A novel cellobiose 2-epimerase from anaerobic halophilic Iocasia fonsfrigidae and its ability to convert lactose in fresh goat milk into epilactose.

BACKGROUND: Cellobiose 2-epimerase (CE) has received great attention due to its potential applications in the food and pharmaceutical industries. In this study, a novel CE from mesophilic anaerobic halophilic bacterium Iocasia fonsfrigidae strain SP3-1 (IfCE) was successfully expressed in Escherichia coli and characterized. RESULTS: Unlike other CEs, the purified IfCE shows only epimerization activity toward ß-1,4-glycosidic linkages of disaccharides, including mannobiose, cellobiose and lactose, but not for monosaccharides, ß-1,4-glycosidic linkages of trisaccharides and α-1,4-glycosidic linkages of disaccharides. Only one epimerization product was obtained from the action of IfCE against mannobiose, cellobiose and lactose. Under optimum conditions, 31.0% of epilactose, a rare and low-calorie prebiotic sweetener with medicinal and pharmacological properties, was obtained from 10 mg mL-1 lactose. IfCE was highly active against lactose under NaCl concentrations up to 500 mmol L-1, possibly due to the excessive basic (arginine and lysine) and acidic (aspartic and glutamic acids) amino acid residues, which are localized on the surface of the halophilic enzyme structure. These residues may protect the enzyme from Cl- and Na+ ions from the environment, respectively. Under normal conditions, IfCE was able to convert lactose present in fresh goat milk to epilactose with a conversion yield of 31% in 10 min. In addition, IfCE has been investigated as a safe enzyme for human allergen. CONCLUSION: The results suggested that IfCE is a promising candidate to increase the quality and value of milk and dairy products by converting lactose that causes digestive problems in people with lactose intolerance into epilactose. © 2024 Society of Chemical Industry.

Substrate-Controlled Bidirectional Pumping by a Bienzymatic Micropump.

The development of autonomous, miniaturized pumps remains a problem of much interest, particularly with a view on microfluidics-based devices with increased portability and simplicity of use by nonspecialists. Spatially localized patches of enzyme imprinted on walls have been shown to induce a hydrodynamic flow when supplied with the corresponding enzyme substrate. Thus, such enzymatic micropumps are seen as a possible way of providing the means for nonmechanical, structurally simple, autonomous pumping. Hereby, we extend the current knowledge of enzymatic micropumps in two ways. First, we introduce ß-glucosidase as an enzyme that facilitates building micropumps with robust inward flows in the presence of cellobiose (e.g., 2.51 ± 0.56 µm s-1 in the presence of 80 mM cellobiose). Second, we embed ß-glucosidase and urease within the same patch and thus obtain a bienzymatic micropump. The latter exhibits the so far missing capability of bidirectional pumping as it produces inward flows in the presence of cellobiose (e.g., 0.95 ± 0.37 µm s-1 in the presence of 20 mM cellobiose) and outward flows in the presence of urea (e.g., 1.46 ± 0.47 µm s-1 in the presence of 20 mM urea). This bienzymatic micropump is a significant step for the development of biocompatible micropumps with versatile, controlled, and on-demand hydrodynamic pumping capabilities.

From agricultural biomass to D form lactic acid in ton scale via strain engineering of Lactiplantibacillus pentosus.

Worsening environmental conditions make lactic acid a sustainable alternative to petroleum-based plastics. This study created a genetically-engineered strain Lactiplantibacillus pentosus PeL containing a disrupted L-lactate dehydrogenase gene to produce high yield and optically pure D-lactic acid. Cellobiose was identified as the optimal sugar in the single carbon source test, yielding the highest lactic acid. In 5-L fermentation tests, pretreated wood chips hydrolysate was the best lignocellulosic substrate for PeL, resulting in a D-lactic acid yield of 900.7 ± 141.4 mg/g of consumed sugars with an optical purity of 99.8 ± 0.0 %. Gradually scaled-up fermentations using this substrate were achieved in 100-, and 9,000-L fermenters; PeL produced remarkably high D-lactic acid yields of 836.3 ± 11.9 and 915.9 ± 4.4 mg/g of consumed sugars, with optical purities of 95.0 ± 0.0 % and 93.8 ± 0.2 %, respectively. This study is the pioneer in demonstrating economical and sustainable ton-scale production of D-lactic acid.

