F]FDG), glucose‐1‐phosphate, and enzyme (cellobiose phosphorylase). A Sep‐Pak cartridge was used to purify the tracer. The overall radiochemical yield was 50%–70% (n = 6, decay corrected) in 75‐min synthesis time with a radiochemical purity of > 98%. This is a highly reliable protocol to produce current good manufacturing practice (cGMP)–compliant [18F]FCB for clinical PET imaging. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[18F]-fluoro-d-glucose ([18F]FDG), the most common tracer used in clinical imaging, to form [18F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [18F]FDG was reacted with β-d-glucose-1-phosphate in the presence of maltose phosphorylase, the α-1,4- and α-1,3-linked products 2-deoxy-[18F]-fluoro-maltose ([18F]FDM) and 2-deoxy-2-[18F]-fluoro-sakebiose ([18F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (β-1,3), and cellobiose (β-1,4) phosphorylases to synthesize 2-deoxy-2-[18F]fluoro-trehalose ([18F]FDT), 2-deoxy-2-[18F]fluoro-laminaribiose ([18F]FDL), and 2-deoxy-2-[18F]fluoro-cellobiose ([18F]FDC). We subsequently tested [18F]FDM and [18F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. Both [18F]FDM and [18F]FSK were stable in human serum with high accumulation in preclinical infection models. The synthetic ease and high sensitivity of [18F]FDM and [18F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of these tracers to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [18F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.

Abstract Formic acid is considered a promising hydrogen carrier. Biomass‐derived formic acid can be obtained by oxidative decomposition of sugars. This study explored the production of formic acid from cellobiose, a disaccharide consisting of d‐glucose linked by β‐glycosidic bonds using heterogeneous catalysts under mild reaction conditions. The use of alkaline earth metal oxide solid base catalysts like CaO and MgO in the presence of hydrogen peroxide could afford formic acid from cellobiose at 343 K. While CaO gave 14 % yield of formic acid, the oxide itself was converted to a harmful metal peroxide, CaO2 after the reaction. In contrast, MgO could produce formic acid without the formation of the metal peroxide. The difficulty in selectively synthesizing formic acid from cellobiose using these solid base catalysts was due to the poor conversion of cellobiose to glucose. Using a combination of solid acid and base catalysts, a high formic acid yield of 33 % was obtained under mild reaction conditions due to the quantitative hydrolysis of cellobiose to glucose by a solid acid followed by the selective decomposition of glucose to formic acid by a solid base.

Low-cost plant substrates, such as soybean hulls, are used for various industrial applications. Filamentous fungi are important producers of Carbohydrate Active enZymes (CAZymes) required for the degradation of these plant biomass substrates. CAZyme production is tightly regulated by several transcriptional activators and repressors. One such transcriptional activator is CLR-2/ClrB/ManR, which has been identified as a regulator of cellulase and mannanase production in several fungi. However, the regulatory network governing the expression of cellulase and mannanase encoding genes has been reported to differ between fungal species. Previous studies showed that Aspergillus niger ClrB is involved in the regulation of (hemi-)cellulose degradation, although its regulon has not yet been identified. To reveal its regulon, we cultivated an A. niger ΔclrB mutant and control strain on guar gum (a galactomannan-rich substrate) and soybean hulls (containing galactomannan, xylan, xyloglucan, pectin and cellulose) to identify the genes that are regulated by ClrB. Gene expression data and growth profiling showed that ClrB is indispensable for growth on cellulose and galactomannan and highly contributes to growth on xyloglucan in this fungus. Therefore, we show that A. niger ClrB is crucial for the utilization of guar gum and the agricultural substrate, soybean hulls. Moreover, we show that mannobiose is most likely the physiological inducer of ClrB in A. niger and not cellobiose, which is considered to be the inducer of N. crassa CLR-2 and A. nidulans ClrB.

Lactobionic acid (LBA) has diverse number of applications in pharmaceutical, food, chemical and nanotechnology fields. Bioproduction of LBA by bi-enzymatic cellobiose dehydrogenase (CDH)/laccase (LAC) is a system where CDH catalyses oxidation of lactose to LBA, whilst LAC has, at the same time, ability to regenerate the redox mediator as electron acceptor for CDH. In this study, CDH mutants derived from Phanerochaete chrysosporium produced in Pichia pastoris were used to determine LBA production rate with varying number of redox mediators. The highest specific productivity was observed for triple mutant CDH (tmCDH) for all redox mediators used in this study. The notable redox mediator was hydroquinone where specific productivity was 29.14 g L−1 kU−1 for tmCDH while specific productivity with DCIP was 27.2 g L−1 kU−1, with ABTS showed 19.1 g L−1 kU−1. Lactobionic acid production using bi-enzymatic system, CDH/LAC. Regeneration of different redox mediators. Recombinant mutants used in reaction system. [ABSTRACT FROM AUTHOR]

