Cellulose-binding Module Research Articles

Acidothermus cellulolyticus E1 endoglucanase expressed in planta undergoes extensive hydroxyproline-O-glycosylation and exhibits enhanced impact on biomass digestibility.

E1 holoenzyme was extensively Hyp-O-glycosylated at the proline rich linker region in plants, which substantially increased the molecular size and improved the enzymatic digestibility of the biomass of transgenic plants. Thermophilic E1 endo-1,4-β-glucanase derived from Acidothermus cellulolyticus has been frequently expressed in planta to reconstruct the plant cell wall to overcome biomass recalcitrance. However, the expressed holoenzyme exhibited a larger molecular size (~ 100kDa) than the theoretical one (57kDa), possibly due to posttranslational modifications in the recombinant enzyme within plant cells. This study investigates the glycosylation of the E1 holoenzyme expressed in tobacco plants and determines its impact on enzyme activity and biomass digestibility. The E1 holoenzyme, E1 catalytic domain (E1cd) and E1 linker (E1Lk) were each expressed in tobacco plants and suspension cells. The accumulation of holoenzyme was 2.0- to 2.3- times higher than that of E1cd. The proline-rich E1Lk region was extensively hydroxyproline-O-glycosylated with arabinogalactan polysaccharides. Compared with E1cd, the holoenzyme displayed a broader optimal temperature range (70 to 85 ºC). When grown in greenhouse, the expression of E1 holoenzyme induced notable phenotypic changes in plants, including delayed flowering and leaf variegation post-flowering. However, the final yield of plant biomass was not significantly affected. Finally, plant biomass engineering with E1 holoenzyme showed 1.7- to 1.8-fold higher saccharification efficiency than the E1cd lines and 2.4- to 2.7-fold higher than the wild-type lines, which was ascribed to the synergetic action of the E1Lk and cellulose binding module in reducing cell wall recalcitrance.

Expression and characterization of a novel β-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater

The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by β-1,4 bonds. The enzyme β-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A β-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a β-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa β-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated β-1,4-endoglucanase had higher activity and stability.

A-365 Carbohydrate-binding Module Proteins to Functionalize Paper for Lateral Flow Applications

Abstract Background Lateral flow (LF) tests have employed nitrocellulose membrane since their introduction. Refinement of nitrocellulose and LF production techniques have led to substantial improvements in reproducibility, sensitivity, clarity, and speed of results. The recent surge in demand for LF tests during the COVID-19 pandemic has drawn increased attention to the consequences of using non-biodegradable or non-recyclable materials in test production, requiring disposal via landfill. The nitrocellulose substrate is a critical component for substitution with an equivalent or superior material to address environmental concerns. Additionally, nitrocellulose and the specific molecular recognition reagents, typically antibodies, contribute significantly to the test cost, and whilst LF tests are less expensive than most other formats, challenges remain with respect to providing affordable tests with adequate performance globally in low resource environments. Despite attempts to substitute cellulose for nitrocellulose, principally for reducing cost and more latterly, environmental impact, cellulose is not widely used due to inferior performance, especially in relation to the clarity and quality of the signal line and reagent requirements. Here, we present preliminary data supporting the use of cellulose membrane (paper) as an alternative substrate to nitrocellulose for LF assays. We demonstrate that cellulose-binding module (CBM)-streptavidin fusion proteins for the immobilisation of capture antibodies on cellulose addresses the performance limitations, yielding sensitivity, clarity and speed of result matching current clinical and industry requirements for a qualitative SARS-CoV-2 antigen LF test. Methods We expressed and purified CBM-streptavidin fusion proteins from E.coli to a purity of >90%. Binding to biotinylated proteins and cellulose material was confirmed. An inhouse SARS-CoV-2 antigen assay was adapted for use with the CBM-streptavidin fusion protein on cellulose material. Using methods compatible with current LF test production, we conjugated and dried down 40nm gold (BBI) with anti-nucleocapsid antibodies (Hytest). The biotinylated anti-nucleocapsid capture antibody was dried down on a separate pad. CBM-streptavidin fusion protein was plotted on Whatman 43 paper at the test line. We evaluated assay performance using SARS-CoV-2 nucleocapsid protein and UV-inactivated SARS-CoV-2 virus (NIBSC) in artificial mucus. We further evaluated accelerated stability of the CBM-paper system at 45°C. Results Plotting of the CBM-fusion protein on the test line (0.3 mg/mL, 1 uL/cm) resulted in a tightly focused line, while the control line antibody, without CBM-fusion protein (1 mg/mL, 1 uL/cm), diffused from its deposition line. Additionally, we were able to substantially reduce the amount of capture antibody needed using the CBM-fusion protein system. The cellulose-based assay exhibited a runtime of 30 minutes and an analytical sensitivity of 10^3 pfu/mL. No significant changes in performance were observed after 9 weeks of incubation at 45°C, representing an approximate accelerated shelf-life of 12 months. Conclusion Our results indicate that paper-based tests match sensitivity, clarity, and speed of current qualitative antigen tests on nitrocellulose, with a shelf-life of at least one year. This preliminary investigation demonstrates the viability of using paper as a replacement for nitrocellulose when coupled with CBM-fusion proteins for antibody immobilisation, offering significantly reduced material cost and environmental impact.

