Metagenomic exploration of cold-active enzymes for detergent applications: Characterization of a novel, cold-active and alkali-stable GH8 endoglucanase from ikaite columns in SW Greenland.

Microbial communities from extreme environments are largely understudied, but are essential as producers of metabolites, including enzymes, for industrial processes. As cultivation of most microorganisms remains a challenge, culture-independent approaches for enzyme discovery in the form of metagenomics to analyse the genetic potential of a community are rapidly becoming the way forward. This study focused on analysing a metagenome from the cold and alkaline ikaite columns in Greenland, identifying 282 open reading frames (ORFs) that encoded putative carbohydrate-modifying enzymes with potential applications in, for example detergents and other processes where activity at low temperature and high pH is desired. Seventeen selected ORFs, representing eight enzyme families were synthesized and expressed in two host organisms, Escherichia coli and Aliivibrio wodanis. Aliivibrio wodanis demonstrated expression of a more diverse range of enzyme classes compared to E. coli, emphasizing the importance of alternative expression systems for enzymes from extremophilic microorganisms. To demonstrate the validity of the screening strategy, we chose a recombinantly expressed cellulolytic enzyme from the metagenome for further characterization. The enzyme, Cel240, exhibited close to 40% of its relative activity at low temperatures (4°C) and demonstrated endoglucanase characteristics, with a preference for cellulose substrates. Despite low sequence similarity with known enzymes, computational analysis and structural modelling confirmed its cellulase-family affiliation. Cel240 displayed activity at low temperatures and good stability at 25°C, activity at alkaline pH and increased activity in the presence of CaCl2, making it a promising candidate for detergent and washing industry applications.

Optimization of Composite Enzymatic Extraction, Structural Characterization and Biological Activity of Soluble Dietary Fiber from Akebia trifoliata Peel.

In order to reduce the waste of Akebia trifoliata peel and maximize its utilization, in this study, on the basis of a single-factor experiment and the response surface method, the optimum technological conditions for the extraction of soluble dietary fiber from Akebia trifoliata peel with the compound enzyme method were obtained. The chemical composition, physical and chemical properties, structural characterization and biological activity of the purified soluble dietary fiber (AP-SDF) from the Akebia trifoliata peel were analyzed. We discovered that that the optimum yield was 20.87% under the conditions of cellulase addition 600 U/g, enzymolysis time 100 min, solid-liquid ratio 1:24 g/mL and enzymolysis temperature 51 °C. At the same time, AP-SDF was a porous network structure cellulose type I acidic polysaccharose mainly composed of arabinoxylan (36.03%), galacturonic acid (27.40%) and glucose (19.00%), which possessed the structural characteristic peaks of the infrared spectra of polysaccharides and the average molecular weight (Mw) was 95.52 kDa with good uniformity. In addition, the AP-SDF exhibited high oil-holding capacity (15.11 g/g), good water-holding capacity and swelling capacity, a certain antioxidant capacity in vitro, hypoglycemic activity in vitro for α-glucosidase inhibition and hypolipidemic activity in vitro for the binding ability of bile acids and cholesterol. These results will provide a theoretical basis for the development of functional products with antioxidant, hypoglycemic and hypolipidemic effects, which have certain application value in related industries.

Influence of Three Modification Methods on the Structure, Physicochemical, and Functional Properties of Insoluble Dietary Fiber from Rosa roxburghii Tratt Pomace.

Rosa roxburghii Tratt pomace is rich in insoluble dietary fiber (IDF). This study aimed to investigate the influence of three modification methods on Rosa roxburghii Tratt pomace insoluble dietary fiber (RIDF). The three modified RIDFs, named U-RIDF, C-RIDF, and UC-RIDF, were prepared using ultrasound, cellulase, and a combination of ultrasound and cellulase methods, respectively. The structure, physicochemical characteristics, and functional properties of the raw RIDF and modified RIDF were comparatively analyzed. The results showed that all three modification methods, especially the ultrasound-cellulase combination treatment, increased the soluble dietary fiber (SDF) content of RIDF, while also causing a transition in surface morphology from smooth and dense to wrinkled and loose structures. Compared with the raw RIDF, the modified RIDF, particularly UC-RIDF, displayed significantly improved water-holding capacity (WHC), oil-binding capacity (OHC), and swelling capacity (SC), with increases of 12.0%, 84.7%, and 91.3%, respectively. Additionally, UC-RIDF demonstrated the highest nitrite ion adsorption capacity (NIAC), cholesterol adsorption capacity (CAC), and bile salt adsorption capacity (BSAC). In summary, the combination of ultrasound and cellulase treatment proved to be an efficient approach for modifying IDF from RRTP, with the potential for developing a functional food ingredient.

Unlocking the potential of Algerian lignocellulosic biomass: exploring indigenous microbial diversity for enhanced enzyme and sugar production.