The Ruminiclostridium thermocellum cellulosome system is one of the most efficient cellulase systems in nature. The main hydrolysis product of cellulosic saccharification by R. thermocellum was cellobiose, which served as a strong feedback inhibitor to the cellulosome. In this study, R. thermocellum M3, which directly hydrolyzes cellulosic biomass into monosaccharides, was used to degrade cellobiose to investigate the kinetic characteristics of cellobiose degradation. Moreover, the transcriptomic characteristics of strain M3 under cellobiose and Avicel culture conditions were compared. The results indicated that strain M3 was able to grow and hydrolyze cellobiose under cellobiose concentrations higher than 20 g/L. The best growth performance and glucose production were obtained under 12 g/L cellobiose. Compared with that of Avicel, cellobiose had greater β-glucosidase activity under cellobiose culture, with a maximum glucose yield of 767.97±19.13 mg/g cellobiose. The transcription results indicated that R. thermocellum M3 resisted the feedback inhibition of cellobiose by regulating genes encoding β-glucosidase, cellobiose phosphorylase and ABC transporters. The high glucose yield of strain M3 was closely related to the expression of the bglX gene. In addition, the gene related to the chemotactic pathway of strain M3 also increased its perception and adaptation to cellobiose. [Display omitted] • The cellulosome of R. thermocellum M3 remains active under cellobiose more than 20 g/L. • R. thermocellum hydrolyzed cellobiose related with BGL and non-BGL genes. • BglX was potential responsible for the cellobiose hydrolysis by R. thermocellum. • The findings provide solutions for the biorefined of cellulosic energy and fuels. [ABSTRACT FROM AUTHOR]

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother's milk. They are synthesized by β-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many β-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable β-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the β-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly β(1 → 3) and β(1 → 6) elongating activity, but also some β(1 → 4) elongation was observed. The β-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose. Key points: • β-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield. • Thermotoga maritima β-glucosidase has high activity and high thermostability. • Thermotoga maritima β-glucosidase GOS contains mainly (β1-3) and (β1-6) linkages. [ABSTRACT FROM AUTHOR]

Background: Cellobiose dehydrogenase (CDH) is an extracellular fungal oxidoreductase with multiple functions in plant biomass degradation. Its primary function as an auxiliary enzyme of lytic polysaccharide monooxygenase (LPMO) facilitates the efficient depolymerization of cellulose, hemicelluloses and other carbohydrate-based polymers. The synergistic action of CDH and LPMO that supports biomass-degrading hydrolases holds significant promise to harness renewable resources for the production of biofuels, chemicals, and modified materials in an environmentally sustainable manner. While previous phylogenetic analyses have identified four distinct classes of CDHs, only class I and II have been biochemically characterized so far. Results: Following a comprehensive database search aimed at identifying CDH sequences belonging to the so far uncharacterized class III for subsequent expression and biochemical characterization, we have curated an extensive compilation of putative CDH amino acid sequences. A sequence similarity network analysis was used to cluster them into the four distinct CDH classes. A total of 1237 sequences encoding putative class III CDHs were extracted from the network and used for phylogenetic analyses. The obtained phylogenetic tree was used to guide the selection of 11 cdhIII genes for recombinant expression in Komagataella phaffii. A small-scale expression screening procedure identified a promising cdhIII gene originating from the plant pathogen Fusarium solani (FsCDH), which was selected for expression optimization by signal peptide shuffling and subsequent production in a 5-L bioreactor. The purified FsCDH exhibits a UV-Vis spectrum and enzymatic activity similar to other characterized CDH classes. Conclusion: The successful production and functional characterization of FsCDH proved that class III CDHs are catalytical active enzymes resembling the key properties of class I and class II CDHs. A detailed biochemical characterization based on the established expression and purification strategy can provide new insights into the evolutionary process shaping CDHs and leading to their differentiation into the four distinct classes. The findings have the potential to broaden our understanding of the biocatalytic application of CDH and LPMO for the oxidative depolymerization of polysaccharides. [ABSTRACT FROM AUTHOR]

The food enzyme cellobiose phosphorylase (cellobiose: phosphate α‐d‐glucosyltransferase; EC 2.4.1.20) is produced with the genetically modified Escherichia coli strain LE1B109‐pPB130 by c‐LEcta GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used in combination with a sucrose phosphorylase in the production of the specialty carbohydrate cellobiose. Since residual amounts of total organic solids are removed by downstream purification steps, the Panel considered that toxicological studies other than assessment of allergenicity were unnecessary and a dietary exposure was not estimated. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single- molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well- characterized exo- cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a Ki of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate- binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme's velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the "back door" product release site to slow activity and to the "front door" substrate- binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy. [ABSTRACT FROM AUTHOR]