A Role of Cellulose Binding Module of the Thermophilic Endoglucanase TbCel12A

Endoglucanases are enzymes that play an important role in hydrolysis of lignocellulose by attacking glycosidic linkages in cellulose fibers and other glucans. The cellulose binding module (CBM) is responsible for binding the enzyme to the substrate. However, CBMs in certain enzymes interfere with substrate hydrolysis resulting in moderate or low activity. In a previous study, the processive endoglucanase TbCel12A including its CBM had low activity towards carboxymethyl cellulose (CMC). To assess the effect of the CBM, the catalytic domain of TbCel12A was produced without the CBM. The TbCel12A catalytic domain without the CBM hydrolyzed CMC 23 times more rapidly, while the pH and temperature optima and thermotolerance remained unchanged compared to full-length TbCel12A. Therefore, TbCel12A does not require the CBM for CMC hydrolysis and its application may be improved without it.

Manipulating microRNA miR408 enhances both biomass yield and saccharification efficiency in poplar

The conversion of lignocellulosic feedstocks to fermentable sugar for biofuel production is inefficient, and most strategies to enhance efficiency directly target lignin biosynthesis, with associated negative growth impacts. Here we demonstrate, for both laboratory- and field-grown plants, that expression of Pag-miR408 in poplar (Populus alba × P. glandulosa) significantly enhances saccharification, with no requirement for acid-pretreatment, while promoting plant growth. The overexpression plants show increased accessibility of cell walls to cellulase and scaffoldin cellulose-binding modules. Conversely, Pag-miR408 loss-of-function poplar shows decreased cell wall accessibility. Overexpression of Pag-miR408 targets three Pag-LACCASES, delays lignification, and modestly reduces lignin content, S/G ratio and degree of lignin polymerization. Meanwhile, the LACCASE loss of function mutants exhibit significantly increased growth and cell wall accessibility in xylem. Our study shows how Pag-miR408 regulates lignification and secondary growth, and suggest an effective approach towards enhancing biomass yield and saccharification efficiency in a major bioenergy crop.

Biofilm inspired fabrication of functional bacterial cellulose through ex-situ and in-situ approaches

Bacterial cellulose (BC) has been explored for use in a range of applications including tissue engineering and textiles. BC can be produced from waste streams, but sustainable approaches are needed for functionalisation. To this end, BslA, a B. subtilis biofilm protein was produced recombinantly with and without a cellulose binding module (CBM) and the cell free extract was used to treat BC either ex-situ, through drip coating or in-situ, by incorporating during fermentation. The results showed that ex-situ modified BC increased the hydrophobicity and water contact angle reached 120°. In-situ experiments led to a BC film morphological change and mechanical testing demonstrated that addition of BslA with CBM resulted in a stronger, more elastic material. This study presents a nature inspired approach to functionalise BC using a biofilm hydrophobin, and we demonstrate that recombinant proteins could be effective and sustainable molecules for functionalisation of BC materials.

