Commercial Cellulase Cocktail Research Articles

Production of Medium Chain Length Polyhydroxyalkanoate from Waste Cannabis sativa Biomass

This works aims to evaluate a process to convert residual medical Cannabis sativa stalks into medium chain length polyhydroxyalkanoate (mcl-PHA), using chemical pretreatment and enzymatic hydrolysis to obtain sugars as a carbon source for a fermentation process with Pseudomonas aeruginosa. Chemical composition analysis revealed a content of structural polysaccharides of 57.64%. Thermochemical pretreatments with 2% sulfuric acid or 2% sodium hydroxide were capable of partially removing hemicellulose and lignin, as well as increasing cellulose crystallinity. Pretreated biomass was subjected to hydrolysis using commercial cellulase cocktails Celluclast® 1.5 L and Cellic® CTec3. Acid pretreatment showed an adverse effect on hydrolysis yield of holocellulose, decreasing to 39.5%; compared to 44.4% of untreated biomass. Alkaline pretreatment increased degree of hydrolysis up to 73.3%. Shake flask fermentation of hydrolysate with Pseudomonas aeruginosa produced cell growth of 1.65 g/L and a mcl-PHA titer of 0.41 g/L. Extracted polymer presented characteristic FTIR bands for PHAs, glass transition temperature of − 50.8 °C, melting temperature of 48.9 °C, possibly allowing its use in the biomedical industry. The developed process represents a potential way to valorize Cannabis waste stalks, using alkaline pretreatment, enzymatic hydrolysis with Celluclast® 1.5 L and fermentation with Pseudomonas aeruginosa. Further work should focus on improving yield of the obtained polyhydroxyalkanoate in fermentation, in order to improve industrial feasibility of the entire process and, in turn, increasing revenue of the medical Cannabis industry.Graphical

A novel AA14 LPMO from Talaromyces rugulosus with bifunctional cellulolytic/hemicellulolytic activity boosted cellulose hydrolysis

BackgroundThe recently discovered PcAA14A and B from white-rot basidiomycete Pycnoporus coccineus enriched our understanding of the oxidative degradation of xylan in fungi, however, the unusual mode of action of AA14 LPMOs has sparked controversy. The substrate specificity and functionality of AA14 LPMOs still remain enigmatic and need further investigation.ResultsIn this study, a novel AA14 LPMO was characterized from the ascomycete Talaromyces rugulosus. TrAA14A has a broad substrate specificity with strong oxidative activity on pure amorphous cellulose and xyloglucan. It could simultaneously oxidize cellulose, xylan and xyloglucan in natural hemi/cellulosic substrate such as fibrillated eucalyptus pulp, and released native and oxidized cello-oligosaccharides, xylo-oligosaccharides and xyloglucan oligosaccharides from this substrate, but its cellulolytic/hemicellulolytic activity became weaker as the contents of xylan increase in the alkaline-extracted hemi/cellulosic substrates. The dual cellulolytic/hemicellulolytic activity enables TrAA14A to possess a profound boosting effect on cellulose hydrolysis by cellulolytic enzymes. Structure modelling of TrAA14A revealed that it exhibits a relatively flat active-site surface similar to the active-site surfaces in AA9 LPMOs but quite distinct from PcAA14B, despite TrAA14A is strongly clustered together with AA14 LPMOs. Remarkable difference in electrostatic potentials of L2 and L3 surfaces was also observed among TrAA14A, PcAA14B and NcLPMO9F. We speculated that the unique feature in substrate-binding surface might contribute to the cellulolytic/hemicellulolytic activity of TrAA14A.ConclusionsThe extensive cellulolytic/hemicellulolytic activity on natural hemi/cellulosic substrate indicated that TrAA14A from ascomycete is distinctively different from previously characterized xylan-active AA9 or AA14 LPMOs. It may play as a bifunctional enzyme to decompose some specific network structures formed between cellulose and hemicellulose in the plant cell walls. Our findings shed new insights into the novel substrate specificities and biological functionalities of AA14 LPMOs, and will contribute to developing novel bifunctional LPMOs as the booster in commercial cellulase cocktails to efficiently break down the hemicellulose-cellulose matrix in lignocellulose.