Cellobiose dehydrogenase (CDH) is applied as a bioelectrocatalyst in biosensors because its mobile cytochrome domain is capable of direct electron transfer. This study investigates the electron transfer mechanism of CDH molecules embedded in the polycation polyethyleneimine (PEI), which has been reported as a current‐boosting component of CDH‐based biosensors. By immobilizing different concentrations of CDH and its isolated cytochrome domain in PEI films, we found that increasing concentrations of cytochrome enhanced the film conductivity (up to 251±8 mS cm−1) through improved electron transfer between the protein redox centers. The increased electrical conductivity of the film contacts CDH molecules at a greater distance from the electrode. The cross‐linker poly(ethylene glycol) diglycidyl ether improves the packing and contacting of the cytochrome domains, whereas glutaraldehyde reduces the current obtained. Deglycosylation of CDH enhances the conductivity of enzyme‐polymer films by up to 34 %, implying a higher number of productive electron‐hopping events between cytochrome domains due to enhanced mobility or reduced shielding. By balancing negative charges on the CDH surface at neutral and alkaline pH, PEI increases the interdomain electron transfer and the electrical film conductivity. The resulting increased current output is relevant for in vivo bioanalytical applications. [ABSTRACT FROM AUTHOR]

Cellobiose dehydrogenase (CDH) is one of the cellulase auxiliary proteins, which is widely used in the field of biomass degradation. However, how to efficiently and cheaply apply it in industrial production still needs further research. Aspergillus niger C112 is a significant producer of cellulase and has a relatively complete lignocellulose degradation system, but its CDH activity was only 3.92 U. To obtain a recombinant strain of A. niger C112 with high cellulases activity, the CDH from the readily available white-rot fungus Grifola frondose had been heterologously expressed in A. niger C112, under the control of the gpdA promoter. After cultivation in the medium with alkali-pretreated poplar fiber as substrate, the enzyme activity of recombinant CDH reached 36.63 U/L. Compared with the original A. niger C112, the recombinant A. niger transformed with Grifola frondosa CDH showed stronger lignocellulase activity, the activities of cellulases, ß-1, 4-glucosidase and manganese peroxidase increased by 28.57, 35.07 and 121.69%, respectively. The result showed that the expression of the gcdh gene in A. niger C112 could improve the activity of some lignocellulose degrading enzymes. This work provides a theoretical basis for the further application of gcdh gene in improving biomass conversion efficiency. [ABSTRACT FROM AUTHOR]

Solar energy is gradually becoming integrated into households, holding the potential to address energy requirements through technologies like PV cells. Ongoing research is actively exploring diverse methods of harnessing solar power, with Photogalvanic cells emerging as a particularly promising alternative to Photovoltaic cells. The advantage lies in the cost-effectiveness and simplified fabrication, coupled with the capability of power storage. The utilization of the economical Dioctyl sulfosuccinate sodium (DOSS) surfactant, widely employed in industry, has yielded impressive electrical performance. The present investigation presents a reliable photogalvanic system composed of the photosensitizer dye Quinoline Yellow, the reductant Cellobiose, and the surfactant Dioctyl sulfosuccinate sodium (DOSS), all in a highly alkaline solution with platinum and graphite electrodes. The platinum electrode employed is notably small, boasting a surface area of 0.03 cm2, which enhances the diffusion characteristics of the dye molecules, it is contributing to an enhanced electrical performance of the photogalvanic cell. The resulting photogalvanic cell demonstrates superior electrical performance, featuring a maximum potential of 870 mV, a maximum current of 8000 µA, power at PowerPoint of 695 µW, a fill factor of 0.11, and a conversion efficiency of 13.78 %. Spectrophotometric analysis has confirmed the stability of the dye within the electrolyte solution. Additionally, conductometric analysis has revealed that the surfactant Dioctyl sulfosuccinate sodium (DOSS) enhances the electrical conductivity of the electrolyte solution.

The hydration structure of cellulose is very important for understanding the hydrolysis of cellulose at the molecular level. In this paper, we report a joint experimental and theoretical study on x-ray absorption spectroscopy (XAS) of aqueous cellobiose, a disaccharide unit of cellulose. In the experimental part, high resolution measurements of the carbon K-edge XAS spectra were taken. In the theoretical part, ab initio molecular dynamics simulations and ensemble calculations of electronic excited states were performed to obtain the continuous XAS spectra. The XAS spectra were found to have three characteristic peaks at 289.3, 290.7, and 293.6 eV, each representing the absorption by carbon atoms of the alcohol group, the hemiacetal group, and both of these functional groups. It was found that the peak heights in the spectrum change considerably over the temperature range of 25–60 °C, which is a reflection of the number of hydrogen bonds between cellobiose and water. We suggest that this spectral change could be useful information for identifying the hydration of cellulose in various environments. [ABSTRACT FROM AUTHOR]

