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Low nutritional value and digestive efficiency restrict the feeding application of brewer's spent grain (BSG). Bacillus velezensis has been increasingly considered a promising fermenting agent in feed production. This study aimed to investigate the fermentative characteristics of Bacillus velezensis K8 on degrading lignocellulose and increasing soluble sugar and protein in BSG. The lignocellulose contents (including cellulose, hemicellulose and lignin) decreased by 36.85 % and 27.68 % in BSG and ultrasonic-pretreated BSG (UBSG), respectively. The reducing sugar contents increased by 226.8 % and 198.1 %, and the soluble protein contents increased by 260.7 % and 258.3 % in BSG and UBSG, respectively. The arabinose, cellobiose, xylose, glucose, fructose, and xylooligosaccharides contents were significantly increased, indicating effective conversion of cellulose and hemicellulose into soluble sugars. High cellulase activity was obtained in BSG fermentation, especially CMCase and filter paper activities. Protease secretion increased but cellulase decreased, and the activities of xylanase and fructofuranosidase remained stable in UBSG fermentation. Genome analysis revealed that B. velezensis K8 possessed more amino acid-metabolism genes than carbohydrate-metabolism genes, illustrating that B. velezensis K8 preferentially utilized protein nutrients but left more sugars. These results provide a promising strategy for improving the value of grain residue in food and feed industrial applications.

Two genes encoding lytic polysaccharide monooxygenases (LPMOs) CsLPMO9A and CsLPMO9B were cloned from the white-rot fungus Ceriporiopsis subvermispora and successfully expressed in Komagataella phaffii. The oxidative degradation of phosphoric acid swollen cellulose by the LPMOs was conducted using ascorbic acid as an electron donor. Analysis with high performance anion-exchange chromatography and matrix assisted laser desorption/ionization-time-of-flight mass spectrometry indicated that the two enzymes cleaved the polysaccharides by oxidization of the C1 carbon atom of the glucose unit. Synergistic effects between CsLPMO9A or CsLPMO9B and a commercial cellulase cocktail from Trichoderma reesei were observed in the saccharification of delignified wheat straw. The amounts of glucose, cellobiose and xylose released from the substrate by the combined enzymes were increased by 83 %, 16 % and 47 % (for CsLPMO9A), or 79 %, 52 % and 81 % (for CsLPMO9B), compared to the cellulase alone, respectively. The two new enzymes displayed promising potential in industrial bioconversion of lignocellulosic biomass.

We previously reported an in vitro enzymatic pathway for conversion of nonfood cellulose to starch (PNAS,110 (18): 7182-7187, 2013), in which the two sequential enzymes cellobiose phosphorylase (CBP) from Clostridium thermocellum and potato alpha-glucan phosphorylase (PGP) from Solanum tuberosum were the two key enzymes responsible for the whole conversion rate. In this work CBP and PGP were fused to form a large enzyme and it turned out that the fusion protein could exhibit a good bifunctionality when PGP moiety was put at the N-terminus and CBP moiety at the C-terminus (designated as PGP-CBP). Although the coupled reaction rate of PGP-CBP was decreased by 23.0% compared with the free enzymes, substrate channeling between the two active sites in PGP-CBP was formed, demonstrated by the introduction of the competing enzyme of PGP to the reaction system. The potential of PGP-CBP fusion enzyme being applied to the conversion of cellulose to amylose was discussed.

Single cultures and co-cultures of Bacillus licheniformis and Bacillus paralicheniformis isolated from compost were evaluated for their carboxymethyl cellulase (CMCase) and filter paperase (FPase) production potential. Using a medium supplemented with microcrystalline cellulose (MCC), in the co-culture, CMCase and FPase activities increased 8.87- and 2.28-fold and 10.15- and 3.20-fold over B. licheniformis and B. paralicheniformis monocultures, respectively. The synergistic behavior of the two isolates might be due to the consumption of hydrolysis product (glucose, cellobiose) by one or both of the isolates, which improved their metabolic performance for cellulase secretion. Optimal conditions for cellulase production by this co-culture were a temperature of 45 °C, and pH 7 at 180 rpm in a medium containing rice bran at 1% (w/v) and chicken manure as nitrogen supplement at 2% (w/v). The maximum CMCase and FPase produced under the above conditions were 79.8 U/mL and 12.5 U/mL, respectively. This corresponds to 257.4- and 59.5-fold enhancement in CMCase and FPase activity, respectively, over B. licheniformis monoculture, and 306.9- and 83.3-fold increase with respect to the B. paralicheniformis monoculture. These results indicate that improved cellulase production can be achieved through co-culture and chicken manure nitrogen-supplement.

Although engineered Saccharomyces cerevisiae fermenting cellobiose is useful for the production of biofuels from cellulosic biomass, cellodextrin accumulation is one of the main problems reducing ethanol yield and productivity in cellobiose fermentation with S. cerevisiae expressing cellodextrin transporter (CDT) and intracellular β-glucosidase (GH1-1). In this study, we investigated the reason for the cellodextrin accumulation and how to alleviate its formation during cellobiose fermentation using engineered S. cerevisiae fermenting cellobiose. From the series of cellobiose fermentation using S. cerevisiae expressing only GH1-1 under several culture conditions, it was discovered that small amounts of GH1-1 were secreted and cellodextrin was generated through trans-glycosylation activity of the secreted GH1-1. As GH1-1 does not have a secretion signal peptide, non-conventional protein secretion might facilitate the secretion of GH1-1. In cellobiose fermentations with S. cerevisiae expressing only GH1-1, knockout of TLG2 gene involved in non-conventional protein secretion pathway significantly delayed cellodextrin formation by reducing the secretion of GH1-1 by more than 50%. However, in cellobiose fermentations with S. cerevisiae expressing both GH1-1 and CDT-1, TLG2 knockout did not show a significant effect on cellodextrin formation, although secretion of GH1-1 was reduced by more than 40%. These results suggest that the development of new intracellular β-glucosidase, not influenced by non-conventional protein secretion, is required for better cellobiose fermentation performances of engineered S. cerevisiae fermenting cellobiose.

High cost of lignocellulases restricts the commercialization of biofuel and bio‐product production from lignocellulosic biomass. Constitutively expressed lignocellulases are considered to degrade cellulose to release small amount of soluble cellodextrins such as cellobiose for further large‐scale production of lignocellulases; however, the underlying mechanism remains to be elucidated. Here, a triple β‐glucosidase mutant of the model fungus Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to perform parallel analyses of proteome and phosphoproteome changes in response to cellobiose and Avicel cellulose. The results revealed shared proteome and phosphoproteome responses to cellobiose and Avicel, corroborating the idea that cellobiose mediates the regulation of lignocellulase expression and secretion. The results further suggest that this regulation is achieved at multiple levels, including epigenetic, transcription, post‐transcription, translation, and post‐translation. Proteome profiling revealed that the proteins upregulated by cellobiose and Avicel were over‐represented in cellulose degradation and degradation product transport pathways. Phosphoproteome profiling revealed that the proteins differentially phosphorylated by cellobiose and Avicel were over‐represented by the pathways such as transcriptional control, protein processing and export, cell wall biogenesis, and cellular signaling. Deletion mutation analysis further suggests that the ER chaperon protein Hsp70‐6, the translocation complex subunit Sec66/Sec71, and the signal peptidase subunit Spc2 are involved in lignocellulase secretion, particularly translocation across the endoplasmic reticulum. Altogether, the results offer a new insight into how cellobiose mediates the regulation of lignocellulase expression and secretion, providing a potential strategy for the strain engineering to improve lignocellulase production.