In planta Production and Validation of Neuraminidase Derived from Genotype 4 Reassortant Eurasian Avian-like H1N1 Virus as a Vaccine Candidate.

Influenza viruses are a major public health threat that causes repetitive outbreaks. In recent years, genotype 4 (G4) reassortant Eurasian avian-like (EA) H1N1 (G4 EA H1N1) has garnered attention as a potential novel pandemic strain. The necessity of developing vaccines against G4 EA H1N1 is growing because of the increasing cases of human infection and the low cross-reactivity of the strain with current immunity. In this study, we produced a G4 EA H1N1-derived neuraminidase (G4NA) as a vaccine candidate in Nicotiana benthamiana. The expressed G4NA was designed to be accumulated in the endoplasmic reticulum (ER). The M-domain of the human receptor-type tyrosine-protein phosphatase C was incorporated into the expression cassette to enhance the translation of G4NA. In addition, the family 3 cellulose-binding module and Brachypodium distachyon small ubiquitin-like modifier sequences were used to enable the cost-effective purification and removal of unnecessary domains after purification, respectively. The G4NA produced in plants displayed high solubility and assembled as a tetramer, which is required for the efficacy of an NA-based vaccine. In a mouse immunization model, the G4NA produced in plants could induce significant humoral immune responses. The plant-produced G4NA also stimulated antigen-specific CD4 T cell activation. These G4NA vaccine-induced immune responses were intensified by the administration of the antigen with a vaccine adjuvant. These results suggest that G4NA produced in plants has great potential as a vaccine candidate against G4 EA H1N1.

Tracking Bacterial Nanocellulose in Animal Tissues by Fluorescence Microscopy.

The potential of nanomaterials in food technology is nowadays well-established. However, their commercial use requires a careful risk assessment, in particular concerning the fate of nanomaterials in the human body. Bacterial nanocellulose (BNC), a nanofibrillar polysaccharide, has been used as a food product for many years in Asia. However, given its nano-character, several toxicological studies must be performed, according to the European Food Safety Agency’s guidance. Those should especially answer the question of whether nanoparticulate cellulose is absorbed in the gastrointestinal tract. This raises the need to develop a screening technique capable of detecting isolated nanosized particles in biological tissues. Herein, the potential of a cellulose-binding module fused to a green fluorescent protein (GFP–CBM) to detect single bacterial cellulose nanocrystals (BCNC) obtained by acid hydrolysis was assessed. Adsorption studies were performed to characterize the interaction of GFP–CBM with BNC and BCNC. Correlative electron light microscopy was used to demonstrate that isolated BCNC may be detected by fluorescence microscopy. The uptake of BCNC by macrophages was also assessed. Finally, an exploratory 21-day repeated-dose study was performed, wherein Wistar rats were fed daily with BNC. The presence of BNC or BCNC throughout the GIT was observed only in the intestinal lumen, suggesting that cellulose particles were not absorbed. While a more comprehensive toxicological study is necessary, these results strengthen the idea that BNC can be considered a safe food additive.

Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana.