Third-generation biosensors use enzymes capable of direct electron transfer (DET) to the sensor surface. They are of interest for continuous glucose monitoring in blood or interstitial fluid, but they are rarely investigated. One reason is the hindered DET of the enzymes to the metallic electrodes. In this publication, a novel method for the immobilization of cellobiose dehydrogenase (CDH) DET enzymes employing conductive poly(3,4-ethylenedioxythiophene)-poly (styrene sulfonate) (PEDOT:PSS) inks and a protective polyethylene glycol dimethacrylate (PEG-DMA) hydrogel layer on gold electrodes is reported. This layer stack showed a glucose-specific current response for voltages between −0.2 and 0.4 V in physiological PBS buffer, and enabled interference-less sensing in a solution of acetaminophen, ascorbic acid, dopamine, and uric acid at 0 V. A Michaelis–Menten fit led to a maximum current density (Imax) of 257 ± 7.9 nA/mm2 and a Michaelis–Menten constant (Km) of 28.4 ± 2.2 mM, with a dynamic range of 0.1–20 mM glucose and a limit of detection of 0.1 mM. After 16 h of continuous measurement of 20 mM glucose, the signal decreased to 60% of its initial value. Storage stability was successfully verified until up to 10 days. In summary, this paper shows a simplified approach for the fabrication of third-generation biosensors using CDH-PEDOT:PSS and PEG-DMA hydrogel inks. [ABSTRACT FROM AUTHOR]

Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin resulting from the enzymatic hydrolysis of cellulose. It is a major source of carbon for soil bacteria. In bacteria, the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), encoded by the cel operon, is responsible for the transport and utilization of cellobiose. In this study, we analyzed the transcription and regulation of the cel operon in Bacillus thuringiensis (Bt). The cel operon is composed of five genes forming one transcription unit. β-Galactosidase assays revealed that cel operon transcription is induced by cellobiose, controlled by Sigma54, and positively regulated by CelR. The HTHAAA+ domain of CelR recognized and specifically bound to three possible binding sites in the celA promoter region. CelR contains two PTS regulation domains (PRD1 and PRD2), which are separated by two PTS-like domains-the mannose transporter enzyme IIA component domain (EIIAMan) and the galactitol transporter enzyme IIB component domain (EIIBGat). Mutations of His-546 on the EIIAMan domain and Cys-682 on the EIIBGat domain resulted in decreased transcription of the cel operon, and mutations of His-839 on PRD2 increased transcription of the cel operon. Glucose repressed the transcription of the cel operon and catabolite control protein A (CcpA) positively regulated this process by binding the cel promoter. In the celABCDE and celR mutants, PTS activities were decreased, and cellobiose utilization was abolished, suggesting that the cel operon is essential for cellobiose utilization. Bt has been widely used as a biological pesticide. The metabolic properties of Bt are critical for fermentation. Nutrient utilization is also essential for the environmental adaptation of Bt. Glucose is the preferred energy source for many bacteria, and the presence of the phosphotransferase system allows bacteria to utilize other sugars in addition to glucose. Cellobiose utilization pathways have been of particular interest owing to their potential for developing alternative energy sources for bacteria. The data presented in this study improve our understanding of the transcription patterns of cel gene clusters. This will further help us to better understand how cellobiose is utilized for bacterial growth. [ABSTRACT FROM AUTHOR]

Cellulose is the most abundant biomass in nature, and the development of high-value utilization technologies for cellulose is of great significance. In this work, a recombinant strain was engineered by introducing Arabidopsis thaliana glycosyltransferase (AtUGT78D2) and the cellobiose phosphorolysis route to produce astragalin from kaempferol. By optimizing the transformation conditions, the production of astragalin increased from 1053 to 3031 mg/L with the addition of cellobiose. Subsequently, the cellulose enzymatic solution was used to replace cellobiose as the carbon source and UDP-glucose precursor for producing astragalin. By optimizing enzymatic conditions and adding additives, the concentration of cellobiose significantly increased, resulting in an increase in the production of astragalin to 2279 mg/L. The adsorption strategy was employed to modulate the composition and activity of cellulase, and the ratio of cellobiose to glucose in the cellulose enzymatic solution increased from 0.73 to 1.62, with the yield of cellobiose reaching up to 5.9 g/L. Finally, the highest production of astragalin in the recombinant strain reached 2654 mg/L when the optimal ratio of cellobiose to glucose was used as the carbon source. This study provides a novel method for producing astragalin by using the enzymatic hydrolysis of microcrystalline cellulose. [Display omitted] • A recombinant strain was constructed to produce astragalin from kaempferol. • Cellulose enzymatic solution was used to replace cellobiose for astragalin production. • Cellobiose concentration significantly increased through optimizing enzymatic conditions. • The ratio of cellobiose to glucose increased with the adsorption strategy. • Astragalin production reached 2654 mg/L with cellulose enzymatic solution as a carbon source. [ABSTRACT FROM AUTHOR]