Stable cellulolytic activity of Clostridium thermocellum against cellulosic biomass pretreated with ionic liquid 1-ethyl 3-methylimidazolium acetate

Using cellulosic biomass has considerable potential for the production of valuable chemicals while contributing to carbon neutrality. This study investigates the effect of biomass pretreatment with ionic liquid 1-ethyl 3-methylimidazolium acetate ([EMIM][Ac]) on the efficiency of enzymatic saccharification using the cellulolytic system of the thermophilic bacterium Clostridium thermocellum. Although [EMIM][Ac] pretreatment improves the efficiency of enzymatic saccharification, it also inhibits the cellulolytic enzyme. Following incubation with 30 % [EMIM][Ac], while the cellulolytic activity of the commercial cellulase cocktail (Celluclast) decreased to 15 % and that of C. thermocellum exoproteome remained stable, suggesting a relative tolerance of C. thermocellum cellulases to [EMIM][Ac]. The reducing sugars released from the pretreated cellulosic biomass corn hull by Celluclast were reduced by 24 % with supplementation of 5 % [EMIM][Ac] but remained unchanged for C. thermocellum exoproteome. These results highlight the potential of [EMIM][Ac] pretreatment of cellulosic biomass as a promising approach for enzymatic saccharification using C. thermocellum.

Optimisation of β-Glucosidase Production in a Crude Aspergillus japonicus VIT-SB1 Cellulase Cocktail Using One Variable at a Time and Statistical Methods and its Application in Cellulose Hydrolysis.

Pulp and paper mill sludge (PPMS) is currently disposed of into landfills which are reaching their maximum capacity. Valorisation of PPMS by enzymatic hydrolysis using cellulases is an alternative strategy. Existing commercial cellulases are expensive and contain low titres of β-glucosidases. In this study, β-glucosidase production was optimised by Aspergillus japonicus VIT-SB1 to obtain higher β-glucosidase titres using the One Variable at a Time (OVAT), Plackett Burman (PBD), and Box Behnken design (BBD)of experiments and the efficiency of the optimised cellulase cocktail to hydrolyse cellulose was tested. β-Glucosidase production was enhanced from 0.4 to 10.13 U/mL, representing a 25.3-fold increase in production levels after optimisation. The optimal BBD production conditions were 6 days of fermentation at 20 °C, 125 rpm, 1.75% soy peptone, and 1.25% wheat bran in (pH 6.0) buffer. The optimal pH for β-glucosidase activity in the crude cellulase cocktail was (pH 5.0) at 50 °C. Optimal cellulose hydrolysis using the crude cellulase cocktail occurred at longer incubation times, and higher substrate loads and enzyme doses. Cellulose hydrolysis with the A. japonicus VIT-SB1 cellulase cocktail and commercial cellulase cocktails resulted in glucose yields of 15.12 and 12.33 µmol/mL glucose, respectively. Supplementation of the commercial cellulase cocktail with 0.25 U/mg of β-glucosidase resulted in a 19.8% increase in glucose yield.

Synergetic effect of lytic polysaccharide monooxygenase from Thermobifida fusca on saccharification of agrowastes

Saccharification is one of the most noteworthy processes in biomass-based biorefineries. In particular, the lytic polysaccharide monooxygenase has recently emerged as an oxidative cleavage-recalcitrant polysaccharide; however, there is insufficient information regarding its application to actual biomass. Accordingly, this study focused optimizing the recombinant expression level of a bacterial lytic polysaccharide monooxygenase from Thermobifida fusca (TfLPMO), which was characterized as a cellulolytic enzyme. Finally, the synergistic effect of the lytic polysaccharide monooxygenase and a commercial cellulase cocktail on the saccharification of agrowaste was investigated. TfLPMO functioned on various cellulosic and hemicellulosic substrates, and the combination of TfLPMO with cellulase exhibited a synergistic effect on the saccharification of agrowastes, resulting in a 19.2% and 14.1% increase in reducing sugars from rice straw and corncob, respectively. The results discussed herein can lead to an in-depth understanding of enzymatic saccharification and suggest viable options for valorizing agrowastes as renewable feedstocks in biorefineries.