The production of recombinant proteins in plant systems is receiving wider attention. Indeed, various plant-produced pharmaceuticals have been shown to be biologically active. However, the production of human growth factors and cytokines in heterologous systems is still challenging because they often act as complex forms, such as homo- or hetero-dimers, and their production is tightly regulated in vivo. In this study, we demonstrated that the mature form of human TGFβ1 produced and purified from Nicotiana benthamiana shows biological activity in animal cells. To produce the mature form of TGFβ1, various recombinant genes containing the mature form of TGFβ1 were generated and produced in N. benthamiana. Of these, a recombinant construct, BiP:M:CBM3:LAP[C33S]:EK:TGFβ1, was expressed at a high level in N. benthamiana. Recombinant proteins were one-step purified using cellulose-binding module 3 (CBM3) as an affinity tag and microcrystalline cellulose (MCC) beads as a matrix. The TGFβ1 recombinant protein bound on MCC beads was proteolytically processed with enterokinase to separate mature TGFβ1. The mature TGFβ1 still associated with Latency Associated Protein, [LAP(C33S)] that had been immobilized on MCC beads was released by HCl treatment. Purified TGFβ1 activated TGFβ1-mediated signaling in the A549 cell line, thereby inducing phosphorylation of SMAD-2, the expression of ZEB-2 and SNAIL1, and the formation of a filopodia-like structure. Based on these results, we propose that active mature TGFβ1, one of the most challenging growth factors to produce in heterologous systems, can be produced from plants at a high degree of purity via a few steps.

Enhanced in situ H2O2 production explains synergy between an LPMO with a cellulose-binding domain and a single-domain LPMO

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that catalyze oxidative depolymerization of recalcitrant substrates such as chitin or cellulose. Recent work has shown that LPMOs catalyze fast peroxygenase reactions and that, under commonly used reaction set-ups, access to in situ generated H2O2 likely limits catalysis. Based on a hypothesis that the impact of a cellulose-binding module (CBM) on LPMO activity could relate to changes in in situ H2O2 production, we have assessed the interplay between CBM-containing ScLPMO10C and its truncated form comprising the catalytic domain only (ScLPMO10CTR). The results show that truncation of the linker and CBM leads to elevated H2O2 production and decreased enzyme stability. Most interestingly, combining the two enzyme forms yields strong synergistic effects, which are due to the combination of high H2O2 generation by ScLPMO10CTR and efficient productive use of H2O2 by the full-length enzyme. Thus, cellulose degradation becomes faster, while enzyme inactivation due to off-pathway reactions with excess H2O2 is reduced. These results underpin the complexity of ascorbic acid-driven LPMO reactions and reveal a potential mechanism for how LPMOs may interact synergistically during cellulose degradation.

Rumen and fecal microbial profiles in cattle fed high lignin diets using metagenome analysis

Coconut coir (a lignin-rich, organic material) is widely used for its commercial and economic benefits. In this study, crossbred (exotic) and Kankrej (indigenous) breeds of cattle were fed diets containing 7 or 14% coconut coir. Metagenomic analyses (16S rRNA gene amplicon and shotgun sequencing) were used to characterize the microbial community in the rumen and fecal samples along with their functional capabilities. Both amplicon and shotgun analyses revealed the predominance of bacterial phyla, Bacteroidetes, Firmicutes, Actinobacteria and Fibrobacter in ruminal liquid, ruminal solid and fecal samples. 16S rRNA gene amplicon sequencing revealed a total of 18 different bacterial taxa were found to be enriched exclusively in the animals fed with 14% coir. The shotgun analysis revealed abundance of bacterial genera, Fibrobacter, Clostridium, Prevotella, Butyrivibrio, and Ruminococcus in both liquid and solid fractions of ruminal contents, while in the fecal sample, Bacteroides, Alistipes, Plaudibacter, Parabacteroides, Porphyromonas, and Victivallis and archaeal genus, Methanocorpusculum were abundant. The functional analysis based on dbCAN database suggested that among the Glycoside hydrolase family, genes that encode oligosaccharide degrading enzymes, GH3, GH13 (p-value < 0.05), and GH43 were abundant in the feces. In ruminal solid, cellulase encoding the GH5 family was abundant. Also, lignocellulosic binding modules encoded by the CBM family, including cellulose (CBM3) and hemicellulose binding modules (CBM32 and CBM67) were abundant. Thus, the study indicated the enrichment of lignocellulosic enzymes in ruminal contents in response to feeding the coconut coir, which could be mined for potential biofuel production and other biotechnological applications.