The inherent complexity of coupled biocatalytic reactions presents a major challenge for process development with one‐pot multienzyme cascade transformations. Kinetic models are powerful engineering tools to guide the optimization of cascade reactions towards a performance suitable for scale up to an actual production. Here, we report kinetic model‐based window of operation analysis for cellobiose production (≥100 g/L) from sucrose and glucose by indirect transglycosylation via glucose 1‐phosphate as intermediate. The two‐step cascade transformation is catalyzed by sucrose and cellobiose phosphorylase in the presence of substoichiometric amounts of phosphate (≤27 mol% of substrate). Kinetic modeling was instrumental to uncover the hidden effect of bulk microviscosity due to high sugar concentrations on decreasing the rate of cellobiose phosphorylase specifically. The mechanistic‐empirical hybrid model thus developed gives a comprehensive description of the cascade reaction at industrially relevant substrate conditions. Model simulations serve to unravel opposed relationships between efficient utilization of the enzymes and maximized concentration (or yield) of the product within a given process time, in dependence of the initial concentrations of substrate and phosphate used. Optimum balance of these competing key metrics of process performance is suggested from the model‐calculated window of operation and is verified experimentally. The evidence shown highlights the important use of kinetic modeling for the characterization and optimization of cascade reactions in ways that appear to be inaccessible to purely data‐driven approaches. [ABSTRACT FROM AUTHOR]

One‐pot cascade reactions of coupled disaccharide phosphorylases enable an efficient transglycosylation via intermediary α‐d‐glucose 1‐phosphate (G1P). Such transformations have promising applications in the production of carbohydrate commodities, including the disaccharide cellobiose for food and feed use. Several studies have shown sucrose and cellobiose phosphorylase for cellobiose synthesis from sucrose, but the boundaries on transformation efficiency that result from kinetic and thermodynamic characteristics of the individual enzyme reactions are not known. Here, we assessed in a step‐by‐step systematic fashion the practical requirements of a kinetic model to describe cellobiose production at industrially relevant substrate concentrations of up to 600 mM sucrose and glucose each. Mechanistic initial‐rate models of the two‐substrate reactions of sucrose phosphorylase (sucrose + phosphate → G1P + fructose) and cellobiose phosphorylase (G1P + glucose → cellobiose + phosphate) were needed and additionally required expansion by terms of glucose inhibition, in particular a distinctive two‐site glucose substrate inhibition of the cellobiose phosphorylase (from Cellulumonas uda). Combined with mass action terms accounting for the approach to equilibrium, the kinetic model gave an excellent fit and a robust prediction of the full reaction time courses for a wide range of enzyme activities as well as substrate concentrations, including the variable substoichiometric concentration of phosphate. The model thus provides the essential engineering tool to disentangle the highly interrelated factors of conversion efficiency in the coupled enzyme reaction; and it establishes the necessary basis of window of operation calculations for targeted optimizations toward different process tasks. [ABSTRACT FROM AUTHOR]

Oxidoreductase (OXR) enzymes are in high demand for biocatalytic applications in the food industry and cosmetics (glucose oxidase (GOx) and cellobiose dehydrogenase (CDH)), bioremediations (horseradish peroxidase (HRP) and laccase (LAC)), and medicine for biosensors and miniature biofuel cells (GOx, CDH, LAC, and HRP). They can be used in a soluble form and/or within the yeast cell walls expressed as chimeras on the surface of yeast cells (YSD), such as P. pastoris and S. cerevisiae. However, most of the current studies suffer from either low yield for soluble enzyme expression or low enzyme activity when expressed as chimeric proteins using YSD. This is always the case in studies dealing with the heterologous expression of oxidoreductase enzymes, since there is a requirement not only for multiple OXR gene integrations into the yeast genome (super transformations), and codon optimization, but also very careful design of fermentation media composition and fermentation conditions during expression due to the need for transition metals (copper and iron) and metabolic precursors of FAD and heme. Therefore, scientists are still trying to find the optimal formula using the above-mentioned approaches; most recently, researcher started using protein engineering and directed evolution to increase in the yield of recombinant enzyme production. In this review article, we will cover all the current state-of-the-art technologies and most recent advances in the field that yielded a high expression level for some of these enzymes in specially designed expression/fermentation systems. We will also tackle and discuss new possibilities for further increases in fermentation yield using cutting-edge technologies such as directed evolution, protein and strain engineering, high-throughput screening methods based on in vitro compartmentalization, flow cytometry, and microfluidics. [ABSTRACT FROM AUTHOR]