BsLPMO10A from Bacillus subtilis boosts the depolymerization of diverse polysaccharides linked via β-1,4-glycosidic bonds

Lytic polysaccharide monooxygenase (LPMO) is known as an oxidatively cleaving enzyme in recalcitrant polysaccharide deconstruction. Herein, we report a novel AA10 LPMO derived from Bacillus subtilis (BsLPMO10A). A substrate specificity study revealed that the enzyme exhibited an extensive active-substrate spectrum, particularly for polysaccharides linked via β-1,4 glycosidic bonds, such as β-(Man1 → 4Man), β-(Glc1 → 4Glc) and β-(Xyl1 → 4Xyl). HPAEC-PAD and MALDI-TOF-MS analyses indicated that BsLPMO10A dominantly liberated native oligosaccharides with a degree of polymerization (DP) of 3–6 and C1-oxidized oligosaccharides ranging from DP3ox to DP6ox from mixed linkage glucans and beechwood xylan. Due to its synergistic action with a variety of glycoside hydrolases, including glucanase IDSGLUC5-38, xylanase TfXYN11-1, cellulase IDSGLUC5-11 and chitinase BtCHI18-1, BsLPMO10A dramatically accelerated glucan, xylan, cellulose and chitin saccharification. After co-reaction for 72 h, the reducing sugars in Icelandic moss lichenan, beechwood xylan, phosphoric acid swollen cellulose and chitin yielded 3176 ± 97, 7436 ± 165, 649 ± 44, and 2604 ± 130 μmol/L, which were 1.47-, 1.56-, 1.44- and 1.25-fold higher than those in the GHs alone groups, respectively (P < 0.001). In addition, the synergy of BsLPMO10A and GHs was further validated by the degradation of natural feedstuffs, the co-operation of BsLPMO10A and GHs released 3266 ± 182 and 1725 ± 107 μmol/L of reducing sugars from Oryza sativa L. and Arachis hypogaea L. straws, respectively, which were significantly higher than those produced by GHs alone (P < 0.001). Furthermore, BsLPMO10A also accelerated the liberation of reducing sugars from Celluclast® 1.5 L, a commercial cellulase cocktail, on filter paper, A. hypogaea L. and O. sativa L. straws by 49.58 % (P < 0.05), 72.19 % (P < 0.001) and 54.36 % (P < 0.05), respectively. This work has characterized BsLPMO10A with a broad active-substrate scope, providing a promising candidate for lignocellulosic biomass biorefinery.

H2 O2 feeding enables LPMO-assisted cellulose saccharification during simultaneous fermentative production of lactic acid.

Simultaneous saccharification and fermentation (SSF) is a well-known strategy for valorization of lignocellulosic biomass. Because the fermentation process typically is anaerobic, oxidative enzymes found in modern commercial cellulase cocktails, such as lytic polysaccharide monooxygenases (LPMOs), may be inhibited, limiting the overall efficiency of the enzymatic saccharification. Recent discoveries, however, have shown that LPMOs are active under anoxic conditions if they are provided with H2 O2 at low concentrations. In this study, we build on this concept and investigate the potential of using externally added H2 O2 to sustain oxidative cellulose depolymerization by LPMOs during an SSF process for lactic acid production. The results of bioreactor experiments with 100 g/L cellulose clearly show that continuous addition of small amounts of H2 O2 (at a rate of 80 µM/h) during SSF enables LPMO activity and improves lactic acid production. While further process optimization is needed, the present proof-of-concept results show that modern LPMO-containing cellulase cocktails such as Cellic CTec2 can be used in SSF setups, without sacrificing the LPMO activity in these cocktails.

