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.

Reductant-independent oxidative cleavage of cellulose by a novel marine fungal lytic polysaccharide monooxygenase.

Among the enzymes derived from fungus that act on polysaccharides, lytic polysaccharide monooxygenase (LPMOs) has emerged as a new member with complex reaction mechanisms and high efficiency in dealing with recalcitrant crystalline polysaccharides. This study reported the characteristics, structure, and biochemical properties of a novel LPMO from Talaromyces sedimenticola (namely MaLPMO9K) obtained from the Mariana Trench. MaLPMO9K was a multi-domain protein combined with main body and a carbohydrate-binding module. It was heterologously expressed in E. coli for analyzing peroxidase activity in reactions with the substrate 2,6-DMP, where H2O2 serves as a co-substrate. Optimal peroxidase activity for MaLPMO9K was observed at pH 8 and 25 °C, achieving the best Vmax value of 265.2 U·g-1. In addition, MaLPMO9K also demonstrated the ability to treat cellulose derivatives, and cellobiose substrates without the presence of reducing agents.

Investigation of the influence of Candida tropicalis on bioethanol production using sugarcane bagasse: stochastic and in silico analysis.

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

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.

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.

Rational Design of Loop Dynamics for a Barrel-Shaped Enzyme by Introducing Disulfide Bonds.

Loop dynamics redesign is an important strategy to manipulate protein function. Cellobiose 2-epimerase (CE) and other members of its superfamily are widely used for diverse industrial applications. The structural feature of the loops connecting barrel helices contributes greatly to the differences in their functional characteristics. Inspired by the in-silico mutation with molecular dynamics (MD) simulation analysis, we propose a strategy for identifying disulfide bond mutation candidates based on the prediction of protein flexibility and residue-residue interaction. The most beneficial mutant with the newly introduced disulfide bond would simultaneously improve both its thermostability and its reaction propensity to the targeting isomerization product. The ratio of the isomerization/epimerization catalytic rate was improved from 4:103 to 9:22. MD simulation and binding free energy calculations were applied to provide insights into molecular recognition upon mutations. The comparative analysis of enzyme/substrate binding modes indicates that the altered catalytic reaction pathway is due to less efficient binding of the native product. The key residue responsible for the observed phenotype was identified by energy decomposition and was further confirmed by the mutation experiment. The rational design of the key loop region might be a promising strategy to alter the catalytic behavior of all (α/α)6-barrel-like proteins.

Tailored green synthesized silymarin-selenium nanoparticles: Topical nanocarrier of promising antileishmanial activity.

Poor drug penetration, emerging drug resistance, and systemic toxicity are among the major obstacles challenging the current treatment of cutaneous leishmaniasis. Hence, developing advanced strategies for effective and targeted delivery of antileishmanial agents is crucial. Several drug delivery carriers have been developed till current date for dermal/transdermal delivery, especially those which are fabricated using eco-friendly synthesis approaches, since they protect the environment from the harmful effects of chemical waste disposal. This work describes the preparation of selenium nanoparticles loaded with silymarin via one-pot green reduction technique, for treatment of cutaneous leishmaniasis. The selected silymarin loaded selenium nanoparticles (SSNs4-0.1) displayed good loading efficiency of 58.22 ± 0.56 %, zeta potential of -30.63 ± 0.40 mV, hydrodynamic diameter of 245.77 ± 11.12 nm, and polydispersity index of 0.19 ± 0.01. It exhibited good physical stability, as well as high ex vivo deposition % in the epidermis (46.98 ± 1.51 %) and dermis (35.23 ± 1.72 %), which was further proven using confocal laser microscopy. It also exhibited significant cytocompatibility and noticeable cellular internalization of 90.02 ± 3.81 % in human fibroblasts, as well as high trypanothione reductase inhibitory effect (97.10 ± 0.30 %). Results of this study confirmed the successful green synthesis of silymarin-loaded selenium nanoparticles; delineating them as one of the promising antileishmanial topical delivery systems.