Hydrogen produced from lignocellulosic biomass via dark fermentation is a promising alternative to fossil fuels. This study assessed fermentative H 2 production from single sugars and sugar mixtures mimicking real lignocellulose hydrolysates during a continuous process. The highest hydrogen production rate (HPR) was obtained with pentose sugars, reaching 8.80 and 8.09 L/L-d for arabinose and xylose, respectively. Glucose showed the lowest HPR (6.19 L/L-d), while in the case of sugar mixtures (glucose, cellobiose, xylose, arabinose), HPR was approximately 7.3 L/L-d and was lower than expected from calculations. Thus, cofermentation reduced the H 2 production potential. It was shown that pentose sugars are preferred for hydrogen production, but their utilization is limited by the presence of glucose, which is utilized preferentially in sugar mixtures. Xylose, arabinose and sugar mixtures showed similar H 2 yields (approximately 0.29 mol H 2 /mol C), so the efficiency of substrate conversion into H 2 was comparable for these substrates. The microbial community distribution showed that the Clostridium genus was dominant regardless of the substrate, with Clostridium beijerinckii and Clostridium guangxiense being the main bacterial species. Different substrates did not significantly affect the type of microorganisms present in the microbiome and changed only their relative abundance. [Display omitted] • Long-term continuous fermentative H 2 production from single sugars and their mixtures. • The highest HPR obtained for arabinose (8.80 L/L-d) with 96% substrate utilization. • Sugar mixtures exhibited lower HPRs due to the low consumption of arabinose. • Pentoses are preferred for H 2 production, but their utilization is limited by glucose. • The lowest substrate consumption for glucose resulted in the lowest HPR. [ABSTRACT FROM AUTHOR]

The discovery that certain cellobiose 2-epimerases (C2E) are capable of synthesizing lactulose and epilactose with high yield and selectivity has renewed the interest in their biological synthesis. In order to provide a valuable tool for the design and optimization of the synthesis of lactulose and epilactose with C2E as biocatalyst, the reaction is modeled here for the first time. Two competing reaction mechanisms, so called extended and reduced mechanisms, were proposed and their corresponding models developed. The extended mechanism was discarded because of the overfitting of the resulting model. The parameters of the reduced model were successfully estimated by no linear regression at 30, 40, 50, 60 and 70 °C, and the temperature dependence of the kinetic parameters was modeled by fitting them to Arrhenius and Vant Hoff-type equations. The reduced model showed a good fit to experimental data within the studied temperature range, well describing the course of reactions. [ABSTRACT FROM AUTHOR]

The present study was undertaken to investigate the difference in carbohydrate metabolization between Listeria ivanovii wild-type strain and a pediocin-resistant variant. The growth and organic acid production of both L. ivanovii strains under different carbohydrate conditions in the presence or absence of pediocin were investigated. At the genetic level, the mannose phosphotransferase system (Man-PTS) related genes were analysed at the transcriptional level. In contrast to the rapid growth of wild-type strain cultured in mannose and glucose, cellobiose appeared to be the most efficiently utilized carbohydrate for the resistant variant. Pediocin addition caused a decline in OD600, which for the resistant variant was especially pronounced in the mannose medium. The resistant variant cultured in cellobiose displayed the highest lactic acid production in the presence of pediocin. Upregulation of mptA, mptC and mptD was detected in the L. ivanovii resistant cells cultured in glucose and mannose media, while downregulation of these genes was observed when cultured in the presence of cellobiose and fructose. These results suggested that there were inherent connection between carbohydrate metabolism and L. ivanovii sensitivity to pediocin, which may provide clue for unraveling mechanism of resistance development. [ABSTRACT FROM AUTHOR]

Cellobiose dehydrogenase (CDH) catalyzes the oxidation of cello‐oligosaccharides and transfers electrons to oxygen or other electron receptors, such as lytic polysaccharide oxygenases (LPMOs). Cellobiose dehydrogenase has the potential to be used as an electron donor for LPMOs. Therefore, it is important to obtain CDHs under reaction conditions that are compatible with those of cellulases and LPMOs. Here, a CDH gene from Myceliophthora thermophila (MtCDH‐A) was constitutively expressed in Trichoderma reesei, and its ability to donate electrons to the LPMO was validated. MtCDH‐A has an optimal pH of 6.0 and maintains relatively high activity over a wide pH range. The optimal temperature was 60°C, and it remained stable at 50°C for a long time. These enzymatic properties were compatible with the catalytic conditions of fungal LPMOs and cellulases. When MtCDH‐A was used as an electron donor, the synergistic effect of LPMO and cellulase improved the degradation efficiency of phosphoric acid‐swollen cellulose by 141%. This work provides a novel insight into finding a stable electron donor for LPMOs and a new direction for finding more efficient thermostable CDHs in newly identified fungal species. © 2023 Society of Industrial Chemistry and John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