Construction, Investigation and Application of TEV Protease Variants with Improved Oxidative Stability.

Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tags, but wild-type TEVp is less stable under oxidized redox state. In this work, we introduced and combined C19S, C110S and C130S into TEVp variants containing T17S, L56V, N68D, I77V and S135G to improve protein solubility, and S219V to inhibit self-proteolysis. The solubility and cleavage activity of the constructed variants in Escherichia coli strains including BL21(DE3), BL21(DE3)pLys, Rossetta(DE3) and Origami(DE3) under the same induction conditions were analyzed and compared. The desirable soluble amounts, activity, and oxidative stability were identified to be reluctantly favored in the TEVp. Unlike C19S, C110S and C130S hardly impacted on decreasing protein solubility in the BL21(DE3), but they contributed to improved tolerance to the oxidative redox state in vivo and in vitro. After two fusion proteins were cleaved by purified TEVp protein containing double mutations under the oxidized redox state, the refolded disulfide-rich bovine enterokinase catalytic domain or maize peroxidase with enhanced yields were released from the regenerated amorphous cellulose via affinity absorption of the cellulose-binding module as the affinity tag.

A Single Mutation in the Carbohydrate-Binding Module Enhances Cellulase Activity in Bacillus Amyloliquefaciens Mutant

From our earlier work, we modified the carbohydrate-binding module (CBM) of Bacillus amyloliquefaciens to increase cellulase activity using cold plasma technology. The cellulase gene (BglC-M) from the mutant was expressed in Escherichia coli BL21(DE3) under the T7 promoter. The hydrolysis activity of the cellulase mutant (BglC-M) was approximately 2.5-fold higher than the control (BglC-W) over a wide range of pH and temperature conditions. The amino acid sequence of the mutant BglC-M contained 471 residues that were almost identical to the control BglC-W. Only a single amino acid, lysine, was replaced by glutamic acid at position 370 (K370E) within the carbohydrate-binding module (CBM). Structure prediction and substrate docking of BglC-M indicated that the single mutation (K370E) might involve cellulose binding of the β-sandwich facilitated by hydrogen bonding. The docking study of cellopentaose with the model structure of BglC-M indicated that the replacement of lysine-370 led to the formation of a hydrogen bond with 436Y, which has a shorter distance (2.6 Å) compared with the control (5.4 Å). As a result, the structure becomes more compact and stable, resulting in increased catalytic efficiency. Finally, the biomass hydrolysis ability of cellulase was investigated on lignocellulosic wastes such as pineapple peel, corncob, and durian peel. The BglC-M enzyme showed a more significant amount of reducing sugar released from all lignocellulosic wastes than the control. This was the first evidence that altering the base composition of the cellulose binding module enhanced the catalytic activity.&#x0D; HIGHLIGHTS&#x0D; &#x0D; Increasing cellulase activity of Bacillus amyloliquefaciens using plasma technology&#x0D; Mutation at cellulose-binding module enhance cellulase hydrolysis activity&#x0D; Greater cellulase activity in the hydrolysis on lignocellulosic wastes&#x0D; &#x0D; GRAPHICAL ABSTRACT

Production of Gloeophyllum trabeum Endoglucanase Cel12A in Nicotiana benthamiana for Cellulose Degradation.