BACKGROUND Epilactose, a potential prebiotics, was derived from lactose through enzymatic catalysis. However, production and purification of epilactose are currently difficult due to powerless enzymes and inefficient downstream processing steps. RESULTS The encoding gene of cellobiose 2-epimerase (CE) from Caldicellulosiruptor sp. Rt8.B8 was cloned and expressed in Escherichia coli BL21(DE3). The enzyme was purified and it was suitable for industrial production of epilactose from lactose without by-products, because of high kcat (197.6 s-1 ) and preferable thermostability. The Rt8-CE gene was further expressed in the Bacillus subtilis strain. We successfully produced epilactose from 700 g L-1 lactose in 30.4% yield by using the recombinant Bacillus subtilis whole cells. By screening of a β-galactosidase from Bacillus stearothermophilus (BsGal), a process for separating epilactose and lactose was established, which showed a purity of over 95% in a total yield of 69.2%. In addition, a mixed rare sugar syrup composed of epilactose and d-tagatose was successfully produced from lactose through the co-expression of l-arabinose isomerase and β-galactosidase. CONCLUSION Our study shed light on the efficient production of epilactose using a food-grade host expressing a novel CE enzyme. Moreover, an efficient and low-cost process was attempted to obtain high purity epilactose. In order to improve the utilization of raw materials, the production process of mixed syrup containing epilactose and d-tagatose with prebiotic properties produced from lactose was also established for the first time. © 2021 Society of Chemical Industry.

Cost-competitive production of lignocellulosic H2 using biological methods is limited by energy-intensive pretreatments and expensive cellulase hydrolysis. Here we report stable two-stage fermentation to convert unpretreated energy sorghum without added cellulase to H2, where Clostridium thermocellum was fed semi-continuously with 20 g/L sorghum in the first stage and then Rhodobacter sphaeroides consumed the effluent wastes in the second stage to achieve residence times (RTs) of 60, 48, and 24 h. Glutamic acid was the preferred nitrogen source for coordinating the two-stage, two-organism fermentation. 25.0 ± 1.1 mL-H2/day in consolidated bioprocessing stage and 133.0 ± 1.5 mL-H2/day in photofermentation stage were obtained for 200 mL cultures at RT 48 h. Propionate is the main product accumulated, with cellobiose, xylose, arabinose, formate, acetate and ethanol also observed in the effluent wastes, which were degraded effectively by Rhodobacter sphaeroides with COD removal of 72.6%. These results provide references for developing low-cost H2 production from lignocellulose.

Enzymatic cellulose hydrolysis is an important step for the production of second-generation biofuels. The filamentous fungus Trichoderma reesei is among the most important organisms for obtaining cellulolytic enzymes. The Cel6A (CBH II) cellulase from T. ressei plays an important role in cellulose hydrolysis and acts on the non-reducing end of cellulose, in contrast to Cel7B (CBH I), which acts on the reducing end of cellulose thus releasing cellobiose. Therefore, Cel6A deficiency becomes a limiting factor in cellulose saccharification. This work attempted to use codon optimization to enhance Cel6A expression in Escherichia coli. A plasmid expression vector, pUCITD04, was designed; this vector contains: the cel6a gene, regulatory regions (the promoter and terminator T7 sequences), the OmpT signal peptide that allows the secretion of proteins into the culture medium, and a 6His tail to allow purification of the protein by affinity chromatography. The protein expression experiment using a strain of E. coli transformed with pUCITD04 resulted in a 31 kDa polypeptide being secreted into the culture medium that did not possess enzymatic activity, meanwhile, the control strain transformed with the empty plasmid did not secrete any protein fragments, indicating that a truncated Cel6A was being produced by the experimental strain. This phenomenon has been reported during the production of recombinant cellulases in E. coli. In this research, we discuss probable causes of this phenomenon, as well as the drawbacks in the production of cellulases by E. coli, directing efforts to elucidate the causes of the production of truncated cellulases by this bacterial factory. Key words: Gene construct, recombinant cellobiohydrolase, Escherichia coli.

β-glucosidases (BGLs) belong to the group of enzymes of cellulases and acts in the last stage of cellulose degradation, releasing glucose molecules, eliminating the inhibitory effect of cellobiose. This study focused on the production, characterization, and application of β-glucosidase from Moniliophthora perniciosa in the hydrolysis of pre-treated sugarcane bagasse (3% NaOH + 6% Na2 SO3 ), with varying enzymatic loads and reaction times. The enzyme showed an optimum pH of 4.5 and 60°C. It was stable at all temperatures analyzed (50-90°C) and retained about 100% of its activity at 50°C after 60 min of incubation. Among the ions analyzed, BaCl2 increased BGL activity 9.04 ± 1.41 times. The maximum production of reducing sugars (89.15%) was achieved after 48 h with 10 mg of protein. This article is protected by copyright. All rights reserved.

To improve the efficiency of isolating and culturing lichen mycobionts, we performed a growth assay on an Australian strain of the soil-crust lichenized fungus Endocarpon pusillum Hedw. This assay determined the preferred nitrogen and carbon sources of the fungus by limiting the available nitrogen or carbon sources to single compounds found in soils, plants and lichen thalli. We found that the non-proteinaceous amino acid, GABA, produced the most growth of all nutrients when provided as the sole nitrogen source but was a poor carbon source. Fructose, glucose, cellobiose and sorbitol produced the most growth of all the carbon sources tested. Ammonium, nitrate and polyamines were poor nutrient sources. These findings correspond with reports of primary metabolite pools in other lichen species and may guide future studies involving growth of recalcitrant lichen mycobionts.

Background β-Glucosidases are essential for cellulose hydrolysis by catalyzing the final cellulolytic degradation of cello-oligomers and cellobiose to glucose. D2-BGL is a fungal glycoside hydrolase family 3 (GH3) β-glucosidase isolated from Chaetomella raphigera with high substrate affinity, and is an efficient β-glucosidase supplement to Trichoderma reesei cellulase mixtures for the saccharification of lignocellulosic biomass. Results We have carried out error-prone PCR to further increase catalytic efficiency of wild-type (WT) D2-BGL. Three mutants, each with substitution of two amino acids on D2-BGL, exhibited increased activity in a preliminary mutant screening in Saccharomyces cerevisiae. Effects of single amino acid replacements on catalysis efficiency and enzyme production have been investigated by subsequent expression in Pichia pastoris. Substitution F256M resulted in enhancing the tolerance to substrate inhibition and specific activity, and substitution D224G resulted in increasing the production of recombinant enzyme. The best D2-BGL mutant generated, Mut M, was constructed by combining beneficial mutations D224G, F256M and Y260D. Expression of Mut M in Pichia pastoris resulted in 2.7-fold higher production of recombinant protein, higher Vmax and greater substrate inhibition tolerance towards cellobiose relative to wild-type enzyme. Surprisingly, Mut M overexpression induced the ER unfolded protein response to a level lower than that with WT D2 overexpression in P. pastoris. When combined with the T. reesei cellulase preparation Celluclast 1.5L, Mut M hydrolyzed acid-pretreated sugarcane bagasse more efficiently than WT D2. Conclusions D2-BGL mutant Mut M was generated successfully by following directed evolution approach. Mut M carries three mutations that are not reported in other directed evolution studies of GH3 β-glucosidases, and this mutant exhibited greater tolerance to substrate inhibition and higher Vmax than wild-type enzyme. Besides the enhanced specific activity, Mut M also exhibited a higher protein titer than WT D2 when it was overexpressed in P. pastoris. Our study demonstrates that both catalytic efficiency and productivity of a cellulolytic enzyme can be enhanced via protein engineering.