Cocoa production in Indonesia is increasing from year to year. Cocoa production increased has an impact on increasing waste cocoa pod and pulp. Cocoa pod contains lignocellulose, while cocoa pulp contains many sugars. The composition of cocoa pod and pulp allows the cellulolytic microbe to grow. This study aims to perform microbial selection from fermented cocoa and sugar cane can grow in cocoa pod and pulp (Theobroma cacao). The selection method using the CMC 1%, pod 1%, pulp 1%, and pod 0,5%+pulp 0,5% media. Then, the analysis of reducing sugar using DNS method, while the total sugar analysis using phenol-H2SO4 method. Sugars of hydrolisis result were analyzed quantitatively using TLC. The results of microbial selection on CMC 1%, pod %, pulp 1%, and pod 0,5%+pulp 0,5% media obtained TBT 3.2 have the largest cellulolytic index on the pod 1% media. Isolates TBT 3.2 grown on pod 1% medium produce reducing sugar is 4,965 mg/mL at 30 hours sampling with highest total sugar at 6 hours sampling is 9,789 mg/mL. The types of sugars identified using the TLC are mannose, galactose, glucose, and selobiose.

Developing recombinant Saccharomyces cerevisiae strains capable of transporting and fermenting cellobiose directly is a promising strategy for second-generation ethanol production from lignocellulosic biomass. In this study, we cloned and expressed in the S. cerevisiae CEN.PK2-1C strain an intracellular β-glucosidase (SpBGL7) from Spathaspora passalidarum and co-expressed the cellobiose transporter SiHXT2.4 from Scheffersomyces illinoinensis, and two putative transporters, one from Candida tropicalis (CtCBT1 gene), and one from Meyerozyma guilliermondii (MgCBT2 gene). While all three transporters allowed cell growth on cellobiose, only the MgCBT2 permease allowed cellobiose fermentation, although cellobiose consumption was incomplete. The analysis of the β-glucosidase and transport activities revealed that the cells stopped consuming cellobiose due to a drop in the transport activity. Since ubiquitinylation of lysine residues at the N- or C-terminal domains of the permease are involved in the endocytosis and degradation of sugar transporters, we constructed truncated versions of the permease lacking lysine residues at the C-terminal domain (MgCBT2ΔC), and at both the C- and N-terminal domain (MgCBT2ΔNΔC) and co-expressed these permeases with the SpBGL7 β-glucosidase in an industrial strain. While the strain harboring the MgCBT2ΔC transporter continued to produce incomplete cellobiose fermentations as the wild-type MgCBT2 permease, the strain with the MgCBT2ΔNΔC permease was able to consume and ferment all the cellobiose present in the medium. Thus, our results highlight the importance of expressing cellobiose transporters lacking lysine at the N- and C-terminal domains for efficient cellobiose fermentation by recombinant S. cerevisiae.

Conversion of delignified cellulose from rice husk biomass, and model compounds of cellobiose and glucose to levulinic acid (LA) over hierarchical Mn3O4/ZSM-5 catalyst was carried out using a household microwave method, and then compared to the established conventional thermos-reaction method. The hierarchical ZSM-5 was prepared using a double template method, aiming for micro and mesoporous systems developed in the structure. The as-prepared ZSM-5 were modified with Mn3O4 through incipient wetness impregnation with Mn2+ solution followed by calcination at 550 °C. The catalysts were characterized using various techniques such as powder XRD, SEM, BET, AAS, and FT-IR which indicated the hierarchical structure of MFI zeolite (Si/Al of 30–34) with Mn loading of 2.14 wt%. The conversion products were analyzed using HPLC, 1H NMR, and 13C NMR instruments. The microwave-assisted reaction using 600 W for 180 s using delignified cellulose, cellobiose, and glucose gave conversion of 37.27%, 46.35%, and 54.29%, respectively which is close to the conversion given by the conventional reaction carried out at 130 °C for 4 h (36.75%, 55.62%, and 60.9%, respectively). Interestingly, the LA yield from the microwave-assisted reaction (4.33%, 6.12%, and 9.57%) is higher than the yield from the conventional reaction, which only produced 5.2%, 4.88%, and 6.93% respectively. The microwave-assisted method is also shown to give less by-products compared to the thermochemical reaction. Therefore, it could be considered an alternative method for converting cellulose to LA.