Lignocellulosic biomass from plants has been used as a biofuel source and the potent acidic endoglucanase GtCel12A has been isolated from Gloeophyllum trabeum, a filamentous fungus. In this study, we established a plant-based platform for the production of active GtCel12A fused to family 3 cellulose-binding module (CBM3). We used the signal sequence of binding immunoglobulin protein (BiP) and the endoplasmic reticulum (ER) retention signal for the accumulation of the produced GtCel12A in the ER. To achieve enhanced enzyme expression, we incorporated the M-domain of the human receptor-type tyrosine-protein phosphatase C into the construct. In addition, to enable the removal of N-terminal domains that are not necessary after protein expression, we further incorporated the cleavage site of Brachypodium distachyon small ubiquitin-like modifier. The GtCel12A-CBM3 fusion protein produced in the leaves of Nicotiana benthamiana exhibited not only high solubility but also efficient endoglucanase activity on the carboxymethyl cellulose substrate as determined by 3,5-dinitrosalicylic acid assay. The endoglucanase activity of GtCel12A-CBM3 was maintained even when immobilized on microcrystalline cellulose beads. Taken together, these results indicate that GtCel12A endoglucanase produced in plants might be used to provide monomeric sugars from lignocellulosic biomass for bioethanol production.

A Multiomic Approach to Understand How Pleurotus eryngii Transforms Non-Woody Lignocellulosic Material.

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.

MicroRNA408 Enhances Growth and Saccharification Efficiency Through Altering the Balance between Vascular Development and Lignification in Hybrid Poplar

Saccharification, the conversion of cellulose to sugar for fermentation-based biofuels, remains problematic for lignocellulosic feedstocks such as wood. Genetic modifications to reduce the amount or change the structure of lignin can improve saccharification efficiency but often result in dwarf plants. Here, we report that overexpression of Pag-MIR408 in hybrid poplar results in a near doubling saccharification efficiency, with no requirement for acid-pretreatment, increased accessibility of secondary cell walls to fungal cellulase and scaffoldin cellulose-binding modules, accompanied by enhanced photosynthesis, and increased number of cambium zone layers, xylem width and vessel diameter. Conversely, Pag-MIR408 loss-of-function plants by CRISPR/Cas9 showed decreased cell wall accessibility. Moreover, miR408 targets LACCASE 19/25, leading to delayed lignification, and modestly reduced lignin content, S/G ratio, as well as the degree of lignin polymerization. This study provides a unique insight into enhanced saccharification efficiency altered wood structure through modulation of the balance between secondary development and lignification.

Facile biosynthesis of synthetic crystalline cellulose nanoribbon from maltodextrin through a minimized two-enzyme phosphorylase cascade and its application in emulsion

Nanocellulose has many promising applications such as a green ingredient for Pickering emulsion. Traditional strategies to produce nanocellulose, which are acid or enzymatic hydrolysis and mechanical methods on natural complicated cellulose, are hard to control and can result in significant pollutants during the processes. Herein, we demonstrated a facile and sustainable method for the biocatalytic production of insoluble synthetic crystalline cellulose nanoribbon (CCNR) from cheap maltodextrin by coupling α-glucan phosphorylase (αGP) and cellodextrin phosphorylase (CDP) using cellobiose as a primer. And by optimizing the combination of different αGP and CDP, it turned out that the optimal enzyme combination is αGP from Thermotoga maritime and CDP from Clostridium thermocellum, in which CDP was attached to a family 9 cellulose-binding module. The product yield and degree of polymerization (DP) of insoluble synthetic CCNR was affected by the primer concentration at a fixed concentration of maltodextrin. After optimization of reaction conditions, the highest product yield of insoluble synthetic CCNR was 44.92 % and the highest DP of the insoluble synthetic CCNR was 24 from 50 g 1−1 maltodextrin. This insoluble synthetic CCNR can be used as a Pickering emulsions stabilizer, showing excellent emulsifiability. This study provides a promising alternative for cost-efficient production of insoluble synthetic CCNR which was used as a green emulsion stabilizer.