BACKGROUND: High‐efficiency saccharification technology is one of the bottlenecks of cellulosic bio‐hydrogen production. Cellulosic feedstocks saccharification currently performed by commercial cellulase, which is composed of different fungal cellulase. Compared with fungi, thermocellulosic bacteria represented by Ruminiclostridium thermocellum have a complete cellulase system, and a higher cellulase catalytic efficiency than fungi; however, R. thermocellum is susceptible to feedback inhibition by cellobiose, which limits the application of R. thermocellum on cellulosic bio‐hydrogen production. In this study, a strain named R. thermocellum M3, which is not subject to feedback inhibition by cellobiose, was used in the bio‐hydrogen production of cellulosic agricultural waste feedstocks to explore the feasibility of bacterial saccharification of cellulosic substrates for biological hydrogen production. RESULTS: Results of batch tests indicate that the combination of domestic sewage sludge and strain M3 promoted the hydrogen production for different lignin content feedstocks (rice straw: from 0.66 to 6.42 mmol H₂/g substrate; corn cob: from 0.61 to 5.55 mmol H₂/g substrate; pine wood waste: from 0.58 to 5.32 mmol H₂/g substrate), which were competitive with the combination of domestic sewage sludge and Trichoderma viride cellulase. Specific activity analysis indicates that compared with the addition of T. viride cellulase, the addition of strain M3 completed the cellulase system in sludge. CONCLUSION: Thermo‐anaerobic bacteria R. thermocellum M3 enhanced the hydrogen production of the consolidated bioprocessing (CBP) of raw lignocellulosic agricultural wastes and, more importantly, provided a promising solution for the CBP strategy in the industrial application of lignocellulose bioconversion. © 2021 Society of Chemical Industry

Ship timber beetles (Coleoptera: Lymexylidae) grow symbiotic fungi of the genus Alloascoidea in wood. The female adults possess fungus-carrying organs (mycetangia) and deposit the symbiont onto wood during oviposition. The larvae acquire the symbiont, excavate a tunnel into wood, and feed on the symbiont growing on the tunnel walls. As lymexylids use wood as a larval habitat, it is possible that the fungal symbionts can utilize indigestible wood-associated sugars. However, their abilities to assimilate wood-associated carbon sources remain unknown. In addition, no lymexylid-associated fungal communities have been reported except for Elateroides dermestoides. Here, I report that the ship timber beetle E. flabellicornis originating from Japan harbored five fungal species. When microbial isolation was conducted from mycetangia of a female adult of E. flabellicornis, colonies of filamentous fungi and yeasts were recovered. DNA analyses revealed that they were Alloascoidea sp., Ambrosiozyma llanquihuensis, Ambrosiozyma sp., Cyberlindnera sp., and Saccharomycopsis sp. When wood-associated carbon assimilation abilities of four of the five fungal species: Alloascoidea sp., Am. llanquihuensis, Cyberlindnera sp., and Saccharomycopsis sp., were tested, the abilities were variable among them. Only Saccharomycopsis sp. assimilated galactose and galacturonic acid. Alloascoidea sp. and Cyberlindnera sp. strongly assimilated xylan from corn. Saccharomycopsis sp. and Cyberlindnera sp. assimilated cellobiose. All of the fungi assimilated glucose, mannose, and xylose. These results suggest that the association with multiple fungal species with various carbon assimilation abilities may help the larvae of E. flabellicornis to achieve efficient nutrient intake in space-limited tunnels within nutrient-poor wood.

Cellobiose 2-epimerase (CE) offers a promising enzymatic approach to produce lactulose. However, its application is limited by the unsatisfactory isomerization activity and thermostability. Our study attempted to optimize the catalytic performances of CEs by flexible loop exchange, for which four mutants were constructed using CsCE (CE from Caldicellulosiruptor saccharolyticus) as a template. As a result, all mutants maintained the same catalytic directions as the templates. Mutant RmC displayed a 2.2- and 1.34-fold increase in the isomerization activity and catalytic efficiency, respectively. According to the results of molecular dynamics (MD) simulations, it was revealed that the loop exchange in RmC enlarged the entrance of the active site for substrate binding and benefited proton transfer involved in the isomerization process. Besides, the t1/2 of mutant StC at 70 °C was increased from 29.07 to 38.29 h, owing to the abundance of rigid residues (proline) within the flexible loop of StC. Our work demonstrated that the isomerization activity and thermostability of CEs were closely related to the flexible loop surrounding the active site, which provides a new perspective to engineer CEs for higher lactulose production.

Microbial biorefinery is a promising route toward sustainable production of glycolic acid (GA), a valuable raw material for various industries. However, inherent microbial GA production has limited substrate consumption using either d-xylose or d-glucose as carbon catabolite repression (CCR) averts their co-utilization. To bypass CCR, a GA-producing strain using d-xylose via Dahms pathway was engineered to allow cellobiose uptake. Unlike glucose, cellobiose was assimilated and intracellularly degraded without repressing d-xylose uptake. The final GA-producing E. coli strain (CLGA8) has an overexpressed cellobiose phosphorylase (cep94A) from Saccharophagus degradans 2–40 and an activated glyoxylate shunt pathway. Expression of cep94A improved GA production reaching the maximum theoretical yield (0.51 g GA g−1 xylose), whereas activation of glyoxylate shunt pathway enabled GA production from cellobiose, which further increased the GA titer (2.25 g GA L−1). To date, this is the highest reported GA yield from d-xylose through Dahms pathway in an engineered E. coli with cellobiose as co-substrate.

Biological hydrogen (H2) is a promising candidate for production of renewable hydrogen. Using entrapped cells rather than conventional suspended cell cultures for the production of H2 offers several advantages, such as improved production yields related to higher cell density, and enhanced resistance to substrate and end-product inhibition. In this study, H2 production by a novel isolate of Clostridium intestinale (strain URNW) was evaluated using cells entrapped within 2% calcium-alginate beads under strictly anaerobic conditions. Both immobilized cells and suspended cultures were studied in sequential batch-mode anaerobic fermentation over 192 h. The production of H2 in the headspace was examined for four different initial cellobiose concentrations (5, 10, 20, and 40 mM). Although a lag period for initiation of the fermentation process was observed for bacteria entrapped within hydrogel beads, the immobilized cells achieved both higher volumetric production rates (mmol H2/(L culture h)) and molar yields (mol H2/mol glucose equivalent) of H2 compared with suspended cultures. In the current study, the maximum cellobiose consumption rate of 0.40 mM/h, corresponding to 133.3 mg/(L h), was achieved after 72 h of fermentation by immobilized cells, generating a high hydrogen yield of 3.57 mol H2/mol cellobiose, whereas suspended cultures only yielded 1.77 mol H2/mol cellobiose. The results suggest that cells remain viable within the hydrogels and proliferated with a slow rate over the course of fermentation. The stable productivity of immobilized cells over 8 days with four changes of medium depicted that the immobilized cells of the isolated strain can successfully yield higher hydrogen and lower soluble metabolites than suspended cells suggesting a feasible process for future applications for bioH2 production.

In order to evaluate the potential prebiotic effects of cellobiose, 10 healthy adult research beagle dogs received a complete diet containing 0, 0.5 and 1 g cellobiose/kg bodyweight (BW)/day. At the end of each feeding period, faeces, urine and blood of the dogs were collected. The results demonstrated a significant increase of faecal lactate concentrations, indicating a bacterial fermentation of cellobiose in the canine intestine. Along with this, a dose-dependent linear increase of the relative abundance of Lactobacillaceae in the faeces of the dogs was observed (p = 0.014). In addition, a dose-dependent increase (p < 0.05) of Alloprevotella, Bacteroides and Prevotella, and a linear decrease for unidentified Lachnospiraceae (p = 0.011) was observed when cellobiose was added to the diet, although the relative abundance of these genera was low (

SUMMARY Certain transglucanases can covalently graft cellulose and mixed‐linkage β‐glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero‐transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero‐transglucosylation, we studied Equisetum fluviatile hetero‐trans‐β‐glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo‐transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia‐produced EfHTG was up to approximately 300% increased by addition of methanol‐boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol‐stable factor is proposed to be expansin‐like, a suggestion supported by observations of pH dependence. Screening numerous low‐molecular‐weight compounds for hetero‐transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero‐transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero‐transglucosylation in planta by identifying factors that govern this reaction., Significance Statement The enzyme HTG can graft segments of cellulose molecules onto xyloglucan (a hemicellulose), thereby re‐structuring the cell wall via hetero‐transglycosylation. In native walls, two endogenous hemicelluloses competed with cellulose (and with each other) as substrate. HTG more readily selected cellulose as substrate if an expansin‐enriched preparation was added. Hetero‐transglycosylation was inhibited by cellobiose – a potential tool for exploring HTG’s biological functions. Interestingly, cellobiose retarded Equisetum stem elongation, suggesting a role for HTG in growth.

