Phosphoric Acid-swollen Cellulose Research Articles

Cassava Bagasse as a Low-Cost Substrate for Cellulase and Organic Acid Production Using Co-Cultivated Fungi

Cassava bagasse has great potency as a substrate in the biorefinery industry. This paper proposes the valorisation of cassava bagasse into organic acids by cellulase through the co-cultivation of Aspergillus violaceofuscus and Trichoderma reesei RUT-C30 at the ratio 1:1. The optimised conditions for β-glucosidase production under submerged fermentation were pH 4.50, a tween 80 concentration of 0.05% (v/v), and a spore concentration of 7.18 × 107 spores·mL−1. We found base steam cassava bagasse (BSCB) to have high cellulose content, making it possible to replace avicel phosphoric acid swollen cellulose (PASC) as a substrate. The co-cultivation with the BSCB substrate had higher levels of β-glucosidase (1.72-fold), cellobiohydrolase (2.83-fold), and endoglucanase (2.82-fold) activity compared to that of the avicel PASC substrate. Moreover, acetic acid (7.41 g·L−1), citric acid (3.54 g·L−1), gluconic acid (0.30 g·L−1), and malic acid (0.37 g·L−1) were detected in the BSCB crude extract. These results demonstrate the considerable prospects of the A. violaceofuscus and T. reesei RUT-C30 co-cultivation approaches in the biorefinery industry.

Functional study of a lytic polysaccharide monooxygenase MsLPMO3 from Morchella sextelata in the oxidative degradation of cellulose

Lytic polysaccharide monooxygenases (LPMOs) can improve the effectiveness with which agricultural waste is utilized. This study described the potent AA9 family protein MsLPMO3, derived from Morchella sextelata. It exhibited strong binding to phosphoric acid swollen cellulose (PASC), and had the considerable binding ability to Cu2+ with a Kd value of 2.70 μM by isothermal titration calorimetry (ITC). MsLPMO3 could also act on PASC at the C1 carbon via MALDI-TOF-MS results. Moreover, MsLPMO3 could boost the hydrolysis efficiency of corncob and wheat bran in combination with glycoside hydrolases. MsLPMO3 also exhibited strong oxidizing ability for 2,6-dimethoxyphenol (2,6-DMP), achieving the best Vmax value of 443.36 U·g−1 for pH 7.4 with a H2O2 concentration of 300 µM. The structure of MsLPMO3 was obtained using AlphaFold2, and the molecular docking results elucidated the specific interactions and key residues involved in the recognition process between MsLPMO3 and cellulose. Altogether, this study expands the knowledge of AA9 family proteins in cellulose degradation, providing valuable insights into the mechanisms of synergistic degradation of lignocellulose with cellulases.

Heterologously Expressed Cellobiose Dehydrogenase Acts as Efficient Electron-Donor of Lytic Polysaccharide Monooxygenase for Cellulose Degradation in Trichoderma reesei.

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

Aspergillus fumigatus Lytic Polysaccharide Monooxygenase AfLPMO9D: Biochemical Properties and Photoactivation of a Multi-Domain AA9 Enzyme

Lytic polysaccharide monooxygenases (LPMOs) are critical players in enzymatic deconstruction of cellulose. A number of LPMOs have been identified at a genomics level; however, they still need to be characterized and validated for use in industrial processes aimed at cellulose deconstruction. In the present study, we biochemically characterized a new LPMO, a member of auxiliary activities family 9 (AA9) from the filamentous fungus Aspergillus fumigatus (AfLPMO9D). This LPMO demonstrated higher efficiency against amorphous cellulose as compared to more recalcitrant forms of cellulose such as bacterial cellulose and Avicel. AfLPMO9D has a capacity to oxidize the substrate at either the C1 or C4 positions, with pH-dependent regioselectivity. Photoactivation experiments demonstrated that light-stimulated chlorophyllin triggers AfLPMO9D activation without requirements of an external electron donor. AfLPMO9D is capable of boosting phosphoric acid-swollen cellulose depolymerization via GH7 endoglucanase and cellobiohydrolase. The results of the present study might help to elucidate the role of different LPMOs in cellulosic fiber deconstruction.