Stimulating biogas production from steam-exploded birch wood using Fenton reaction and fungal pretreatment

Delignification of steam-exploded birch wood (SEBW) was stimulated using a pretreatment method including Fenton reaction (FR) and fungi. SEBW was employed as a substrate to optimize the Fe(III) and Fe(II) dosage in FR. Maximum iron-binding to SEBW was obtained at pH 3.5. FR pretreatment increased biological methane yields from 257 mL/g vS in control to 383 and 352 mL/ g vS in samples with 0.5 mM Fe(II) and 1.0 mM Fe(III), respectively. Further enzymatic pretreatment using a commercial cellulase cocktail clearly improved methane production rate but only increased the final methane yields by 2–9 %. Finally, pretreatments with the fungi Pleurotus ostreatus (PO) and Lentinula edodes (LE), alone or in combination with FR, were carried out. SEBW pretreated with only LE and samples pretreated with PO and1 mM Fe(III) + H2O2 increased the methane production yield to 420 and 419 mL/g vS respectively. These pretreatments delignified SEBW up to 25 %.

Abatement of microfibre pollution and detoxification of textile dye - Indigo by engineered plant enzymes.

Microfibres (diameter <5 mm) and textile dyes released from textile industries are ubiquitous, cause environmental pollution, and harm aquatic flora, fauna, animals and human life. Therefore, enzymatic abatement of microfibre pollution and textile dye detoxification is essential. Microbial enzymes for such application present major challenges of scale and affordability to clean up large scale pollution. Therefore, enzymes required for the biodegradation of microfibres and indigo dye were expressed in transplastomic tobacco plants through chloroplast genetic engineering. Integration of laccase and lignin peroxidase genes into the tobacco chloroplast genomes and homoplasmy was confirmed by Southern blots. Decolorization (up to 86%) of samples containing indigo dye (100 mg/L) was obtained using cp-laccase (0.5% plant enzyme powder). Significant (8-fold) reduction in commercial microbial cellulase cocktail was achieved in pretreated cotton fibre hydrolysis by supplementing cost effective cellulases (endoglucanases, ß-glucosidases) and accessory enzymes (swollenin, xylanase, lipase) and ligninases (laccase lignin peroxidase) expressed in chloroplasts. Microfibre hydrolysis using cocktail of Cp-cellulases and Cp-accessory enzymes along with minimal dose (0.25% and 0.5%) of commercial cellulase blend (Ctec2) showed 88%-89% of sugar release from pretreated cotton and microfibres. Cp-ligninases, Cp-cellulases and Cp-accessory enzymes were stable in freeze dried leaves up to 15 and 36 months respectively at room temperature, when protected from light. Use of plant powder for decolorization or hydrolysis eliminated the need for preservatives, purification or concentration or cold chain. Evidently, abatement of microfibre pollution and textile dye detoxification using Cp-enzymes is a novel and cost-effective approach to prevent their environmental pollution.

Effect of enzymes adsorption on enzymatic hydrolysis of coffee silverskin: Kinetic characterization and validation

The use of waste lignocellulose feedstock for sugar-based biorefineries is attracting the interest of the scientific and industrial communities. The aim is to develop efficient sustainable processes to produce fuels and chemicals via the biotechnological route. However, the rational design of processes aimed at fermentable sugar recovery from lignocellulosic wastes asks for reliable quantitative data of a wide spectrum of biomass. Waste biomasses from food industries are among the most studied potential carbon sources. In the present study, the kinetics of enzymatic hydrolysis of coffee silverskin has been characterized. Coffee silverskin is one of the most abundant fractions of the coffee industry waste and it has been already used for saccharification purposes. The experimental investigation provided kinetic parameters of a semi-mechanistic model of glucose production rate from coffee silverskin catalyzed by a commercial cellulase cocktail. In addition, the adsorption of the enzymes on the solid biomass substrate has been characterized according to a Langmuir type model. The effect of enzymes adsorption on cellulose conversion and the process dynamics have been highlighted by validation tests. The optimization of biocatalyst use has been provided via a two-step hydrolysis procedure. The developed procedure can be successfully applied in the future to several biomasses to describe a wide range of possible substrates.