β-Glucosidase (EC 3.2.1.21) plays an essential role in the hydrolysis of the β-1,4 linkage of cellobiose. Accumulated glucose during saccharification leads to the inhibition of the production of β-glucosidase, which causes an accumulation of cellobiose and inhibition of other cellulolytic enzymes. Thus, glucose tolerant and active β-glucosidase is required for the efficient saccharification of biomass. Freshwater is an essential ingredient of biotechnological processes and contributes to the environmental and economic cost of processes. Relatively few biocatalytic processes have been developed to utilize seawater, which is more abundant. Towards both the requirements, we set out to characterize a hyperthermophilic enzyme, B9L147, and evaluate its activity in seawater, and compared it with the industrial enzyme benchmarks. B9L147 from Thermomicrobium roseum was cloned and expressed in Escherichia coli. The overexpressed and purified wild-type showed a high specific activity of 280 ± 5.2 μmol min−1 mg−1 on pNPGlc when assayed at pH 7 and 84 °C. B9L147 retains at least 80% relative specific activity across a wide pH range from 5.5 to 10.0. The enzyme is glucose tolerant and remains fully active until 3 M glucose. The kinetic properties, stability, and glucose tolerance remain identical in seawater, unlike commercial enzymes. An engineered variant, V169C, showed a 15% enhanced specific activity and almost twice the half-life compared to the wild type (B9L147). Both B9L147 and V169C show very high synergistic activity when supplemented with commercial cellulases and enzymes cloned and overexpressed in our lab. To the best of our knowledge, B9L147 is the first β-glucosidase that can hydrolyze cellulose in seawater at elevated temperatures and thus may be of value for industrial applications. Our studies offer a framework for developing seawater tolerant in vitro saccharification systems for biomass hydrolysis towards the sustainable production of biofuels and chemicals from biomass.

Background One of the sustainable development goals focuses on the biomass-based production as a replacement for fossil-based commodities. A novel feedstock with vast potentials is tunicate biomass, which can be pretreated and fermented in a similar way to lignocellulose. Ciona intestinalis is a marine filter feeder that is cultivated to produce fish feed. While the inner tissue body is used for feed production, the surrounding tunic remains as a cellulose-rich by-product, which can be further separated into outer and inner tunic. Ethanol production from organosolv-pretreated whole-tunic biomass was recently validated. The aim of the present study was to evaluate the potential of organosolv pretreated outer-tunic biomass for the production of biofuels and cellobiose that is a disaccharide with prebiotic potential. Results As a result, 41.4 g/L of ethanol by Saccharomyces cerevisiae, corresponding to a 90.2% theoretical yield, was achieved under the optimal conditions when the tunicate biomass was pretreated at 195 °C for 60 min at a liquid-to-solid ratio of 50. In addition, cellobiose production by enzymatic hydrolysis of the pretreated tunicate biomass was demonstrated with a maximum conversion yield of 49.7 wt. %. Conclusions The utilisation of tunicate biomass offers an eco-friendly and sustainable alternative for value-added biofuels and chemicals. The cultivation of tunicate biomass in shallow coastal sea improves the quality of the water and ensures sustainable production of fish feed. Moreover, there is no competition for arable land, which leaves the latter available for food and feed production.

Background The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber > 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance. Results The rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that > 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners—Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum—exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%. Conclusions This study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading.

This study reports covalent immobilization of β-glucosidase (BGL) from Bacillus subtilis PS on magnetically recyclable iron nanoparticles for enhancing robustness, facile recovery and reuse of enzyme. Immobilized BGL iron nanoparticles (BGL-INPs) were characterized by various biophysical techniques viz. TEM, DLS, FTIR and CD spectroscopy. The efficiency and yield of immobilization were 89.78 and 84.80%, respectively. After immobilization, optimum pH remained 6.0 whereas optimum temperature upraised to 70 °C whereas apparent Km and Vmax shifted from 0.819 mM to 0.941 mM and 54.46 to 57.67 μmole/min/mg, respectively. Immobilization conferred lower activation energy and improved pH and thermal stabilities. The BGL-INPs retained 85% activity up to 10th cycle of reuse and hydrolyzed more than 90% of cellobiose to glucose within 30 min. Conclusively, improved pH, thermal stability and excellent reusability over free enzyme make BGL-INPs a promising candidate for sustainable bioethanol production and other industrial applications.

Increased demand for biofuels promotes the search for new biomass-degrading fungi. Acremonium strictum is an environmentally widespread filamentous fungi found on plant debris; that secretes lignocellulose-degrading enzymes. A recently isolated A. strictum strain, AAJ6; native to the Brazilian Cerrado biome was evaluated for its capacity to degrade lignocellulosic substrates. In this study, whole-genome sequencing of AAJ6 was performed and 775 CAZy domains were identified which correlated to those of A. strictum strain DS1bioAY4a and other lignocellulolytic fungi; suggesting AAJ6 is a high CAZyme producer. We expressed the glycoside hydrolase families GH74 and GH3 from plasmid or genome-integrated to evaluate the ethanol production from cellulosic substrates in Brazilian industrial Saccharomyces cerevisiae strains (PE-2 and SA-1) evolved for thermotolerance (AMY12 and AMY35). Those expressing the genome-integrated enzymes showed the highest β-glucosidase activity and growth in medium with cellobiose at 40°C. The strain AGY005 (integrated cassettes) showed 19, 23 and 46% higher ethanol production in SHF, pSSF (partial hydrolysis SSF) and SSF processes, respectively, using Avicel, and ∼50% more ethanol using pre-treated sugarcane bagasse, compared to the strain with a plasmid-based expression. These results indicate the improved performance of thermotolerant industrial strains with genome-integrated CAZymes in the SSF process for 2G ethanol.

Cellobiose 2-epimerase (CE) is commonly recognized as an epimerase as most CEs mainly exhibit an epimerization activity towards disaccharides. In recent years, several CEs have been found to possess bifunctional epimerization and isomerization activities. They can convert lactose into lactulose, a high-value disaccharide that is widely used in the food and pharmaceutical industries. However, the factors that determine the catalytic direction in CEs are still not clear. In this study, the crystal structures of three newly discovered CEs, CsCE (a bifunctional CE from Caldicellulosiruptor saccharolyticus), StCE (a bifunctional CE from Spirochaeta thermophila DSM 6578) and BtCE (a monofunctional CE from Bacillus thermoamylovorans B4166), were determined at 1.54, 2.05 and 1.80 Å resolution, respectively, in order to search for structural clues to their monofunctional/bifunctional properties. A comparative analysis of the hydrogen-bond networks in the active pockets of diverse CEs, YihS and mannose isomerase suggested that the histidine corresponding to His188 in CsCE is uniquely required to catalyse isomerization. By alignment of the apo and ligand-bound structures of diverse CEs, it was found that bifunctional CEs tend to have more flexible loops and a larger entrance around the active site, and that the flexible loop 148–181 in CsCE displays obvious conformational changes during ligand binding. It was speculated that the reconstructed molecular interactions of the flexible loop during ligand binding helped to motivate the ligands to stretch in a manner beneficial for isomerization. Further site-directed mutagenesis analysis of the flexible loop in CsCE indicated that the residue composition of the flexible loop did not greatly impact epimerization but affects isomerization. In particular, V177D and I178D mutants showed a 50% and 80% increase in isomerization activity over the wild type. This study provides new information about the structural characteristics involved in the catalytic properties of CEs, which can be used to guide future molecular modifications.