Recombinant lactase with a cellulose binding domain permits facile immobilization onto cellulose with retained activity

Because of their potential impact in food and bioprocessing, diagnostics, green(er) chemistry, and waste remediation, new enzyme immobilization technologies continue to be explored. In this work, a recombinant lactase (β-galactosidase) (LacZ) presenting a carbohydrate binding module (LacZ-CBM), was engineered, expressed, and immobilized onto cellulose (LacZ-CBM:cell). Binding density, activity, and pH and temperature activity profiles were characterized in comparison to wild type lactase (LacZ). LacZ-CBM effectively self-immobilized onto cellulose (Sigmacell®) in less than 2 h, demonstrating that recombinant enzymes with cellulose binding modules can enable immobilization onto solid supports without the need for chemical crosslinking agents. The immobilized recombinant LacZ-CBM:cell retained over 30% of its initial activity over 9 cycles. Both immobilized and non-immobilized LacZ-CBM presented similar optimum reaction conditions as wild type lactase, which demonstrates that the addition of a carbohydrate binding module (CBM) to lactase does not alter its optimum reaction conditions. This work has direct relevance to immobilized lactase applications (e.g. lactose reduced milk; oligosaccharide production) and serves as a model for other greener enzyme immobilization applications.

Engineered LPMO Significantly Boosting Cellulase-Catalyzed Depolymerization of Cellulose.

Lytic polysaccharide monooxygenases (LPMOs) play a crucial role in the enzymatic depolymerization of cellulose through oxidative cleavage of the glycosidic bond in the highly recalcitrant crystalline cellulose region. Improving the activity of LPMOs is of considerable importance for second-generation biorefinery. In this study, we identified a beneficial amino acid substitution (N526S) located in the cellulose binding module (CBM) of HcLPMO10 (LPMO of Hahella chejuensis) using directed evolution. The improved variant HcLPMO10 M1 (N526S) exhibits 2.1-fold higher activity for the H2O2 production, 2.7-fold higher oxidation activity, and 1.9-fold higher binding capacity toward cellulose compared with those of the wild type (WT). Furthermore, M1 shows 2.1-fold higher activity for degradation of crystalline cellulose in synergy with cellulase, compared to the WT. Structural analysis through molecular modeling and molecular dynamics (MD) simulation revealed that the substitution N526S located in the CBM likely stabilizes the cellulose binding surface and enhances the binding capacity of HcLPMO10 to cellulose, thereby enhancing enzyme activity. These findings demonstrate the important role of the CBM in the catalytic function of LPMO.

Multienzyme Cellulose Films as Sustainable and Self-Degradable Hydrogen Peroxide-Producing Material.

The use of hydrogen peroxide-releasing enzymes as a component to produce alternative and sustainable antimicrobial materials has aroused interest in the scientific community. However, the preparation of such materials requires an effective enzyme binding method that often involves the use of expensive and toxic chemicals. Here, we describe the development of an enzyme-based hydrogen peroxide-producing regenerated cellulose film (RCF) in which a cellobiohydrolase (TrCBHI) and a cellobiose dehydrogenase (MtCDHA) were efficiently adsorbed, 90.38 ± 2.2 and 82.40 ± 5.7%, respectively, without making use of cross-linkers. The enzyme adsorption kinetics and binding isotherm experiments showed high affinity of the proteins possessing cellulose-binding modules for RCF, suggesting that binding on regenerated cellulose via specific interactions can be an alternative method for enzyme immobilization. Resistance to compression and porosity at a micrometer scale were found to be tunable by changing cellulose concentration prior to film regeneration. The self-degradation process, triggered by stacking TrCBHI and MtCDHA (previously immobilized onto separate RCF), produced 0.15 nmol/min·cm2 of H2O2. Moreover, the production of H2O2 was sustained for at least 24 h reaching a concentration of ∼2 mM. The activity of MtCDHA immobilized on RCF was not affected by reuse for at least 3 days (1 cycle/day), suggesting that no significant enzyme leakage occurred in that timeframe. In the material herein designed, cellulose (regenerated from a 1-ethyl-3-methylimidazolium acetate/dimethyl sulfoxide (DMSO) solution) serves both as support and substrate for the immobilized enzymes. The sequential reaction led to the production of H2O2 at a micromolar-millimolar level revealing the potential use of the material as a self-degradable antimicrobial agent.