Safety evaluation of the food enzyme cellobiose phosphorylase from the genetically modified Escherichia coli strain LE1B109-pPB130.

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

Efficacious bioconversion of alginate/cellulose to value-added oligosaccharides by alginate-degrading GH5 endoglucanase from Trichoderma asperellum.

Enzymatic degradation of lignocellulosic biomass provides an eco-friendly approach to produce value-added macromolecules, e.g., bioactive polysaccharides. A novel acidophilic GH5 ß-1,4-endoglucanase (termed TaCel5) from Trichoderma asperellum ND-1 was efficiently expressed in Komagataella phaffii (∼1.5-fold increase, 38.42 U/mL). TaCel5 displayed both endoglucanase (486.3 U/mg) and alginate lyase (359.5 U/mg) enzyme activities. It had optimal pH 3.0 and strong pH stability (exceed 86 % activity retained over pH range 3.0-5.0). 80 % activity (both endoglucanase and alginate lyase) was retained in the presence of 15 % ethanol or 3.42 M NaCl. Analysis of action mode revealed that hydrolytic activity of TaCel5 required at least three glucose (cellotriose) residues, yielding mainly cellobiose. Glu241 and Glu352 are essential catalytic residues, while Asp106, Asp277 and Asp317 play auxiliary roles in cellulose degradation. TaCel5 displayed high hydrolysis efficiency for glucan and alginate substrates. ESI-MS analysis indicated that the enzymatic hydrolysates of alginate mainly contained disaccharides and heptasaccharides. This is the first detailed report of a bifunctional GH5 endoglucanase/alginate lyase enzyme from T. asperellum. Thus TaCel5 has strong potential in food and feed industries as a catalyst for bioconversion of cellulose- and alginate-containing waste materials into value-added products oligosaccharides, which was of great benefit both for the economy and environment.

Exploring class III cellobiose dehydrogenase: sequence analysis and optimized recombinant expression.

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

Engineering Bacillus subtilis for highly efficient production of functional disaccharide lactulose from lactose.

Bioconversion of lactose to functional lactose derivatives attracts increasing attention. Lactulose is an important high-value lactose derivative, which has been widely used in pharmaceutical, nutraceutical, and food industries. Lactulose can be enzymatically produced from lactose by cellobiose 2-epimerase (CEase). Several studies have already focused on the food-grade expression of CEase, but they are all aimed at the biosynthesis of epilactose. Herein, we reported for the first time the biosynthesis of lactulose using the recombinant food-grade Bacillus subtilis. Lactulose biosynthesis was optimized by varying lactulose-producing CEases and expression vectors. Caldicellulosiruptor saccharolyticus CEase and pP43NMK were determined to be the optimal CEase and expression vector. Fine-tuning of CEase expression was investigated by screening a beneficial N-terminal coding sequence. After fed-batch cultivation, the highest fermentation isomerization activity reached 11.6 U/mL. Lactulose was successfully produced by the broth of the engineered B. subtilis with a yield of 52.1 %.

Expression of cellobiose dehydrogenase gene in Aspergillus niger C112 and its effect on lignocellulose degrading enzymes.

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

Transcriptional regulation of cellobiose utilization by PRD-domain containing Sigma54-dependent transcriptional activator (CelR) and catabolite control protein A (CcpA) in Bacillus thuringiensis.

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

Surface display system of Bacillus subtilis: A promising approach for improving the stability and applications of cellobiose dehydrogenase.