Heterologous expression and characterization of a MoAA16 polysaccharide monooxygenase from the rice blast fungus Magnaporthe oryzae

BackgroundCellulose is an organic carbon source that can be used as a sustainable alternative for energy, materials, and chemicals. However, the substantial challenge of converting it into soluble sugars remains a major obstacle in its use as a biofuel and chemical feedstock. A new class of enzymes knowns as copper-dependent polysaccharide monooxygenases (PMOs) or lytic polysaccharide monooxygenases (LPMOs) can break down polysaccharides such as cellulose, chitin, and starch through oxidation. This process enhances the efficiency of cellulose degradation by cellulase. ResultsThe genome of the fungus Magnaporthe oryzae, the causal agent of rice blast disease, contains the MGG_00245 gene, which encodes a putative PMO referred to as MoAA16. MoAA16 has been found to be highly expressed in planta during the early stages of fungal infection. The gene was optimized for heterologous expression in Pichia pastoris, and its oxidative cleavage activity on cellulose was characterized by analyzing soluble oligosaccharide products using highperformance anion exchange chromatography (HPAEC-PAD). The reaction catalyzed by MoAA16 requires 2 electrons from an electron donor, such as ascorbic acid, and aerobic conditions. It primarily produces Glc1 to Glc4 oligosaccharides, as well as oxidized cellobionic and cellotrionic acids. MoAA16 has been observed to enhance cellulase hydrolysis on phosphoric acid swollen cellulose (PASC) substrate, resulting in the production of more monosaccharide products. ConclusionsOur findings reveal the successful heterologous expression of MoAA16 in P. pastoris and its cellulose-active PMO properties. These results highlight the potential of MoAA16 as a promising candidate for applications in biofuel production and chemical synthesis.How to cite: Nguyen HM, Le LQ, Sella L, et al. Heterologous expression and characterization of a MoAA16 polysaccharide monooxygenase from the rice blast fungus Magnaporthe oryzae. Electron J Biotechnol 2023. https://doi.org/10.1016/j.ejbt.2023.06.002.

Biochemical Characterization of Novel GH6 Endoglucanase from Myxococcus sp. B6-1 and Its Effects on Agricultural Straws Saccharification.

Cellulase has been widely used in many industrial fields, such as feed and food industry, because it can hydrolyze cellulose to oligosaccharides with a lower degree of polymerization. Endo-β-1,4-glucanase is a critical speed-limiting cellulase in the saccharification process. In this study, endo-β-1,4-glucanase gene (CelA257) from Myxococcus sp. B6-1 was cloned and expressed in Escherichia coli. CelA257 contained carbohydrate-binding module (CBM) 4-9 and glycosyl hydrolase (GH) family 6 domain that shares 54.7% identity with endoglucanase from Streptomyces halstedii. The recombinant enzyme exhibited optimal activity at pH 6.5 and 50 °C and was stable over a broad pH (6-9.5) range and temperature < 50 °C. CelA257 exhibited broad substrate specificity to barley β-glucan, lichenin, CMC, chitosan, laminarin, avicel, and phosphoric acid swollen cellulose (PASC). CelA257 degraded both cellotetrose (G4) and cellppentaose (G5) to cellobiose (G2) and cellotriose (G3). Adding CelA257 increased the release of reducing sugars in crop straw powers, including wheat straw (0.18 mg/mL), rape straw (0.42 mg/mL), rice straw (0.16 mg/mL), peanut straw (0.16 mg/mL), and corn straw (0.61 mg/mL). This study provides a potential additive in biomass saccharification applications.

Improving the Enzymatic Activity and Stability of a Lytic Polysaccharide Monooxygenase.