Expression of a β-glucosidase from Trichoderma reesei in Escherichia coli using a synthetic optimized gene and stability improvements by immobilization using magnetite nano-support

The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low β-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized β-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The β-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the β-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, β-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.

Digestibility of Bacillus firmus K-1 pretreated rice straw by different commercial cellulase cocktails

Removal of xylan in plant biomass is believed to increase cellulose hydrolysis by uncovering cellulose surfaces for cellulase adsorption and, in turn, catalysis reaction. Herein, we describe an eco-friendly method by culturing a xylanolytic Bacillus firmus K-1 on rice straw to remove xylan. The bacterium was grown on 2.5% (w/v) rice straw with different biomass particle sizes for two days at 37 °C. We found that the particle sizes ranged from <1 to 5 mm gave a similar xylan removal degree (about 21%). Besides, the porosity and disintegration of the rice straw fibers were observed at the molecular level. The digestibility of pretreated rice straw was tested with different commercial cellulase cocktails. We found that the pretreated rice straw was more susceptible to enzymatic hydrolysis, giving 30–70% glucan conversion than the untreated one. The degree of cellulose hydrolysis depended strongly on the kinds of enzyme and their formulations. Highlight Culturing B. firmus K-1 on rice straw yielded about 21% removal of xylan. Particle sizes (of 1–5 mm) had negligible effects on xylan removal efficiency. The degree of glucan conversion in pretreated biomass relied on enzyme formulation.

Two C1-oxidizing lytic polysaccharide monooxygenases from Ceriporiopsis subvermispora enhance the saccharification of wheat straw by a commercial cellulase cocktail

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

A thermostable bacterial lytic polysaccharide monooxygenase with high operational stability in a wide temperature range

BackgroundLytic polysaccharide monooxygenases (LPMOs) are oxidative, copper-dependent enzymes that function as powerful tools in the turnover of various biomasses, including lignocellulosic plant biomass. While LPMOs are considered to be of great importance for biorefineries, little is known about industrial relevant properties such as the ability to operate at high temperatures. Here, we describe a thermostable, cellulose-active LPMO from a high-temperature compost metagenome (called mgLPMO10).ResultsMgLPMO10 was found to have the highest apparent melting temperature (83 °C) reported for an LPMO to date, and is catalytically active up to temperatures of at least 80 °C. Generally, mgLPMO10 showed good activity and operational stability over a wide temperature range. The LPMO boosted cellulose saccharification by recombinantly produced GH48 and GH6 cellobiohydrolases derived from the same metagenome, albeit to a minor extent. Cellulose saccharification studies with a commercial cellulase cocktail (Celluclast®) showed that the performance of this thermostable bacterial LPMO is comparable with that of a frequently utilized fungal LPMO from Thermoascus aurantiacus (TaLPMO9A).ConclusionsThe high activity and operational stability of mgLPMO10 are of both fundamental and applied interest. The ability of mgLPMO10 to perform oxidative cleavage of cellulose at 80 °C and the clear synergy with Celluclast® make this enzyme an interesting candidate in the development of thermostable enzyme cocktails for use in lignocellulosic biorefineries.