Biosynthesis and one-step enrichment process of potentially prebiotic cello-oligosaccharides produced by ß-glucosidase from Fusarium solani.

Cello-oligosaccharides (COS) become a new type of functional oligosaccharides. COS transglycosylation reactions were studied to enhance COS yield production. Seeking the ability of the free form of Fusarium solani ß-glucosidase (FBgl1) to synthesize COS under low substrate concentrations, we found out that this biocatalyst initiates this reaction with only 1 g/L of cellobiose, giving rise to the formation of cellotriose. Cellotriose and cellopentaose were detected in biphasic conditions with an immobilized FBgl1 and when increased to 50 g/L of cellobiose as a starter concentration. After the biocatalyst recycling process, the trans-glycosylation yield of COS was maintained after 5 cycles, and the COS concentration was 6.70 ± 0.35 g/L. The crude COS contained 20.15 ± 0.25 g/L glucose, 23.15 ± 0.22 g/L non-reacting substrate cellobiose, 5.25 ± 0.53 g/L, cellotriose and 1.49 ± 0.32 g/L cellopentaose. A bioprocess was developed for cellotriose enrichment, using whole Bacillus velezensis cells as a microbial purification tool. This bacteria consumed glucose, unreacted cellobiose, and cellopentaose while preserving cellotriose in the fermented medium. This study provides an excellent enzyme candidate for industrial COS production and is also the first study on the single-step COS enrichment process.

Rational Engineering of a ß-Glucosidase (H0HC94) from Glycosyl Hydrolase Family I (GH1) to Improve Catalytic Performance on Cellobiose.

The conversion of lignocellulosic feedstocks by cellulases to glucose is a critical step in biofuel production. ß-Glucosidases catalyze the final step in cellulose breakdown, producing glucose, and are often the rate-limiting step in biomass hydrolysis. The specific activity of most natural and engineered ß-glucosidase is higher on the artificial substrate p-nitrophenyl ß-d-glucopyranoside (pNPGlc) than on the natural substrate, cellobiose. We report an engineered ß-glucosidase (Q319A H0HC94) with a 1.8-fold higher specific activity (366.3 ± 36 µmol/min/mg), a 1.5-fold increase in kcat (340.8 ± 27 s-1), and a 3-fold increase in catalytic efficiency (236.65 mM-1 s-1) over H0HC94 (WT) on cellobiose. Molecular dynamic simulations and protein structure network analysis indicate that the Q319A H0HC94 active site pocket is significantly remodeled compared to the WT, leading to changes in enzyme conformation, better accessibility of cellobiose inside the active site pocket, and higher enzymatic activity. This study shows the impact of rational engineering of a nonconserved residue to increase ß-glucosidase substrate accessibility and catalytic efficiency by reducing crowding interaction between cellobiose and active site pocket residues near the gatekeeper region and increasing pocket volume and surface area. Thus, rational engineering of previously characterized enzymes could be an excellent strategy to improve cellulose hydrolysis.

Combinational manipulation of transcription factors, CreA and ClbR, is a viable strategy to improve cellulolytic enzyme production in Aspergillus aculeatus.

The production of cellulolytic enzymes in response to inducible carbon sources is mainly regulated at the transcriptional level in filamentous fungi. We have identified a cellobiose-response regulator (ClbR) controlling the expression of cellulolytic enzyme-encoding genes in Aspergillus aculeatus. However, the engineering potential of combining the deletion of transcriptional repressors with the overexpression of transcriptional activators to enhance enzyme production has not been analyzed. Here, we investigated the effect of the deletion of the transcriptional repressor creA and the overexpression of the transcriptional activator clbR in enzyme production in A. aculeatus. Here, we verified that a combination of creA deletion and clbR overexpression (Δc&OE) improved cellulase, ß-1,4-xylanase, and ß-glucosidase production. Cellulase and ß-1,4-xylanase production increased 3.4- and 8.0-fold in Δc&OE compared with the host strain (MR12) at 96-h incubation, respectively. ß-Glucosidase production in ΔcreA and Δc&OE increased approximately 5.0-fold compared with that in MR12 at 240-h incubation. Transcriptional analysis revealed that the increase in enzyme production was due to increased expression of cellobiohydrolase, endo-ß-1,4-glucanase, ß-1,4-xylanase, and ß-glucosidase 1 (bgl1). Interestingly, bgl1 expression in ΔcreA increased in a dose-dependent manner in response to glucose. Thus, combinational manipulation of transcription factors improved cellulase, xylanase, and ß-glucosidase production in A. aculeatus.