Lytic Polysaccharide Monooxygenases (LPMOs) are copper-dependent enzymes that play a pivotal role in the enzymatic conversion of the most recalcitrant polysaccharides, such as cellulose and chitin. Hence, protein engineering is highly required to enhance their catalytic efficiencies. To this effect, we optimized the protein sequence encoding for an LPMO from Bacillus amyloliquefaciens (BaLPMO10A) using the sequence consensus method. Enzyme activity was determined using the chromogenic substrate 2,6-Dimethoxyphenol (2,6-DMP). Compared with the wild type (WT), the variants exhibit up to a 93.7% increase in activity against 2,6-DMP. We also showed that BaLPMO10A can hydrolyze p-nitrophenyl-β-D-cellobioside (PNPC), carboxymethylcellulose (CMC), and phosphoric acid-swollen cellulose (PASC). In addition to this, we investigated the degradation potential of BaLPMO10A against various substrates such as PASC, filter paper (FP), and Avicel, in synergy with the commercial cellulase, and it showed up to 2.7-, 2.0- and 1.9-fold increases in production with the substrates PASC, FP, and Avicel, respectively, compared to cellulase alone. Moreover, we examined the thermostability of BaLPMO10A. The mutants exhibited enhanced thermostability with an apparent melting temperature increase of up to 7.5 °C compared to the WT. The engineered BaLPMO10A with higher activity and thermal stability provides a better tool for cellulose depolymerization.

Reduced cellulose accessibility slows down enzyme-mediated hydrolysis of cellulose

Enzyme-mediated hydrolysis of cellulose always starts with an initial rapid phase, which gradually slows down, sometimes resulting in incomplete cellulose hydrolysis even after prolonged incubation. Although mechanisms such as end-product inhibition are known to play a role, the predominant mechanism appears to be reduced cellulose accessibility to the enzymes. When using Simon’s stain to quantify accessibility, the accessibility of mechanically disintegrated and phosphoric acid-swollen cellulose substrates decreased as hydrolysis proceeded. In contrast, the poor initial accessibility of Avicel remained low throughout hydrolysis. However, washing the residual cellulose increased cellulose accessibility, likely due to the removal of tightly bound but non-productive enzymes which blocked access to more active enzymes in solution. Atomic force microscopy (AFM) analysis of the initial and residual cellulose collected when the hydrolysis plateaued, showed an increase in the roughness of the cellulose surface, possibly resulting in the tighter binding of less active cellulases.

Enzymatic debranching is a key determinant of the xylan-degrading activity of family AA9 lytic polysaccharide monooxygenases

BackgroundPrevious studies have revealed that some Auxiliary Activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) oxidize and degrade certain types of xylans when incubated with mixtures of xylan and cellulose. Here, we demonstrate that the xylanolytic activities of two xylan-active LPMOs, TtLPMO9E and TtLPMO9G from Thermothielavioides terrestris, strongly depend on the presence of xylan substitutions.ResultsUsing mixtures of phosphoric acid-swollen cellulose (PASC) and wheat arabinoxylan (WAX), we show that removal of arabinosyl substitutions with a GH62 arabinofuranosidase resulted in better adsorption of xylan to cellulose, and enabled LPMO-catalyzed cleavage of this xylan. Furthermore, experiments with mixtures of PASC and arabinoglucuronoxylan from spruce showed that debranching of xylan with the GH62 arabinofuranosidase and a GH115 glucuronidase promoted LPMO activity. Analyses of mixtures with PASC and (non-arabinosylated) beechwood glucuronoxylan showed that GH115 action promoted LPMO activity also on this xylan. Remarkably, when WAX was incubated with Avicel instead of PASC in the presence of the GH62, both xylan and cellulose degradation by the LPMO9 were impaired, showing that the formation of cellulose–xylan complexes and their susceptibility to LPMO action also depend on the properties of the cellulose. These debranching effects not only relate to modulation of the cellulose–xylan interaction, which influences the conformation and rigidity of the xylan, but likely also affect the LPMO–xylan interaction, because debranching changes the architecture of the xylan surface.ConclusionsOur results shed new light on xylanolytic LPMO9 activity and on the functional interplay and possible synergies between the members of complex lignocellulolytic enzyme cocktails. These findings will be relevant for the development of future lignocellulolytic cocktails and biomaterials.

Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris.