Cellobiose dehydrogenase (CDH) plays a crucial role in lignocellulose degradation and bioelectrochemical industries, making it highly in demand. However, the production and purification of CDH through fungal heterologous expression methods is time-consuming, costly, and challenging. In this study, we successfully displayed Pycnoporus sanguineus CDH (psCDH) on the surface of Bacillus subtilis spores for the first time. Enzymatic characterization revealed that spore surface display enhanced the tolerance of psCDH to high temperature (80 °C) and low pH levels (3.5) compared to free psCDH. Furthermore, we found that glycerol, lactic acid, and malic acid promoted the activity of immobilized spore-displayed psCDH; glycerol has a more significant stimulating effect, increasing the activity from 16.86 ± 1.27 U/mL to 46.26 ± 3.25 U/mL. After four reuse cycles, the psCDH immobilized with spores retained 48% of its initial activity, demonstrating a substantial recovery rate. In conclusion, the spore display system, relying on cotG, enables the expression and immobilization of CDH while enhancing its resistance to adverse conditions. This system demonstrates efficient enzyme recovery and reuse. This approach provides a novel method and strategy for the immobilization and stability enhancement of CDH.

Engineering of Ogataea polymorpha strains with ability for high-temperature alcoholic fermentation of cellobiose.

Successful conversion of cellulosic biomass into biofuels requires organisms capable of efficiently utilizing xylose as well as cellodextrins and glucose. Ogataea (Hansenula) polymorpha is the natural xylose-metabolizing organism and is one of the most thermotolerant yeasts known, with a maximum growth temperature above 50°C. Cellobiose-fermenting strains, derivatives of an improved ethanol producer from xylose O. polymorpha BEP/cat8∆, were constructed in this work by the introduction of heterologous genes encoding cellodextrin transporters (CDTs) and intracellular enzymes (ß-glucosidase or cellobiose phosphorylase) that hydrolyze cellobiose. For this purpose, the genes gh1-1 of ß-glucosidase, CDT-1m and CDT-2m of cellodextrin transporters from Neurospora crassa and the CBP gene coding for cellobiose phosphorylase from Saccharophagus degradans, were successfully expressed in O. polymorpha. Through metabolic engineering and mutagenesis, strains BEP/cat8∆/gh1-1/CDT-1m and BEP/cat8∆/CBP-1/CDT-2mAM were developed, showing improved parameters for high-temperature alcoholic fermentation of cellobiose. The study highlights the need for further optimization to enhance ethanol yields and elucidate cellobiose metabolism intricacies in O. polymorpha yeast. This is the first report of the successful development of stable methylotrophic thermotolerant strains of O. polymorpha capable of coutilizing cellobiose, glucose, and xylose under high-temperature alcoholic fermentation conditions at 45°C.

Unveiling a classical mutant in the context of the GH3 ß-glucosidase family in Neurospora crassa.

Classical fungal mutant strains obtained by mutagenesis have helped to elucidate fundamental metabolic pathways in the past. In the filamentous fungus Neurospora crassa, the gluc-1 strain was isolated long ago and characterized by its low level of ß-glucosidase activity, which is essential for the degradation of cellulose, the most abundant biopolymer on Earth and the main polymeric component of the plant cell wall. Based on genomic resequencing, we hypothesized that the causative mutation resides in the ß-glucosidase gene gh3-3 (bgl6, NCU08755). In this work, growth patterns, enzymatic activities and sugar utilization rates were analyzed in several mutant and overexpression strains related to gluc-1 and gh3-3. In addition, different mutants affected in the degradation and transport of cellobiose were analyzed. While overexpression of gh3-3 led to the recovery of ß-glucosidase activity in the gluc-1 mutant, as well as normal utilization of cellobiose, the full gene deletion strain Δgh3-3 was found to behave differently than gluc-1 with lower secreted ß-glucosidase activity, indicating a dominant role of the amino acid substitution in the point mutated gh3-3 gene of gluc-1. Our results furthermore confirm that GH3-3 is the major extracellular ß-glucosidase in N. crassa and demonstrate that the two cellodextrin transporters CDT-1 and CDT-2 are essential for growth on cellobiose when the three main N. crassa ß-glucosidases are absent. Overall, these findings provide valuable insight into the mechanisms of cellulose utilization in filamentous fungi, being an essential step in the efficient production of biorefinable sugars from agricultural and forestry plant biomass.

The application of bacteria-derived dehydrogenases and oxidases in the synthesis of gold nanoparticles.