Computer-aided mining of a psychrophilic cellobiose 2-epimerase from the Qinghai-Tibet Plateau gene catalogue.

Cellobiose 2-epimerase (CE) catalyzes the conversion of the lactose into its high-value derivatives, epilactose and lactulose, which has great prospects in food applications. In this study, CE sequences from the Qinghai-Tibet Plateau gene catalogue, we screened these for structural flexibility through molecular dynamics simulation to identify potential psychrophilic CE candidates. One such psychrophilic CE we termed psyCE demonstrated exceptional epimerization activity, achieving an optimum activity of 122.2 ± 1.6 U/mg. Its kinetic parameters (Kcat and Km) for epimerization activity were 219.9 ± 5.6 s-1 and 261.9 ± 18.1 mM, respectively, representing the highest Kcat recorded among known cold-active CEs. Notably, this is the first report of a psychrophilic CE. The psyCE can effectively produce epilactose at 8 °C, converting 20.3 % of 200 mM lactose into epilactose within four hours. These findings suggest that psyCE is highly suitable for cryogenic food processing, and glaciers may serve as a valuable repository of psychrophilic enzymes.

Development and preclinical validation of 2-deoxy 2-[18F]fluorocellobiose as an Aspergillus-specific PET tracer.

The global incidence of invasive fungal infections (IFIs) has increased over the past few decades, mainly in immunocompromised patients, and is associated with high mortality and morbidity. Aspergillus fumigatus is one of the most common and deadliest IFI pathogens. Major hurdles to treating fungal infections remain the lack of rapid and definitive diagnosis, including the frequent need for invasive procedures to provide microbiological confirmation, and the lack of specificity of structural imaging methods. To develop an Aspergillus-specific positron emission tomography (PET) imaging agent, we focused on fungal-specific sugar metabolism. We radiolabeled cellobiose, a disaccharide known to be metabolized by Aspergillus species, and synthesized 2-deoxy-2-[18F]fluorocellobiose ([18F]FCB) by enzymatic conversion of 2-deoxy-2-[18F]fluoroglucose ([18F]FDG) with a radiochemical yield of 60 to 70%, a radiochemical purity of >98%, and 1.5 hours of synthesis time. Two hours after [18F]FCB injection in A. fumigatus pneumonia as well as A. fumigatus, bacterial, and sterile inflammation myositis mouse models, retained radioactivity was only seen in foci with live A. fumigatus infection. In vitro testing confirmed production of ß-glucosidase enzyme by A. fumigatus and not by bacteria, resulting in hydrolysis of [18F]FCB into glucose and [18F]FDG, the latter being retained by the live fungus. The parent molecule was otherwise promptly excreted through the kidneys, resulting in low background radioactivity and high target-to-nontarget ratios at A. fumigatus infectious sites. We conclude that [18F]FCB is a promising and clinically translatable Aspergillus-specific PET tracer.

One-pot synthesis of cellobiose from sucrose using sucrose phosphorylase and cellobiose phosphorylase co-displaying Pichia pastoris as a reusable whole-cell biocatalyst.

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.

Encapsulation of Immobilized ß-Glucosidase with Calcium Metal-Organic Frameworks for Enhanced Stability in Hydrolysis of Cellobiose.