4-hydroxybenzoic acid (4-HBA) is an industrially important aromatic compound, and there is an urgent need to establish a bioprocess to produce this compound in a sustainable and environmentally friendly manner from renewable feedstocks such as cellulosic biomass. Here, we developed a bioprocess to directly produce 4-HBA from cellulose using a recombinant Pichia pastoris strain that displays heterologous cellulolytic enzymes on its cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system. β-glucosidase (BGL) from Aspergillus aculeatus, endoglucanase (EG) from Trichoderma reesei, and cellobiohydrolase (CBH) from Talaromyces emersonii were co-displayed on the cell surface of P. pastoris using an appropriate GPI-anchoring domain for each enzyme. The cell-surface cellulase activity was further enhanced using P. pastoris SPI1 promoter- and secretion signal sequences. The resulting strains efficiently hydrolyzed phosphoric acid swollen cellulose (PASC) to glucose. Then, we expressed a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from Providencia rustigianii in the cellulase-displaying strain. This strain produced 975 mg/L of 4-HBA from PASC, which corresponding to 36.8% of the theoretical maximum yield, after 96 h of batch fermentation without the addition of commercial cellulase. This 4-HBA yield was over two times higher than that obtained from glucose (12.3% of the theoretical maximum yield). To our knowledge, this is the first report on the direct production of an aromatic compound from cellulose using cellulase-displaying yeast.

Genomics and cellulolytic, hemicellulolytic, and amylolytic potential of Iocasia fonsfrigidae strain SP3-1 for polysaccharide degradation.

BackgroundCellulolytic, hemicellulolytic, and amylolytic (CHA) enzyme-producing halophiles are understudied. The recently defined taxon Iocasia fonsfrigidae consists of one well-described anaerobic bacterial strain: NS-1T. Prior to characterization of strain NS-1T, an isolate designated Halocella sp. SP3-1 was isolated and its genome was published. Based on physiological and genetic comparisons, it was suggested that Halocella sp. SP3-1 may be another isolate of I. fronsfrigidae. Despite being geographic variants of the same species, data indicate that strain SP3-1 exhibits genetic, genomic, and physiological characteristics that distinguish it from strain NS-1T. In this study, we examine the halophilic and alkaliphilic nature of strain SP3-1 and the genetic substrates underlying phenotypic differences between strains SP3-1 and NS-1T with focus on sugar metabolism and CHA enzyme expression.MethodsStandard methods in anaerobic cell culture were used to grow strains SP3-1 as well as other comparator species. Morphological characterization was done via electron microscopy and Schaeffer-Fulton staining. Data for sequence comparisons (e.g., 16S rRNA) were retrieved via BLAST and EzBioCloud. Alignments and phylogenetic trees were generated via CLUTAL_X and neighbor joining functions in MEGA (version 11). Genomes were assembled/annotated via the Prokka annotation pipeline. Clusters of Orthologous Groups (COGs) were defined by eegNOG 4.5. DNA-DNA hybridization calculations were performed by the ANI Calculator web service.ResultsCells of strain SP3-1 are rods. SP3-1 cells grow at NaCl concentrations of 5-30% (w/v). Optimal growth occurs at 37 °C, pH 8.0, and 20% NaCl (w/v). Although phylogenetic analysis based on 16S rRNA gene indicates that strain SP3-1 belongs to the genus Iocasia with 99.58% average nucleotide sequence identity to Iocasia fonsfrigida NS-1T, strain SP3-1 is uniquely an extreme haloalkaliphile. Moreover, strain SP3-1 ferments D-glucose to acetate, butyrate, carbon dioxide, hydrogen, ethanol, and butanol and will grow on L-arabinose, D-fructose, D-galactose, D-glucose, D-mannose, D-raffinose, D-xylose, cellobiose, lactose, maltose, sucrose, starch, xylan and phosphoric acid swollen cellulose (PASC). D-rhamnose, alginate, and lignin do not serve as suitable culture substrates for strain SP3-1. Thus, the carbon utilization profile of strain SP3-1 differs from that of I. fronsfrigidae strain NS-1T. Differences between these two strains are also noted in their lipid composition. Genomic data reveal key differences between the genetic profiles of strain SP3-1 and NS-1T that likely account for differences in morphology, sugar metabolism, and CHA-enzyme potential. Important to this study, I. fonsfrigidae SP3-1 produces and extracellularly secretes CHA enzymes at different levels and composition than type strain NS-1T. The high salt tolerance and pH range of SP3-1 makes it an ideal candidate for salt and pH tolerant enzyme discovery.

A processive GH9 family endoglucanase of Bacillus licheniformis and the role of its carbohydrate-binding domain.