In this work the green synthesis of gold nanoparticles (Au-NPs) using the oxidoreductive enzymes Myriococcum thermophilum cellobiose dehydrogenase (Mt CDH), Glomerella cingulata glucose dehydrogenase (Gc GDH), and Aspergillus niger glucose oxidase (An GOX)) as bioreductants was investigated. The influence of reaction conditions on the synthesis of Au-NPs was examined and optimised. The reaction kinetics and the influence of Au ions on the reaction rate were determined. Based on the kinetic study, the mechanism of Au-NP synthesis was proposed. The Au-NPs were characterized by UV-Vis spectroscopy and transmission electron microscopy (TEM). The surface plasmon resonance (SPR) absorption peaks of the Au-NPs synthesised with Mt CDH and Gc GDH were observed at 535 nm, indicating an average size of around 50 nm. According to the image analysis performed on a TEM micrograph, the Au-NPs synthesized with Gc GDH have a spherical shape with an average size of 2.83 and 6.63 nm after 24 and 48 h of the reaction, respectively. KEY POINTS: • The Au NPs were synthesised by the action of enzymes CDH and GDH. • The synthesis of Au-NPs by CDH is related to the oxidation of cellobiose. • The synthesis of Au-NPs by GDH was not driven by the reaction kinetic.

Conversion of Deproteinized Cheese Whey to Lactobionate by an Engineered Neurospora crassa Strain F5.

We report a novel production process for lactobionic acid (LBA) production using an engineered Neurospora crassa strain F5. The wild-type N. crassa strain produces cellobiose dehydrogenase (CDH) and uses lactose as a carbon source. N. crassa strain F5, which was constructed by deleting six out of the seven ß-glucosidases in the wild type, showed a much slower lactose utilization rate and produced a much higher level of cellobiose dehydrogenase (CDH) than the wild type. Strain N. crassa F5 produced CDH and laccase simultaneously on the pretreated wheat straw with 3 µM of cycloheximide added as the laccase inducer. The deproteinized cheese whey was added directly to the shake flasks with the fungus present to achieve LBA production. Strain F5 produced about 37 g/L of LBA from 45 g/L of lactose in 27 h since deproteinized cheese whey addition. The yield of LBA from consumed lactose was about 85%, and the LBA productivity achieved was about 1.37 g/L/h.

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production II: Model-based multiobjective optimization.

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

Discovery of a Novel Cellobiose Dehydrogenase from Cellulomonas palmilytica EW123 and Its Sugar Acids Production.

Cellobiose dehydrogenases (CDHs) are a group of enzymes belonging to the hemoflavoenzyme group, which are mostly found in fungi. They play an important role in the production of acid sugar. In this research, CDH annotated from the actinobacterium Cellulomonas palmilytica EW123 (CpCDH) was cloned and characterized. The CpCDH exhibited a domain architecture resembling class-I CDH found in Basidiomycota. The cytochrome c and flavin-containing dehydrogenase domains in CpCDH showed an extra-long evolutionary distance compared to fungal CDH. The amino acid sequence of CpCDH revealed conservative catalytic amino acids and a distinct flavin adenine dinucleotide region specific to CDH, setting it apart from closely related sequences. The physicochemical properties of CpCDH displayed optimal pH conditions similar to those of CDHs but differed in terms of optimal temperature. The CpCDH displayed excellent enzymatic activity at low temperatures (below 30°C), unlike other CDHs. Moreover, CpCDH showed the highest substrate specificity for disaccharides such as cellobiose and lactose, which contain a glucose molecule at the non-reducing end. The catalytic efficiency of CpCDH for cellobiose and lactose were 2.05 x 105 and 9.06 x 104 (M-1 s-1), respectively. The result from the Fourier-transform infrared spectroscopy (FT-IR) spectra confirmed the presence of cellobionic and lactobionic acids as the oxidative products of CpCDH. This study establishes CpCDH as a novel and attractive bacterial CDH, representing the first report of its kind in the Cellulomonas genus.