ß-Glucosidase (ß-G) holds promising applications in various fields, such as biomass energy, food, pharmaceuticals, and environmental protection, yet its industrial application is still limited by issues of stability and recycling. Herein, we first immobilized ß-G onto the surface of magnetic chitosan nanoparticles (MCS/ß-G) through adsorption methods. Subsequently, utilizing the metal-organic framework (MOF), CaBDC, which possesses good stability under acidic conditions, we encapsulated MCS/ß-G. The resulting biocatalyst (MCS/ß-G@CaBDC) exhibited excellent activity and recyclability. MCS/ß-G@CaBDC can convert 91.5% of cellobiose to glucose in 60 min and maintained 81.9% activity after 10 cycles. The apparent Km value of MCS/ß-G@CaBDC was 0.148 mM, lower than free ß-G (0.166 mM) and MCS/ß-G (0.173 mM). The CaBDC layer increased the mass transfer resistance of the reaction but also triggered structural rearrangement of ß-G during the encapsulation process. This resulted in the ß-sheet content rising to 68.4%, which, in turn, contributed to enhancing the rigidity of ß-G. Moreover, the saturated magnetic strength of this biocatalyst could reach 37.3 emu/g, facilitating its magnetic recovery. The biocatalyst prepared in this study exhibits promising application prospects, and the immobilization method can provide valuable insights into the field of enzyme immobilization.

Fully Automated Cassette-Based Synthesis of 2-Deoxy-2-[18F]Fluorocellobiose Using Trasis AllInOne Module.

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.

Efficient heterologous expression of cellobiose 2-epimerase gene in Escherichia coli under the control of T7 lac promoter without addition of IPTG and lactose.

In this study, the cellobiose 2-epimerase gene csce from Caldicellulosiruptor saccharolyticus was expressed in Escherichia coli using TB medium containing yeast extract Oxoid and tryptone Oxoid. Interesting, it was found that when the concentration of isopropyl-beta-d-thiogalactopyranoside (IPTG) and lactose was 0 (no addition), the activity of cellobiose 2-epimerase reached 5.88 U/mL. It was 3.70-fold higher than the activity observed when 1.0 mM IPTG was added. When using M9 medium without yeast extract Oxoid and tryptone Oxoid, cellobiose 2-epimerase gene could not be expressed without IPTG and lactose. However, cellobiose 2-epimerase gene could be expressed when yeast extract Oxoid or tryptone Oxoid was added, indicating that these supplements contained inducers for gene expression. In the absence of IPTG and lactose, the addition of soy peptone Angel-1 or yeast extract Angel-1 to M9 medium significantly upregulated the expression of cellobiose 2-epimerase gene in E. coli BL21 pET28a-csce, and these inductions led to higher expression levels compared to tryptone Oxoid or yeast extract Oxoid. The relative transcription level of csce was consistent with its expression level in E. coli BL21 pET28a-csce. In the medium TB without IPTG and lactose and containing yeast extract Angel-1 and soy peptone Angel-1, the activity of cellobiose 2-epimerase reached 6.88 U/mL, representing a 2.2-fold increase compared to previously reported maximum activity in E. coli. The significance of this study lies in its implications for efficient heterologous expression of recombinant enzyme proteins in E. coli without the need for IPTG and lactose addition.

Fibrobacter sp. HC4, a newly isolated strain, demonstrates a high cellulolytic activity as revealed by enzymatic measurements and in vitro assay.