One of the critical steps in lignocellulosic deconstruction is the hydrolysis of crystalline cellulose by cellulases. Endoglucanases initially facilitate the breakdown of cellulose in lignocellulosic biomass and are further aided by other cellulases to produce fermentable sugars. Furthermore, if the endoglucanase is processive, it can adsorb to the smooth surface of crystalline cellulose and release soluble sugars during repeated cycles of catalysis before dissociating. Most glycoside hydrolase family 9 (GH9) endoglucanases have catalytic domains linked to a CBM (carbohydrate-binding module) (mostly CBM3) and present the second-largest cellulase family after GH5. GH9 endoglucanases are relatively less characterized. Bacillus licheniformis is a mesophilic soil bacterium containing many glycoside hydrolase (GH) enzymes. We identified an endoglucanase gene, gh9A, encoding the GH9 family enzyme H1AD14 in B. licheniformis and cloned and overexpressed H1AD14 in Escherichia coli. The purified H1AD14 exhibited very high enzymatic activity on endoglucanase substrates, such as β-glucan, lichenan, Avicel, CMC-Na (sodium carboxymethyl cellulose) and PASC (phosphoric acid swollen cellulose), across a wide pH range. The enzyme is tolerant to 2 M sodium chloride and retains 74% specific activity on CMC after 10 days, the highest amongst the reported GH9 endoglucanases. The full-length H1AD14 is a processive endoglucanase and efficiently saccharified sugarcane bagasse. The deletion of the CBM reduces the catalytic activity and processivity. The results add to the sparse knowledge of GH9 endoglucanases and offer the possibility of characterizing and engineering additional enzymes from B. licheniformis toward developing a cellulase cocktail for improved biomass deconstruction. KEY POINTS: • H1AD14 is a highly active and processive GH9 endoglucanase from B. licheniformis. • H1AD14 is thermostable and has a very long half-life. • H1AD14 showed higher saccharification efficiency than commercial endoglucanase.

Comparison of the Biochemical Properties and Roles in the Xyloglucan-Rich Biomass Degradation of a GH74 Xyloglucanase and Its CBM-Deleted Variant from Thielavia terrestris.

Xyloglucan is closely associated with cellulose and still retained with some modification in pretreated lignocellulose; however, its influence on lignocellulose biodegradation is less understood. TtGH74 from Thielavia terrestris displayed much higher catalytic activity than previously characterized fungal GH74 xyloglucanases. The carbohydrate-binding module 1 (CBM1) deleted variant (TtGH74ΔCBM) had the same optimum temperature and pH but an elevated thermostability. TtGH74 displayed a high binding affinity on xyloglucan and cellulose, while TtGH74ΔCBM completely lost the adsorption capability on cellulose. Their hydrolysis action alone or in combination with other glycoside hydrolases on the free xyloglucan, xyloglucan-coated phosphoric acid-swollen cellulose or pretreated corn bran and apple pomace was compared. CBM1 might not be essential for the hydrolysis of free xyloglucan but still effective for the associated xyloglucan to an extent. TtGH74 alone or synergistically acting with the CBH1/EG1 mixture was more effective in the hydrolysis of xyloglucan in corn bran, while TtGH74ΔCBM showed relatively higher catalytic activity on apple pomace, indicating that the role and significance of CBM1 are substrate-specific. The degrees of synergy for TtGH74 or TtGH74ΔCBM with the CBH1/EG1 mixture reached 1.22–2.02. The addition of GH10 xylanase in TtGH74 or the TtGH74ΔCBM/CBH1/EG1 mixture further improved the overall hydrolysis efficiency, and the degrees of synergy were up to 1.50–2.16.