A cellobiohydrolase gene, cbhC16, isolated from the cDNA library of a rumen fungus. The recombinant CbhC16 demonstrated a specific activity of 7.0 U mg−1 protein against phosphoric acid-swollen avicel, and cellobiose was the main product in the reaction mixture. Glucose was further released from cellobiose by the action of CbhC16 and fermented to ethanol by Saccharomyces cerevisiae. This phenomenon, possessing activities of both cellobiohydrolase and β-glucosidase, is rarely observed in the hydrolysis by typical cellobiohydrolases. Yeast cells displaying CbhC16 were inoculated into YNB–CAA broth containing carboxymethyl cellulose, β-glucan, ammonia fibre explosion-treated rice straw, or wheat bran, and incubated at 30 °C for 3 d. Under the same conditions, 0.20, 0.61, 0.15, and 0.61 g/L of ethanol were detected in the culture supernatant of the strain with the recombinant plasmid containing cbhC16, whereas wild-type yeasts without the recombinant plasmid containing cbhC16 did not produce detectable ethanol levels. The results indicated that yeast strains with cbhC16 could directly saccharify and ferment cellulose to produce ethanol in singular step. This gene encoding both cellobiohydrolase and β-glucosidase activities will enable developing a simple approach for producing a consolidated microorganism that will directly convert cellulosic feedstocks to ethanol.

Biohydrogen is one of the most potential alternative fuels for the future. Among various substances from agricultural factory waste, molasses and wastewater from ethanol distillation plants are the main focus of this study. To overcome the limitation of bio-H2 production, the direct screening of waste substrates for an efficient producer using co-culture free technique is an interesting choice. Competent species with H2-producing ability and valuable volatile acid such as acetic acid were selected. Several bacteria were isolated and had their species identified by microbial 16SrDNA sequence analysis and phylogenetic analysis by Maximum Likelihood method. Bacillus coaglulans, B. circulans, Sporolactobacillus inulinus, Lactobacillus spp., Pediococcus acidilactici, Paenibacillus thermophilus, Chronobacter sakazaki and Clostridium beijerinckii were found in both reducing-sugar rich waste and wastewater. For bio-H2 application in this criteria, potent natural hydrogen producer B. coagulans MO11 and C. beijerinckii CN were picked to test the growth ability on different substrates (carbon-dioxide, l -arabinose, d -xylose, d -glucose, d -fructose, sucrose, maltose, cellobiose, starch and others). Wastewater from sugarcane factories and other agricultural factories in Thailand were tested for physical and biochemical characteristics which would make them suitable renewable substrates. Among all, Bacillus coagulans MO11 could produce hydrogen gas using molasses and ethanol refinery wastewater effectively (1.634 molH2/mol hexose) as detected by Drager tube, which was the maximum yield in this study. In comparison, the experiment performed by using B. coagulans MO11 and C. beijerinckii CN fed with 40X diluted molasses wastewater showed better production of H2 gas in Clostridium beijerinckii CN during the final 72-hr gas collection (79 mL/L and 125 mL/L respectively). The results pointed in the same direction when replacing 40X diluted molasses with 5X diluted refinery wastewater supplemented with trace elements.

Clostridium (Ruminiclostridium) thermocellum is recognized for its ability to ferment cellulosic biomass directly, but it cannot naturally grow on xylose. Recently, C. thermocellum (KJC335) was engineered to utilize xylose through expressing a heterologous xylose catabolizing pathway. Here, we compared KJC335′s transcriptomic responses to xylose versus cellobiose as the primary carbon source and assessed how the bacteria adapted to utilize xylose. Our analyses revealed 417 differentially expressed genes (DEGs) with log2 fold change (FC) >|1| and 106 highly DEGs (log2 FC >|2|). Among the DEGs, two putative sugar transporters, cbpC and cbpD, were up-regulated, suggesting their contribution to xylose transport and assimilation. Moreover, the up-regulation of specific transketolase genes (tktAB) suggests the importance of this enzyme for xylose metabolism. Results also showed remarkable up-regulation of chemotaxis and motility associated genes responding to xylose feeding, as well as widely varying gene expression in those encoding cellulosomal enzymes. For the down-regulated genes, several were categorized in gene ontology terms oxidation–reduction processes, ATP binding and ATPase activity, and integral components of the membrane. This study informs potentially critical, enabling mechanisms to realize the conceptually attractive Next-Generation Consolidated BioProcessing approach where a single species is sufficient for the co-fermentation of cellulose and hemicellulose.

Cellobiose 2-epimerase (CE) is a promising industrial enzyme that can catalyze bioconversion of lactose to its high-value derivatives, namely epilactose and lactulose. A need exists in the dairy industry to catalyze lactose bioconversions at low temperatures to avoid microbial growth. We focused on the discovery of cold-active CE in this study. A genome mining method based on computational prediction was used to screen the potential genes encoding cold-active enzymes. The CE-encoding gene from Roseburia intestinalis, with a predicted high structural flexibility, was expressed heterologously in Escherichia coli. The catalytic property of the recombinant enzyme was extensively studied. The optimum temperature and pH of the enzyme were 45°C and 7.0, respectively. The specific activity of this enzyme to catalyze conversion of lactose to epilactose was measured to be 77.3 ± 1.6 U/mg. The kinetic parameters, including turnover number (kcat), Michaelis constant (Km), and catalytic efficiency (kcat/Km) using lactose as a substrate were 117.0 ± 7.7 s-1, 429.9 ± 57.3 mM, and 0.27 mM-1s-1, respectively. In situ production of epilactose was carried out at 8°C: 20.9% of 68.4 g/L lactose was converted into epilactose in 4 h using 0.02 mg/mL (1.5 U/mL, measured at 45°C) of recombinant enzyme. The enzyme discovered by this in silico method is suitable for low-temperature applications.

Phytophthora capsici Leonian is an important oomycete pathogen of crop vegetables, causing significant economic losses each year. Its cell wall, rich in cellulose, is vital for cellular integrity and for interactions with the host organisms. Predicted cellulose synthase (CesA) proteins are expected to catalyze the polymerization of cellulose, but this has not been biochemically demonstrated in an oomycete. Here, we present the properties of the four newly identified CesA proteins from P. capsici and compare their domain organization with that of CesAs from other lineages. Using a newly constructed glucosyltransferase-deficient variant of Saccharomyces cerevisiae with low residual background activity, we have achieved successful heterologous expression and biochemical characterization of a CesA protein from P. capsici (PcCesA1). Our results demonstrate that the individual PcCesA1 enzyme produces cellobiose as the major reaction product. Co-immunoprecipitation studies and activity assays revealed that several PcCesA proteins interact together to form a complex whose multiproteic nature is most likely required for cellulose microfibril formation. In addition to providing important insights into cellulose synthesis in the oomycetes, our data may assist the longer term identification of cell wall biosynthesis inhibitors to control infection by pathogenic oomycetes.

Four novel strains (592T, S592, MF47T and SMF47) were isolated from Tibetan antelopes (Pantholops hodgsonii) and plateau pikas (Ochotona curzoniae), respectively. The cells were aerobic, non-motile, Gram-stain- and catalase-positive, rod-shaped bacteria. The 16S rRNA gene sequences of the four strains showed highest similarities to Aeromicrobium fastidiosum DSM 10552T (98.1, 98.6, 98.7 and 98.7 %, respectively), and the phylogenetic analyses based on 16S rRNA gene and genomic sequences indicated that strains 592T and MF47T represent two novel species. The four isolates produced acid from l-rhamnose, d-xylose and cellobiose, but were unable to reduce nitrate. The DNA G+C contents of strains 592T and MF47T were 70.3 and 69.8 mol%, respectively. The digital DNA–DNA hybridization value between strains 592T and MF47T was 32.6 %, lower than the threshold of 70 %, indicating they belong to different species. The four strains’ genomes displayed less than 24.6 % DNA–DNA relatedness with all available genomes of the genus Aeromicrobium in the NCBI database, including Aeromicrobium fastidiosum NBRC 14897T and Aeromicrobium ginsengisoli JCM 14732T. The major fatty acids of the four strains were C18 : 1 ω9c and C18 : 0 10-methyl, and the main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. The predominant respiratory quinones were MK-9(H4) and MK-8(H4). The cell-wall peptidoglycan contained ll-diaminopimelic acid. Based on these genotypic, phenotypic and biochemical analyses, it is proposed that the four unidentified bacteria be classified as two novel species, Aeromicrobium chenweiae sp. nov. and Aeromicrobium yanjiei sp. nov. The type strains are 592T (=CGMCC1.16526T=DSM 106289T) and MF47T (=CGMCC 1.17444T=JCM 33790T), respectively.