The conversion of lignocellulosic biomass to second-generation biofuels through enzymes is achieved at a high cost. Filamentous fungi through a combination of oxidative enzymes can easily disintegrate the glycosidic bonds of cellulose. The combination of cellobiose dehydrogenase (CDH) with lytic polysaccharide monooxygenases (LPMOs) enhances cellulose degradation in many folds. CDH increases cellulose deconstruction via coupling the oxidation of cellobiose to the reductive activation of LPMOs by catalyzing the addition of oxygen to C-H bonds of the glycosidic linkages. Fungal LPMOs show different regio-selectivity (C1 or C4) and result in oxidized products through modifications at reducing as well as nonreducing ends of the respective glucan chain. T. reesei LPMOs have shown great potential for oxidative cleavage of cellobiose at C1 and C4 glucan bonds, therefore, the incorporation of heterologous CDH further increases its potential for biofuel production for industrial purposes at a reduced cost. We introduced CDH of Phanerochaete chrysosporium (PcCDH) in Trichoderma reesei (which originally lacked CDH). We purified CDH through affinity chromatography and analyzed its enzymatic activity, electron-donating ability to LPMO, and the synergistic effect of LPMO and CDH on cellulose deconstruction. The optimum temperature of the recombinant PcCDH was found to be 45 °C and the optimum pH of PcCDH was observed as 4.5. PcCDH has high cello-oligosaccharide kcat, Km, and kcat/Km values. The synergistic effect of LPMO and cellulase significantly improved the degradation efficiency of phosphoric acid swollen cellulose (PASC) when CDH was used as the electron donor. We also found that LPMO undergoes auto-oxidative inactivation, and when PcCDH is used an electron donor has the function of a C1-type LPMO electron donor without additional substrate increments. This work provides novel insights into finding stable electron donors for LPMOs and paves the way forward in discovering efficient CDHs for enhanced cellulose degradation. [ABSTRACT FROM AUTHOR]

Polysaccharide oxidative depolymerization is highly desirable to achieve recalcitrant biomass valorization. Inspired by recently discovered Lytic Polysaccharide Monooxygenases, mononuclear copper complexes have been prepared and studied in the literature. However, the activities were evaluated on different substrates and under various conditions. In this work we intended to establish a robust and reproducible activity assay, in aqueous solution at a pH close from neutrality and under mild conditions. We have evaluated several complexes on substrates of increasing complexity: the model substrate para‐nitrophenyl‐β‐D‐glucopyranoside (p‐NPG), cellobiose (glucose dimer), as well as on extended substrates (chitin, cellulose and bagasse from agave). The different assays were compared and proof‐of‐concept that bioinspired complexes can oxidatively promote polysaccharide depolymerization was obtained. Finally, we measured level of hydroxyl radicals released by the complexes under comparable experimental conditions and mechanistic pathways are discussed. [ABSTRACT FROM AUTHOR]

Overcoming key bottlenecks for 2G ethanol production, such as the most suitable biomass pretreatment, process layout, and appropriate microorganism, is crucial to achieve a technically and economically feasible technology. The consolidated bioprocessing (CBP) is an approach that embodies one single step of enzyme production, biomass hydrolysis, and sugars fermentation. In this work, the CBP using S. cerevisiae AC14, a recombinant strain capable of secreting 7 different lignocellulosic enzymes and ferment xylose and glucose, was investigated in synthetic medium (cellobiose, corncob xylan, glucose, and xylose) and in industrial medium consisting of the solid fraction of hydrothermally pretreated sugarcane bagasse and its liquor. Results obtained were promising, with ethanol productivity of 4.46 g/L/h and complete hydrolysis of cellobiose and corncob xylan. In real industrial medium, 1.86 g/L/h of ethanol was reached, with partial conversion of both solid and liquid fractions. Further strategies to enhance enzymes access to complex substrates can improve process feasibility. [ABSTRACT FROM AUTHOR]

Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose into α-glucose 1-phosphate and glucose. A CBP with a broadened substrate specificity would be more desirable when utilized to convert cellulose into amylose (PNAS, 110: 7182–7187, 2013) and to construct yeast that can phosphorolytically use cellodextrin to produce ethanol. Based on the structure differences in the catalytic loops of CBP and cellodextrin phosphorylase from Clostridium thermocellum (named CtCBP and CtCDP, respectively), CtCBP was mutated to change its substrate specificity. A single-site mutant S497G was identified to exhibit a 5.7-fold higher catalytic efficiency with cellotriose as a substrate in the phosphorolytic reaction compared to the wild type, without any loss of catalytic efficiency on its natural substrate, cellobiose. When the S497G variant was used in the transformation of mixed cellodextrin (cellobiose + cellotriose) to amylose, the amylose yield was significantly increased compared to that of wild-type CtCBP. A structure change in the substrate-binding pocket of the S497G variant accounted for its capacity to accept longer cellodextrins than cellobiose. Taken together, the modified CtCBP, S497G was confirmed to acquire a promising feature favorable to those application scenarios involving cellodextrin's phosphorolysis. [ABSTRACT FROM AUTHOR]