Structural and functional study of a novel lytic polysaccharide monooxygenase cPMO2 from compost sample in the oxidative degradation of cellulose

Lytic polysaccharide monooxygenases (LPMOs) are the major groups of oxidizing the recalcitrant polysaccharides. Here, a novel AA9 family protein cPMO2 originated from composting was characterized, and it had a better binding capacity to phosphoric acid swollen cellulose (PASC) and Avicel, as well the isothermal titration calorimetry (ITC) analysis showed that it owned a considerable capacity of binding Cu2+ with the Kd value of 1.58 ± 0.78 μM. Interestingly, the kinetics of cPMO2 by using 2, 6-dimethoxyphenol (2, 6-DMP) as substrate indicated that cPMO2 was a novel member of LPMOs family, with the Vmax of 21.30 ± 0.83 U·g−1 at pH 7.5. In addition, cPMO2 owned a broad substrate specificity, which could act on PASC and xylan at the C1 site based on the MALDI-TOF-MS analysis results. Simultaneously, cPMO2 could also act on Avicel directly resulting the decrease of crystallinity by 4% and further to synergistic degradation of corncob and rice straw with glycoside hydrolases. The structure of the functional region of cPMO2 was obtained by using single-crystal X-ray diffraction, and the enzymatic properties and molecular docking analysis results showed that His1 and Tyr175 were the critical active sites of the cPMO2. Altogether, this study will be a supplement for the AA9 LPMOs in cellulose degradation.

A sensitive, accurate, and high-throughput gluco-oligosaccharide oxidase-based HRP colorimetric method for assaying lytic polysaccharide monooxygenase activity

BackgroundThe AA9 (auxiliary activities) family of lytic polysaccharide monooxygenases (AA9 LPMOs) is a ubiquitous and diverse group of enzymes in the fungal kingdom. They catalyse the oxidative cleavage of glycosidic bonds in lignocellulose and exhibit great potential for biorefinery applications. Robust, high-throughput and direct methods for assaying AA9 LPMO activity, which are prerequisites for screening LPMOs with excellent properties, are still lacking. Here, we present a gluco-oligosaccharide oxidase (GOOX)-based horseradish peroxidase (HRP) colorimetric method for assaying AA9 LPMO activity.ResultsWe cloned and expressed a GOOX gene from Sarocladium strictum in Trichoderma reesei, purified the recombinant SsGOOX, validated its properties, and developed an SsGOOX-based HRP colorimetric method for assaying cellobiose concentrations. Then, we expressed two AA9 LPMOs from Thielavia terrestris, TtAA9F and TtAA9G, in T. reesei, purified the recombinant proteins, and analysed their product profiles and regioselectivity towards phosphoric acid swollen cellulose (PASC). TtAA9F was characterized as a C1-type (class 1) LPMO, while TtAA9G was characterized as a C4-type (class 2) LPMO. Finally, the SsGOOX-based HRP colorimetric method was used to quantify the total concentration of reducing lytic products from the LPMO reaction, and the activities of both the C1- and C4-type LPMOs were analysed. These LPMOs could be effectively analysed with limits of detection (LoDs) less than 30 nmol/L, and standard curves between the A515 and LPMO concentrations with determination coefficients greater than 0.994 were obtained.ConclusionsA novel, sensitive and accurate assay method that directly targets the main activity of both C1- and C4-type AA9 LPMOs was established. This method is easy to use and could be performed on a microtiter plate for high-throughput screening of AA9 LPMOs with desirable properties.

An effective strategy for improving the specific activity and saccharification efficiency of cellulase by pre-incubation with phenolic acids

This short communication analyzed the effects of lignin-derived phenolic acid compounds on cellulase. Vanillic acid, syringic acid, ferulic acid, and isovanillic acid improved cellulase specific activity and saccharification efficiency. In the enzymatic hydrolysis process, the promotion effect of phenolic acid was concentration-dependent. The effect of low concentration of phenolic acids (less than 5 mM) was negligible. After pre-incubating 1 g cellulase with 5 mmol phenolic acid, FPase-specific activity, CMCase-specific activity, and pNPGase-specific activity increased by 57.06%, 136.79%, and 110.61%, respectively. After digestion with pre-incubated cellulase, the saccharification efficiency of phosphoric acid-swollen cellulose increased by 45.13%. Pre-incubation with phenolic acid improved the saccharification efficiency of cellulase. It might be helpful to enhance the comprehensive utilization capacity of lignin-derived compounds.