Increasing energy demands call for sustainable alternative sources. Solid state fermentation (SSF) of raw oil palm frond leaves (OPFL) as the substrate to produce extracellular cellulases and xylanase by a novel Rhizopus oryzae UC2 (GenBank accession no. MF767597) was optimized. Optimum SSF conditions (30 °C, 40% moisture content, 2.0 × 108 spores/g inoculum size) yielded the maximum carboxymethyl cellulase (CMCase) (94.68 U/g), filter paperase (FPase) (25.46 U/g), β-glucosidase (145.47 U/g) and xylanase (213.99 U/g) activities, showing a broad pH range of between 6.0 and 12.0. Proteome analysis of crude enzyme cocktail revealed three β-glucosidases, as well as one endo-β-1,4-glucanase, exoglanase and endo-β-1,4-xylanase each. Activities of the enzyme complex were maximal at an acidic pH and temperature that ranged between pH 3.0–5.0 and 50–60 °C, respectively. In situ hydrolysis of OPFL released various concentrations of sugars viz. glucose (26.74 mg/g), xylose (1.44 mg/g), fructose (50.8 mg/g) and cellobiose (58.31 mg/g). Moreover, CMCase, FPase, β-glucosidase and xylanase exhibited half-lives of 5.13, 1.48, 18.81, 9.23 h when incubated at 60 °C, respectively. Thus, the desirable qualities of R. oryzae UC2 seen here supported its prospective biocatalytic role for timely and safe production of digestible carbohydrates from agro-industrial biomass for the subsequent biotransformation into biofuel.

Biological degradation of cellulose from dead plants in nature and plant biomass from agricultural and food-industry waste is important for sustainable carbon recirculation. This study aimed at searching diverse cellulose-degrading systems of wild filamentous fungi and obtaining fungal lines useful for cellooligosaccharide production from agro-industrial wastes. Fungal lines with cellulolytic activity were screened and isolated from stacked rice straw and soil in subtropical fields. Among 13 isolated lines, in liquid culture with a nutrition-limited cellulose-containing medium, four lines of Aspergillus spp. secreted 50–60 kDa proteins as markedly dominant components and gave clear activity bands of possible endo-β-1,4-glucanase in zymography. Mass spectroscopy (MS) analysis of the dominant components identified three endo-β-1,4-glucanases (GH5, GH7 and GH12) and two cellobiohydrolases (GH6 and GH7). Cellulose degradation by the secreted proteins was analysed by LC-MS-based measurement of derivatized reducing sugars. The enzymes from the four Aspergillus spp. produced cellobiose from crystalline cellulose and cellotriose at a low level compared with cellobiose. Moreover, though smaller than that from crystalline cellulose, the enzymes of two representative lines degraded powdered rice straw and produced cellobiose. These fungal lines and enzymes would be effective for production of cellooligosaccharides as cellulose degradation-intermediates with added value other than glucose.

A novel endoglucanase gene (1425 bp), designated thrcel5A, was cloned from Thermoactinospora rubra YIM 77501Tand determined to be a member of glycoside hydrolase family 5. The putative amino acid sequence displayed 76% conservation with reported endoglucanases (GenBank: SCG57304.1) from Micromonospora siamensis. Thrcel5A was expressed in Escherichia coli BL 21 (DE3) and purified using Ni2+-affinity chromatography. The resulting purified protein displayed high hydrolytic activity against the sodium salt of carboxymethyl cellulose and β-(1, 3; 1, 4)-glucans from barley and beechwood xylan, with specific activities of 85.7 ± 1.5, 120.3 ± 2.6, and 22.9 ± 1.1 U/mg, respectively. The optimal pH and temperature for the recombinant enzyme were determined to be 8.5 and 60 °C, respectively. Additionally, ThrCel5A was thermotolerant as it retained more than 60% of its original activity after an incubation at 60 °C for 2 h. Moreover, ThrCel5A can hydrolyze β-(1, 3; 1, 4)-glucan into prebiotics, such as cellobiose, cellotriose, and cellotetrose. Its endoglucanase activity was significantly affected by link sequences and CBM2. Due to being an alkali-tolerant, thermostable, and multifunctional cellulolytic enzyme, ThrCel5A is an attractive candidate for use in production of prebiotics.

In this study, the selection of target sugars in lignocellulosic hydrolysate and the implementation of a bioprocess strategy led to efficient production of a catalyst for chitinous bioresource conversion. Streptomyces sp. K5, a chitinase producer in the presence of glucose, was isolated at 50 °C on an agar plate with glucose and colloidal chitin. The K5 was found to produce chitinase with a maximum activity of 70 U/L in a medium containing glucose and xylose as well as colloidal chitin as an inducer. The assimilation of glucose and xylose, however, was extremely slow, with significant residual being present even after 168 h of incubation. Assimilation tests of glucose, xylose, and cellobiose confirmed that K5 produces chitinase without chitinous inducer and assimilates cellobiose much more rapidly than either glucose or xylose. At the same time, the complete exhaustion of each sugar initiated chitinase inactivation. A pulse- feeding strategy was adopted for the cellobiose, taking its rapid assimilation, β-glucosidase activity, and chitinase inactivation into account, and a maximum chitinase activity of 235 U/L was achieved under pulse- feeding conditions that included four pulses.

The aggregation behavior of two model proteins- i) bovine serum albumin (BSA) and ii) β-galactosidase (β-gal), was investigated by micro-flow imaging (MFI) during freeze-thaw cycling in phosphate buffered solutions. The pH shift was measured upon cooling the solutions from 20 to −25 °C. When the buffer concentration was 100 mM, cooling caused a pH decrease of 3.1 and 2.7 units (for BSA and β-gal, respectively) attributed to selective crystallization of disodium hydrogen phosphate as a dodecahydrate. The crystallizing solute phase was characterized by low temperature powder X-ray diffractometry. The pH shift resulted in protein aggregation, evident from the pronounced increase in particle count (by MFI). The addition of cellobiose attenuated the pH shift on cooling (pH decrease of ~1.0 unit), and no evidence of either buffer salt crystallization or protein aggregation was observed. Decreasing the buffer concentration to 10 mM, also prevented protein aggregation. The protein, by inhibiting buffer crystallization, prevented the pH shift and then the buffer, by maintaining the pH, enhanced protein stability.

Thermoanaerobacterium thermosaccharolyticum GSU5 was isolated from animal dung collected in a pasture plain in Buenos Aires, Argentina. This thermophilic and anaerobic microorganism was able to produce butanol and ethanol, but not acetone, using sugars such as xylose, arabinose, glucose, galactose, fructose, sucrose, and cellobiose. Key metabolic enzymes leading to solvent production were identified in its genome. A detailed analysis of the solvent and organic acid biosynthetic pathway genes of sequenced strains revealed new insights into the unique metabolic features of this species. Genes required for the synthesis of acetone are absent in the genomes of all sequenced Thermoanaerobacterium, suggesting that it is a general trait of the genus. Strains able to produce butanol synthesize butyrate through the one step pathway catalyzed by the butyryl-CoA:acetate-CoA transferase (But). The large range of fermentable substrates and the ability to produce both ethanol and butanol without acetone makes this species an interesting candidate for second generation biofuel production.