Despite their low quantity and abundance, the cellulolytic bacteria that inhabit the equine large intestine are vital to their host, as they enable the crucial use of forage-based diets. Fibrobacter succinogenes is one of the most important intestinal cellulolytic bacteria. In this study, Fibrobacter sp. HC4, one cellulolytic strain newly isolated from the horse cecum, was characterized for its ability to utilize plant cell wall fibers. Fibrobacter sp. HC4 consumed only cellulose, cellobiose, and glucose and produced succinate and acetate in equal amounts. Among genes coding for CAZymes, 26% of the detected glycoside hydrolases (GHs) were involved in cellulolysis. These cellulases belong to the GH5, GH8, GH9, GH44, GH45, and GH51 families. Both carboxymethyl cellulase and xylanase activities of Fibrobacter sp. HC4 were detected using the Congo red method and were higher than those of F. succinogenes S85, the type strain. The in vitro addition of Fibrobacter sp. HC4 to a fecal microbial ecosystem of horses with large intestinal acidosis significantly enhanced fibrolytic activity as measured by the increase in gas and volatile fatty acids production during the first 48 h. According to this, the pH decreased and the disappearance of dry matter increased at a faster rate with Fibrobacter sp. HC4. Our data suggest a high specialization of the new strain in cellulose degradation. Such a strain could be of interest for future exploitation of its probiotic potential, which needs to be further determined by in vivo studies.IMPORTANCECellulose is the most abundant of plant cell wall fiber and can only be degraded by the large intestine microbiota, resulting in the production of volatile fatty acids that are essential for the host nutrition and health. Consequently, cellulolytic bacteria are of major importance to herbivores. However, these bacteria are challenged by various factors, such as high starch diets, which acidify the ecosystem and reduce their numbers and activity. This can lead to an imbalance in the gut microbiota and digestive problems such as colic, a major cause of mortality in horses. In this work, we characterized a newly isolated cellulolytic strain, Fibrobacter sp. HC4, from the equine intestinal microbiota. Due to its high cellulolytic capacity, reintroduction of this strain into an equine fecal ecosystem stimulates hay fermentation in vitro. Isolating and describing cellulolytic bacteria is a prerequisite for using them as probiotics to restore intestinal balance.

Efficient production of polyhydroxybutyrate using lignocellulosic biomass derived from oil palm trunks by the inhibitor-tolerant strain Burkholderia ambifaria E5-3.

Lignocellulosic biomass is a valuable, renewable substrate for the synthesis of polyhydroxybutyrate (PHB), an ecofriendly biopolymer. In this study, bacterial strain E5-3 was isolated from soil in Japan; it was identified as Burkholderia ambifaria strain E5-3 by 16 S rRNA gene sequencing. The strain showed optimal growth at 37 °C with an initial pH of 9. It demonstrated diverse metabolic ability, processing a broad range of carbon substrates, including xylose, glucose, sucrose, glycerol, cellobiose, and, notably, palm oil. Palm oil induced the highest cellular growth, with a PHB content of 65% wt. The strain exhibited inherent tolerance to potential fermentation inhibitors derived from lignocellulosic hydrolysate, withstanding 3 g/L 5-hydroxymethylfurfural and 1.25 g/L acetic acid. Employing a fed-batch fermentation strategy with a combination of glucose, xylose, and cellobiose resulted in PHB production 2.7-times that in traditional batch fermentation. The use of oil palm trunk hydrolysate, without inhibitor pretreatment, in a fed-batch fermentation setup led to significant cell growth with a PHB content of 45% wt, equivalent to 10 g/L. The physicochemical attributes of xylose-derived PHB produced by strain E5-3 included a molecular weight of 722 kDa, a number-average molecular weight of 191 kDa, and a polydispersity index of 3.78. The amorphous structure of this PHB displayed a glass transition temperature of 4.59 °C, while its crystalline counterpart had a melting point of 171.03 °C. This research highlights the potential of lignocellulosic feedstocks, especially oil palm trunk hydrolysate, for PHB production through fed-batch fermentation by B. ambifaria strain E5-3, which has high inhibitor tolerance.

Discovery of novel cellobiose lipid gene clusters from Basidiomycetes: How chemical variation is reflected in gene cluster architecture.

Cellobiose lipids are surface-active compounds or biological detergents produced by distinct Basidiomycetes yeasts, of which the most and best-described ones belong to the Ustilaginomycetes class. The molecules display slight variation in congener type, which is linked to the hydroxylation position of the long fatty acid, acetylation profile of the cellobiose unit, and presence or absence of the short fatty acid. In general, this variation is strain specific. Although cellobiose lipid biosynthesis has been described for about 11 yeast species, hitherto only two types of biosynthetic gene clusters are identified, and this for only three species. This work adds six more biosynthetic gene clusters and describes for the first time a novel type of cellobiose lipid biosynthetic cluster with a simplified architecture related to specific cellobiose lipids synthesized by Trichosporonaceae family members.