On the interaction of lytic polysaccharide monooxygenases (LPMOs) with phosphoric acid-swollen cellulose (PASC)

• Mt LPMO9H is immobilized and direct electron transfer is achieved. • The interaction of Mt LPMO9H with PASC is studied with FTacV. • The kinetic model for the interaction of Mt LPMO9H with PASC with FTacV is developed. • The kinetic constant for the interaction of Mt LPMO9H with PASC is extracted. In this work, a lytic polysaccharide monooxygenase (LPMO) belonging to the AA9 family from the filamentous fungus Thermothelomyces thermophilus , Mt LPMO9H, is immobilized on cobalt functionalized multi-walled carbon nanotubes modified glassy carbon electrode. The step of the binding of phosphoric acid swollen cellulose on this LPMO is studied using large amplitude fast Fourier transform alternating current voltammetry (FTacV) at different temperatures, extracting the corresponding kinetic constants, based on a model developed for the interaction of immobilized enzyme with free, in the solution, species.

Metal Affinity Immobilization of the Processive Endoglucanase EG5C-1 from Bacillus subtilis on a Recyclable pH-Responsive Polymer

Establishing a suitable immobilization strategy to improve the accessibility of immobilized cellulase for insoluble cellulose and subsequently recover the enzyme from the remaining substrate is crucial to promote the industrialization of biofuels. Here, using iminodiacetic acid (IDA) and Ni2+ ions, a novel metal-chelated copolymer of methacrylic acid and methyl methacrylate was prepared for the immobilization of His-tagged processive endoglucanase EG5C-1 via affinity interaction between polyhistidine tag and Ni2+. The optimum loading capacity of the functionalized polymer (Eud-IDA-Ni2+) for EG5C-1 was about 280 mg/g, where more than 80% of the activity was recovered. Immobilized EG5C-1 exhibited improved thermal and pH stability and better reusability than the free one, and after five cycles of usage, the hydrolysis productivity remained above 70% of the initial value. The Eud-IDA-Ni2+/EG5C-1 biocomposite displayed reversibly soluble–insoluble characteristics with pH change, which was in the soluble state during the enzyme reaction process but could be recovered in an insoluble form by lowering the pH after the reaction. Thus, the yield obtained from the hydrolysis of an insoluble phosphoric acid-swollen cellulose substrate was similar for the free and immobilized form of EG5C-1. Our combined results suggested that a metal-ion-chelated, pH-responsive polymer had the potential for His-tagged cellulase immobilization to improve both the operational efficiency and economic benefit of the cellulosic biorefinery industry.

Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species

BackgroundThe discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10).ResultsHere, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation.ConclusionsTogether, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.

Characterization of an AA9 LPMO from Thielavia australiensis, TausLPMO9B, under industrially relevant lignocellulose saccharification conditions

BackgroundThe discovery of lytic polysaccharide monooxygenases (LPMO) has changed our perspective on enzymatic degradation of plant biomass. Through an oxidative mechanism, these enzymes are able to cleave and depolymerize various polysaccharides, acting not only on crystalline substrates such as chitin and cellulose, but also on other polysaccharides, such as xyloglucan, glucomannan and starch. Despite their widespread use, uncertainties related to substrate specificity and stereospecificity, the nature of the co-substrate, in-process stability, and the nature of the optimal reductant challenge their exploitation in biomass processing applications.ResultsIn this work, we studied the properties of a novel fungal LPMO from the thermophilic fungus Thielavia australiensis, TausLPMO9B. Heterologous expression of TausLPMO9B in Aspergillus niger yielded a glycosylated protein with a methylated N-terminal histidine showing LPMO activity. High sequence identity of the AA9 domain to that of MtLPMO9B (MYCTH_80312) from Myceliophthora thermophila (84%) indicated strictly C1-oxidizing activity on cellulose, which was confirmed experimentally by the analysis of products released from cellulose using HPAEC. The enzyme was stable and active at a pH ranging from 4 to 6, thus matching the conditions commonly used in industrial biomass processing, where a low pH (between 4 and 5) is used due to the pH-optima of commercial cellulases and a desire to limit microbial contamination.ConclusionWhile the oxidative cleavage of phosphoric acid swollen cellulose (PASC) by TausLPMO9B was boosted by the addition of H2O2 as a co-substrate, this effect was not observed during the saccharification of acid pretreated corn stover. This illustrates key differences between the lab-scale tests with artificial, lignin-free substrates and industrial settings with lignocellulosic biomass as substrate.