Cellobiose dehydrogenase (CDH) is a flavocytochrome catalyzing oxidation of the reducing end of cellobiose and cellooligosaccharides, and has a key role in the degradation of cellulosic biomass by filamentous fungi. Here, we use a lineup of glucose/xylose-mixed β-1,4-linked disaccharides and trisaccharides, enzymatically synthesized by means of the reverse reaction of cellobiose phosphorylase and cellodextrin phosphorylase, to investigate the substrate recognition of CDH. We found that CDH utilizes β-D-xylopyranosyl-(1→4)-D-glucopyranose (Xyl-Glc) as an electron donor with similar K m and k cat values to cellobiose. β-D-Glucopyranosyl-(1→4)-D-xylopyranose (Glc-Xyl) shows a higher K m value, while xylobiose does not serve as a substrate. Trisaccharides show similar behavior; i.e., trisaccharides with cellobiose and Xyl-Glc units at the reducing end show similar kinetics, while the enzyme was less active towards those with Glc-Xyl, and inactive towards those with xylobiose. We also use docking simulation to evaluate substrate recognition of the disaccharides, and we discuss possible molecular mechanisms of substrate recognition by CDH.

Co-production of two or more desirable compounds from low-cost substrates by a single microbial catalyst could greatly improve the economic competitiveness of many biotechnological processes. However, reports demonstrating the adoption of such co-production strategy are still scarce. In this study, the ability of genome-edited strain Psudomonas putida EM42 to simultaneously valorise D-xylose and D-cellobiose - two important lignocellulosic carbohydrates - by converting them into the platform chemical D-xylonic acid and medium chain length polyhydroxyalkanoates, respectively, was investigated. Biotransformation experiments performed with P. putida resting cells showed that promiscuous periplasmic glucose oxidation route can efficiently generate extracellular xylonate with high yield reaching 0.97 g per g of supplied xylose. Xylose oxidation was subsequently coupled to the growth of P. putida with cytoplasmic beta-glucosidase BglC from Thermobifida fusca on D-cellobiose. This disaccharide turned out to be a better co-substrate for xylose-to-xylonate biotransformation than monomeric glucose. This was because unlike glucose, cellobiose did not block oxidation of the pentose by periplasmic glucose dehydrogenase Gcd, but, similarly to glucose, it was a suitable substrate for polyhydroxyalkanoate formation in P. putida. Co-production of extracellular xylose-born xylonate and intracellular cellobiose-born medium chain length polyhydroxyalkanoates was established in proof-of-concept experiments with P. putida grown on the disaccharide. This study highlights the potential of P. putida EM42 as a microbial platform for the production of xylonic acid, identifies cellobiose as a new substrate for mcl-PHA production, and proposes a fresh strategy for the simultaneous valorisation of xylose and cellobiose.

Bacillus amyloliquefaciens SS35 was subjected to ultraviolet irradiation to improve the enzymatic hydrolysis of lignocellulosic biomass. The resulting mutant, UV2, produced endoglucanase, carboxymethyl cellulase, CMCase-UV2 with 1.6–4.1-fold higher activity against cellulosic substrates than the wild type, CMCase-WT. CMCase-UV2 exhibited wider pH stability in the acidic range than CMCase-WT. The TLC analysis showed that the hydrolysis of CMC-Na and β-glucan by CMCase-UV2 produced glucose along with cello-oligosaccharides and cellobiose in 45 min, whereas, CMCase-WT produced, only cello-oligosaccharides and cellobiose in 120 min by endolytic mode of action. The hydrolysis of pretreated Pennisetum purpureum by CMCase-UV2 gave total reducing sugar yield 154.2 mg/g pretreated biomass in 48 h, which was 1.8-fold higher than CMCase-WT. CMCase-UV2 was the promising endoglucanase which improves the saccharification of lignocellulosic biomass therefore, it will improve the efficiency of the process, lignocellulose-based biorefineries for bioethanol production. The gene encoding cellulase was amplified from wild-type and UV2 strains using degenerate primers designed from phylogenetically related spp. Bacillus amyloliquefaciens KHG19 for family 5 glycoside hydrolase. Sequences analysis of genes from wild-type and UV2 strains showed the mutation, D233G. These results will provide information for protein engineering in designing mutant of endoglucanase for improved catalytic efficiency and pH stability.

Hierarchically self-assembled structures are common in biology, but it is often challenging to design and fabricate synthetic analogs. The archetypal cell is defined by hierarchically organized multicompartmentalized structures with boundaries that delineate the interior from exterior environments and is an inspiration for complex functional materials. Here, we have demonstrated an approach to the design and construction of a nested protein cage system that can additionally incorporate the packing of other functional macromolecules and exhibit some of the features of a minimal synthetic cell-like material. We have demonstrated a strategy for controlled co-packaging of subcompartments, ferritin (Fn) cages, together with active cellobiose-hydrolyzing β-glycosidase enzyme macromolecules, CelB, inside the sequestered volume of the bacteriophage P22 capsid. Using controlled

Second-generation bioethanol is a sustainable energy source that can be produced from different renewable materials. However, there is a challenge we must overcome to significantly enhance bioethanol production: the hydrolysis of lignocellulosic biomass to fermentable sugars. Synergistic enzymes, such as endoglucanases, β-glucosidases, cellobiohydrolases, and, more recently, lytic polysaccharide monooxygenases and cellobiose dehydrogenases have been used with great success to hydrolyze pretreated biomass. Further advances in the field of second-generation bioethanol production will likely depend on an increased understanding of the interactions between enzymes and lignocellulosic substrates, the development of enzyme engineering, and the optimization of enzyme mixtures to enhance cellulose hydrolysis.

A Gram-stain-positive, coccus-shaped, non-motile bacterium, designated CF-49T, was isolated from the cloacal content of a snow finch, which was incidentally captured in a plateau pika burrow on the Qinghai–Tibet Plateau, PR China. Analysis of the 16S rRNA gene sequence showed that strain CF-49T was closely related to Vagococcus elongatus CCUG 51432T (96.5 % similarity), Vagococcus fluvialis NCFB 2497T (96.0 %) and Vagococcus lutrae CCUG 39187T (95.9 %), whereas the similarity to another isolate (CF-210) was 99.9 %. Strains CF-49T and CF-210 grew optimally at 37 °C and pH 7.0 and in the presence of 0.5 % (w/v) NaCl. Acid was produced from N-acetylglucosamine, cellobiose, d-fructose, d-glucose, d-mannose, d-mannitol, maltose, d-ribose and salicin. The cell-wall peptidoglycan type was A4α (l-Lys–d-Asp). The major cellular fatty acids (>10 %) were C16 : 0 (35.6 %), C14 : 0 (17.3 %), C18 : 1 ω9c (16.2 %) and C16 : 1 ω9c (10.6 %). The predominant respiratory quinone was menaquinone MK-7 (68.8 %). The G+C content of the genomic DNA was 35.9 mol%. Digital DNA–DNA hybridization of strain CF-49T with V. fluvialis DSM 5731T, V. elongatus CCUG 51432Tand V. lutrae CCUG 39187T resulted in relatedness values of 21.4, 23.3 and 24.6 %, respectively. Based on results from polyphasic analyses, our two isolates are proposed to represent a novel species in the genus Vagococcus , with the name Vagococcus xieshaowenii. The type strain is CF-49T (=CGMCC 1.6436T=GDMCC 1.1588T=JCM 33477T).

Listeria ivanovii is one of the two pathogenic species within the genus Listeria, the other being L. monocytogenes. In this study, we generated a stable pediocin resistant mutant Liv-r1 of a L. ivanovii strain, compared phenotypic differences between the wild-type and the mutant, localised the pediocin-induced mutations in the chromosome, and analysed the mechanisms behind the bacteriocin resistance. In addition to pediocin resistance, Liv-r1 was also less sensitive to nisin. The growth of Liv-r1 was significantly reduced with glucose and mannose, but less with cellobiose. The cells of Liv-r1 adsorbed less pediocin than the wild-type cells. Consequently, with less pediocin on the cell surface, the mutant was also less leaky, as shown as the release of intracellular lactate dehydrogenase to the supernatant. The surface of the mutant cells was more hydrophobic than that of the wild-type. Whole genome sequencing revealed numerous changes in the Liv-r1 chromosome. The mutations were found e.g., in genes encoding sigma-54-dependent transcription regulator and internalin B, as well as in genes involved in metabolism of carbohydrates such as glucose and cellobiose. Genetic differences observed in the mutant may be responsible for resistance to pediocin but no direct evidence is provided.