Adaptive evolution of Clostridium thermocellum ATCC 27405 on alternate carbon sources leads to altered fermentation profiles.

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.

PoSnf1 affects cellulose utilization through interaction with cellobiose transporter in Pleurotus ostreatus.

Pleurotus ostreatus is one of the most cultivated edible fungi worldwide, but its lignocellulose utilization efficiency is relatively low (<50 %), which eventually affects the biological efficiency of P. ostreatus. Improving cellulase production and activity will contribute to enhancing the lignocellulose-degrading capacity of P. ostreatus. AMP-activated/Snf1 protein kinase plays important roles in regulating carbon and energy metabolism. The Snf1 homolog (PoSnf1) in P. ostreatus was obtained and analyzed using bioinformatics. The cellulose response of PoSnf1, the effect of the phosphorylation level of PoSnf1 on the expression of cellulose degradation-related genes, the putative proteins that interact with the phosphorylated PoSnf1 (P-PoSnf1), the cellobiose transport function of two sugar transporters (STP1 and STP2), and the interactions between PoSnf1 and STP1/STP2 were studied in this research. We found that cellulose treatment improved the phosphorylation level of PoSnf1, which further affected cellulase activity and the expression of most cellulose degradation-related genes. A total of 1, 024 proteins putatively interacting with P-PoSnf1 were identified, and they were enriched mainly in the substances transport and metabolism. Most of the putative cellulose degradation-related protein-coding genes could respond to cellulose. Among the P-PoSnf1-interacting proteins, the functions of two sugar transporters (STP1 and STP2) were further studied, and the results showed that both could transport cellobiose and were indirectly regulated by P-PoSnf1, and that STP2 could directly interact with PoSnf1. The results of this study indicated that PoSnf1 plays an important role in regulating the expression of cellulose degradation genes possibly by affecting cellobiose transport.

Production of lactulose from lactose using a novel cellobiose 2-epimerase from Clostridium disporicum.

Lactulose is a semisynthetic nondigestive sugar derived from lactose, with wide applications in the food and pharmaceutical industries. Its biological production routes which use cellobiose 2-epimerase (C2E) as the key enzyme have attracted widespread attention. In this study, a set of C2Es from different sources were overexpressed in Escherichia coli to produce lactulose. We obtained a novel and highly efficient C2E from Clostridium disporicum (CDC2E) to synthesize lactulose from lactose. The effects of different heat treatment conditions, reaction pH, reaction temperature, and substrate concentrations were investigated. Under the optimum biotransformation conditions, the final concentration of lactulose was up to 1.45 M (496.3 g/L), with a lactose conversion rate of 72.5 %. This study provides a novel C2E for the biosynthesis of lactulose from low-cost lactose.

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

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

A chemo-enzymatic pathway to expand cellooligosaccharide chemical space through amine bond introduction.

Enzymatic functionalization of oligosaccharides is a useful and environmentally friendly way to expand their structural chemical space and access to a wider range of applications in the health, food, feed, cosmetics and other sectors. In this work, we first tested the laccase/TEMPO system to generate oxidized forms of cellobiose and methyl ß-D-cellobiose, and obtained high yields of novel anionic disaccharides (>60 %) at pH 6.0. Laccase/TEMPO system was then applied to a mix of cellooligosaccharides and to pure D-cellopentaose. The occurrence of carbonyl and carboxyl groups in the oxidation products was shown by LC-HRMS, MALDI-TOF and reductive amination of the carbonyl groups was attempted with p-toluidine a low molar mass amine to form the Schiff base, then reduced by 2-picoline borane to generate a more stable amine bond. The new grafted products were characterized by LC-HRMS, LC-UV-MS/MS and covalent grafting was evidenced. Next, the same procedure was adopted to successfully graft a dye, the rhodamine 123, larger in size than toluidine. This two-step chemo-enzymatic approach, never reported before, for functionalization of oligosaccharides, offers attractive opportunities to anionic cellooligosaccharides and derived glucoconjugates of interest for biomedical or neutraceutical applications. It also paves the way for more environmentally-friendly cellulose fabric staining procedures.