A comparison of different oxidative pretreatments on polysaccharide hydrolyzability and cell wall structure for interpreting the greatly improved enzymatic digestibility of sugarcane bagasse by delignification

In order to confirm the contribution of delignification to the increase in lignocellulosic cellulose digestibility, several laboratory oxidative pretreatments under mild conditions, including alkaline-hydrogen peroxide (AP), two-step alkaline/peracetic acid (APAA) and sodium chlorite (SC) pretreatments were employed to achieve selective delignification of sugarcane bagasse and retained most of the hemicelluloses (xylan) in the pretreated solids. Four commercial cellulase cocktails were used to test the enzymatic hydrolyzability of pretreated substrates. Results revealed that delignification indeed could greatly improve the final (120 h) cellulose hydrolysis with relatively high final (120 h) glucan conversion (> 90%) by different cellulase cocktails even if the substrates still had a high hemicelluloses content. However, the xylan conversion seemed to be more greatly dependent on the pretreatments and cellulase cocktails used. AP and APAA pretreatments resulted in the disappearance of middle lamella and liberation of cellulose fibers with significant etching, deformation and fracture of cell wall structure. SC pretreatment greatly modified the sugar bagasse surface morphology to make the surface much coarser. The cell wall also underwent serious fracture and deformation with some middle lamella disappearing. However, no significant alteration on the structure of pure cellulose was observed by SC oxidative pretreatment of filter paper. Oxidative pretreatment might also modify lignin structure and surface properties thus greatly reducing the non-specific adsorption of enzymes. The obtained results strongly support the conclusion that delignification under mild pretreatment condition can be very helpful to improve the enzymatic hydrolysis of lignocellulosic cellulose by commercial cellulase cocktails even if the substrates has a high hemicelluloses content.

Demonstration‐scale enzymatic saccharification of sulfite‐pulped spruce with addition of hydrogen peroxide for LPMO activation

AbstractThe saccharification of lignocellulosic materials like Norway spruce is challenging due to the recalcitrant nature of the biomass, and it requires optimized and efficient pretreatment and enzymatic hydrolysis processes to make it industrially feasible. In this study, we report successful enzymatic saccharification of sulfite‐pulped spruce (Borregaard's BALI™ process) at demonstration scale, achieved through the controlled delivery of hydrogen peroxide (H2O2) for the activation of lytic polysaccharide monooxygenases (LPMOs) present in the cellulolytic enzyme preparation. We achieved 85% saccharification yield in 4 days using industrially relevant conditions – that is, an enzyme dose of 4% (w/w dry matter of substrate) of the commercial cellulase cocktail Cellic CTec3 and a substrate loading of 12% (w/w). Addition of H2O2 and the resulting controlled and high LPMO activity had a positive effect on the rate of saccharification and the final sugar titer. Clearly, the high LPMO activity was dependent on feeding the reactors with the LPMO co‐substrate H2O2, as in situ generation of H2O2 from molecular oxygen was limited. These demonstration‐scale experiments provide a solid basis for the use of H2O2 to improve enzymatic saccharification of lignocellulosic biomass at large industrial scale.© 2020 The Authors. Biofuels, Bioproducts, and Biorefining published by Society of Chemical Industry and John Wiley &amp; Sons, Ltd.

Identification and Characterization of a Novel Hyperthermostable Bifunctional Cellobiohydrolase- Xylanase Enzyme for Synergistic Effect With Commercial Cellulase on Pretreated Wheat Straw Degradation.

The novel cellobiohydrolase gene ctcel7 was identified from Chaetomium thermophilum, and its recombinant protein CtCel7, a member of glycoside hydrolase family 7, was heterologously expressed in Pichia pastoris and biochemically characterized. Compared with commercial hydrolases, purified CtCel7 exhibited superior bifunctional cellobiohydrolase and xylanase activities against microcrystalline cellulose and xylan, respectively, under optimal conditions of 60°C and pH 4.0. Moreover, CtCel7 displayed remarkable thermostability with over 90% residual activity after heat (60°C) treatment for 180 min. CtCel7 was insensitive to most detected cations and reagents and preferentially cleaved the β-1,4-glycosidic bond to generate oligosaccharides through the continuous saccharification of lignocellulosic substrates, which are crucial for various practical applications. Notably, the hydrolysis effect of a commercial cellulase cocktail on pretreated wheat straw was substantively improved by its combination with CtCel7. Taken together, these excellent properties distinguish CtCel7 as a robust candidate for the biotechnological production of biofuels and biobased chemicals.