Fermentation has recently re-emerged as an approach for improved functionality of food products in addition to the traditional roles such as shelf life, taste, and texture. Here, we report dynamic changes in the metabolite profiles of Achyranthes japonica Nakai by Lactobacillus plantarum fermentation, primarily, the significant increases in representative functional ingredients, 20-hydroxyecdysone and 25S-inokosterone. Additionally, untargeted metabolite profiling showed 58% of metabolites underwent significant alteration. The most dynamic change was observed in cellobiose, which showed a 56-fold increase. Others were sugar alcohols and amino acids, while lyxitol and erythritol that were among the most dynamically down-regulated.

The inhibitory effect of xylan on enzymatic hydrolysis of cellulose with different ultrastructure is not clear. In this work, cellulose II, cellulose III, and amorphous cellulose were prepared from cellulose I by NaOH, ethylenediamine, and ball-milling treatment, respectively. Their crystalline structure and enzymatic hydrolyzability were systematically characterized and compared with cellulose I. Furthermore, the inhibitory effect of xylan on cellulose with different ultrastructure was investigated in detail. Results suggested that the enzymatic hydrolyzability of different cellulose increased in sequence from cellulose I (25.4%), cellulose III (41.7%), cellulose II (56.2%) to amorphous cellulose (77.4%) at enzyme loadings of 5.56 mg protein/g substrate. The enzymatic hydrolyzability of allomorphic cellulose was strongly inhibited by xylan, while a little inhibition was observed for amorphous cellulose. It was found that cellobiose released from different cellulose was decreased after the addition of xylan. At the initial hydrolysis stage, the inhibition degree was obviously increased for all of different cellulose, and then, the inhibition degree of cellulose I and cellulose II gradually decreased as enzymatic hydrolysis proceeded, while the inhibition degree of cellulose III and amorphous cellulose had changed a little. In addition, at a longer time of hydrolysis, the inhibition degree was decreased with the increase of cellulase dosage except for cellulose III, which remained approximately unchanged. Overall, this study demonstrated that the inhibitory effect of xylan on cellulose hydrolysis was substantially affected by the ultrastructure of cellulose, and thus it provides new insights for relieving the inhibitory effect of xylan by altering the ultrastructure of cellulose.

In this study, a non-sterile (open) continuous fermentation (OCF) process with no-carbon loss was developed to improve lactic acid (LA) productivity and operational stability from the co-utilization of lignocellulose-derived sugars by thermophilic Enterococcus faecium QU 50. The effects of different sugar mixtures on LA production were firstly investigated in conventional OCF at 50°C, pH 6.5 and a dilution rate of 0.20 hr-1 . The xylose consumption ratio was greatly lower than that of glucose in fermentations with glucose/xylose mixtures, indicating apparent carbon catabolite repression (CCR). However, CCR could be efficiently eliminated by feeding solutions containing the cellobiose/xylose mixture. In OCF at a dilution rate ca. 0.10 hr-1 , strain QU 50 produced 42.6 g L-1 of l-LA with a yield of 0.912 g g-1 -consumed sugars, LA yield of 0.655 g g-1 based on mixed sugar-loaded, and a productivity of 4.31 g L-1 hr-1 from simulated energy cane hydrolyzate. In OCF with high cell density by cell recycling, simultaneous and complete co-utilization of sugars was achieved with stable LA production at 60.1 ± 3.25 g L-1 with LA yield of 0.944 g g-1 -consumed sugar and LA productivity of 6.49 ± 0.357 g L-1 hr-1 . Besides this, a dramatic increase in LA yield of 0.927 g g-1 based on mixed sugar-loaded with prolonged operational stability for at least 500 hr (>20 days) was established. This robust system demonstrates an initial green step with a no-carbon loss under energy-saving toward the feasibility of sustainable LA production from lignocellulosic sugars.

Three strains originating from insect frass in South Africa, yellow foxglove in Hungary and soil in France, were characterised phenotypically and by sequencing of the D1/D2 domain of the large subunit and the ITS1-5.8S-ITS2 (ITS)-region of the rRNA gene. The strains have identical D1/D2 domain sequences and only one strain shows a 1 bp indel in a 9 bp homopolymer A/T repeat within the ITS-region. Based on sequence analysis Hyphopichia burtonii is the closest related species. The investigated strains differ from the type strain of H. burtonii by 1.9% (9 substitutions and an indel) in the D1/D2 domain and by 23 substitutions and 21-22 indels in the ITS-region. Since the sequence variability is very low among the three strains and the sequence divergence with the closely related H. burtonii exceeds the level generally encountered between species we propose the new species Hyphopichia lachancei f.a., sp. nov. to accommodate the three novel strains. From H. burtonii the new species can be distinguished phenotypically by its inability to ferment cellobiose and by the formation of endospores (Holotype: CBS 5999T; Isotype: NCAIM Y.02228T; MycoBank no.: MB833616).

Biological pretreatment is a safe and environmentally friendly method for disrupting recalcitrant lignocellulose structures. Expansin and expansin-like proteins are used to open up the cellulose structure and display significant synergism when mixed with cellulases that catalyze the breakdown of (hemi)cellulose into sugars. However, the adsorption behavior of expansin in the presence of sugar products is yet unknown. In this work, we monitored the effects of various sugars on the real-time adsorption of Bacillus subtilis expansin (BsEXLX1) onto cellulose films using a quartz crystal microbalance with dissipation. Cellobiose and xylose at low concentrations enhanced BsEXLX1 adsorption, whereas they disrupted adsorption at higher concentrations. Arabinose and mannose continuously inhibited expansin adsorption with increasing concentration. No obvious influence of glucose and galactose on BsEXLX1 adsorption was found. Contact angle measurements and atomic force microscopy of cellulose upon BsEXLX1 adsorption in the presence of sugars showed that both hydrophilicity and roughness increased with BsEXLX1 treatment. These results give us the ability to modulate and control expansin adsorption and provide insights into effective expansin use during enzymatic hydrolysis of lignocellulose in biorefineries.

One bacterial strain U8 having starch hydrolysing activity was isolated from the rhizospheric soil of rice fields of Purba Bardhaman district in West Bengal, India. This bacterial sample was screened on nutrient agar plates (NA) and checked for starch hydrolysing activity in starch agar media. Colonies of this bacterial isolate were white and opaque with rough margins. Various tests were performed on this isolate including catalase, methyl red test, voges–proskauer, indole, citrate utilization and nitrate reduction test. Positive results were obtained for the former two tests and negative results for the latter tests. The presence of exoenzymes like gelatinase, amylase, oxidase and lipase was observed in the isolate; however, no urease was found. 16S rRNA sequencing and phylogenetic tree analyses indicated the present bacterial strain U8, was Bacillus flexus. Sugar fermentation tests revealed that Bacillus flexus U8 utilized fructose, xylose, lactose, cellobiose, dextrose and mannose as carbon sources. Antibiotic susceptibility tests showed that Bacillus flexus U8 was resistant to penicillin (10 µg/disc) and ampicillin (10 µg/disc). No haemolysis was observed on the blood agar media containing the bacterial isolate, indicating its non-pathogenic nature. The GC content of the isolate was 55% and AT content was 45%. Restriction mapping of the Bacillus flexus U8 16S rRNA gene sequence was also performed. Due to the abundance of Bacillus flexus U8 in the soil, its starch hydrolysing properties can be explored on agricultural land to potentially improve the health of the soil and plants.