β-Mannanase Production Using Coffee Industry Waste for Application in Soluble Coffee Processing.

Soluble coffee offers the combined benefits of high added value and practicality for its consumers. The hydrolysis of coffee polysaccharides by the biochemical route, using enzymes, is an eco-friendly and sustainable way to improve the quality of this product, while contributing to the implementation of industrial processes that have lower energy requirements and can reduce environmental impacts. This work describes the production of hydrolytic enzymes by solid-state fermentation (SSF), cultivating filamentous fungi on waste from the coffee industry, followed by their application in the hydrolysis of waste coffee polysaccharides from soluble coffee processing. Different substrate compositions were studied, an ideal microorganism was selected, and the fermentation conditions were optimized. Cultivations for enzymes production were carried out in flasks and in a packed-bed bioreactor. Higher enzyme yield was achieved in the bioreactor, due to better aeration of the substrate. The best β-mannanase production results were found for a substrate composed of a mixture of coffee waste and wheat bran (1:1 w/w), using Aspergillus niger F12. The enzymatic extract proved to be very stable for 24 h, at 50 °C, and was able to hydrolyze a considerable amount of the carbohydrates in the coffee. The addition of a commercial cellulase cocktail to the crude extract increased the hydrolysis yield by 56%. The production of β-mannanase by SSF and its application in the hydrolysis of coffee polysaccharides showed promise for improving soluble coffee processing, offering an attractive way to assist in closing the loops in the coffee industry and creating a circular economy.

OrganoCat Fractionation of Empty Fruit Bunches from Palm Trees into Lignin, Sugars, and Cellulose-Enriched Pulp.

The palm oil industry produces large amounts of empty fruit bunches (EFB) as waste. EFB are very recalcitrant toward further processing, although their valorization could create novel incentives and bio-economic opportunities for the industries involved. Herein, EFB have been successfully subjected to the OrganoCat pretreatment—using 2,5-furandicarboxylic acid as the biogenic catalyst—to fractionate and separate this lignocellulosic material into its main components in a single step. The pretreatment of EFB leads to the deacetylation and depolymerization of noncellulosic polysaccharides and to the partial delignification of the cellulosic fiber. The OrganoCat processing of EFB yielded 45 ± 0.5 wt % cellulose-enriched pulp, 20 ± 0.7 wt % extracted lignin, 3.8 ± 0.2 wt % furfural, and 11 ± 0.6 wt % hydrolyzed sugars. The obtained EFB-pulp showed high accessibility to cellulases, resulting in a glucan conversion of 73 ± 2% after 72 h (15 ± 2% after 1 h) with commercial cellulase cocktail (Accellerase 1500). Overall, the results suggest that the treatment of the EFB material using OrganoCat may create promising paths for the full valorization of EFBs.

Production, Characterization, and Application of an Alginate Lyase, AMOR_PL7A, from Hot Vents in the Arctic Mid-Ocean Ridge.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. We describe a thermostable alginate lyase belonging to Polysaccharide Lyase family 7 (PL7), which can be used to degrade brown seaweed, Saccharina latissima, at conditions also suitable for a commercial cellulase cocktail (Cellic CTec2). This enzyme, AMOR_PL7A, is a β-d-mannuronate specific (EC 4.2.2.3) endoacting alginate lyase, which degrades alginate and poly mannuronate within a broad range of pH, temperature and salinity. At 65 °C and pH 6.0, its Km and kcat values for sodium alginate are 0.51 ± 0.09 mg/mL and 7.8 ± 0.3 s-1 respectively. Degradation of seaweed with blends of Cellic CTec2 and AMOR_PL7A at 55 °C in seawater showed that the lyase efficiently reduces viscosity and increases glucose solublization. Thus, AMOR_PL7A may be useful in development of efficient protocols for enzymatic seaweed processing.