Nowadays, natural resources like lignocellulosic biomass are gaining more and more attention. This study was conducted to analyse chemical composition of dried and ground samples (500 µm) of various Algerian bioresources including alfa stems (AS), dry palms (DP), olive pomace (OP), pinecones (PC), and tomato waste (TW). AS exhibited the lowest lignin content (3.60 ± 0.60%), but the highest cellulose (58.30 ± 2.06%), and hemicellulose (20.00 ± 3.07%) levels. DP, OP, and PC had around 30% cellulose, and 10% hemicellulose. OP had the highest lignin content (29.00 ± 6.40%), while TW contained (15.70 ± 2.67% cellulose, 13.70 ± 0.002% hemicellulose, and 17.90 ± 4.00% lignin). Among 91 isolated microorganisms, nine were selected for cellulase, xylanase, and/or laccase production. The ability of Bacillus mojavensis to produce laccase and cellulase, as well as B. safensis to produce cellulase and xylanase, is being reported for the first time. In submerged conditions, TW was the most suitable substrate for enzyme production. In this conditions, T. versicolor K1 was the only strain able to produce laccase (4,170 ± 556 U/L). Additionally, Coniocheata hoffmannii P4 exhibited the highest cellulase activity (907.62 ± 26.22 U/L), and B. mojavensis Y3 the highest xylanase activity (612.73 ± 12.73 U/L). T. versicolor K1 culture showed reducing sugars accumulation of 18.87% compared to initial concentrations. Sucrose was the predominant sugar detected by HPLC analysis (13.44 ± 0.02 g/L). Our findings suggest that T. versicolor K1 holds promise for laccase production, while TW represents a suitable substrate for sucrose production.

Expression and characterization of a novel ß-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 ß-1,4 bonds. The enzyme ß-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A ß-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 ß-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 ß-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated ß-1,4-endoglucanase had higher activity and stability.

Simulation and experimental study of a cold atmospheric pressure plasma and comparison of efficiency in boosting recombinant Endoglucanase II production in Pichia pastoris.

Recombinant proteins are essential in various industries, and scientists employ genetic engineering and synthetic biology to enhance the host cell's protein production capacity. Stress response pathways have been found effective in augmenting protein secretion. Cold atmospheric pressure plasma (CAP) can induce oxidative stress and enhance protein production. Previous studies have confirmed the applicability of CAP jets on Phytase and green fluorescent protein (GFP) production in Pichia pastoris hosts. This study investigates the effect of CAP treatment on another valuable recombinant protein, Endoglucanase II (EgII), integrated into the Pichia pastoris genome. The results demonstrated that plasma induction via two different ignition modes: sinusoidal alternating current (AC) and pulsed direct current (DC) for 120, 180, and 240 s has boosted protein secretion without affecting cell growth and viability. The AC-driven jet exhibited a higher percentage increase in secretion, up to 45%. Simulation of plasma function using COMSOL software provided a pattern of electron temperature (Te) and density distribution, which determine the plasma cocktail's chemistry and reactive species production. Furthermore, electron density (ne) and temperature were estimated from the recorded optical spectrum. The difference in electron properties may explain the moderately different impressions on expression capability. However, cell engineering to improve secretion often remains a trial-and-error approach, and improvements are, at least partially, specific to the protein produced.

Self-assembly of choline-based surface-active ionic liquids and concentration-dependent enhancement in the enzymatic activity of cellulase in aqueous medium.

The micellization of choline-based anionic surface-active ionic liquids (SAILs) having lauroyl sarcosinate [Sar]-, dodecylsulfate [DS]-, and deoxycholate [Doc]- as counter-ions was investigated in an aqueous medium. Density functional theory (DFT) was employed to investigate the net interactional energy (Enet), extent of non-covalent interactions, and band gap of the choline-based SAILs. The critical micelle concentration (cmc) along with various parameters related to the surface adsorption, counter-ion binding (ß), and polarity of the cores of the micelles were deduced employing surface tension measurements, conductometric titrations and fluorescence spectroscopy, respectively. A dynamic light scattering (DLS) system equipped with zeta-potential measurement set-up and small-angle neutron scattering (SANS) were used to predict the size, zeta-potential, and morphology, respectively, of the formed micelles. Thermodynamic parameters such as standard Gibb's free energy and standard enthalpy change of micellization were calculated using isothermal titration calorimetry (ITC). Upon comparing with sodium salt analogues, it was established that the micellization was predominantly governed by the extent of hydration of [Cho]+, the head groups of the respective anions, and the degree of counter-ion binding (ß). Considering the concentration dependence of the enzyme-SAIL interactions, aqueous solutions of the synthesized SAILs at two different concentrations (below and above the cmc) were utilized as the medium for testing the enzymatic activity of cellulase. The activity of cellulase was found to be ∼7- to ∼13-fold higher compared to that observed in buffers in monomeric solutions of the SAILs and followed the order: [Cho][Sar] > [Cho][DS] > [Cho][Doc]. In the micellar solution, a ∼4- to 5-fold increase in enzymatic activity was observed.

Heterologous expression and characterization of mutant cellulase from indigenous strain of Aspergillus niger.

The purpose of current research work was to investigate the effect of mutagenesis on endoglucanase B activity of indigenous strain of Aspergillus niger and its heterologous expression studies in the pET28a+ vector. The physical and chemical mutagens were employed to incorporate mutations in A. niger. For determination of mutations, mRNA was isolated followed by cDNA synthesis and cellulase gene was amplified, purified and sequenced both from native and mutant A. niger. On comparison of gene sequences, it was observed that 5 nucleotide base pairs have been replaced in the mutant cellulase. The mutant recombinant enzyme showed 4.5 times higher activity (428.5 µmol/mL/min) as compared to activity of native enzyme (94 µmol/mL/min). The mutant gene was further investigated using Phyre2 and I-Tesser tools which exhibited 71% structural homology with Endoglucanase B of Thermoascus aurantiacus. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent accessible surface area (SASA), radius of gyration (Rg) and hydrogen bonds analysis were carried at 35°C and 50°C to explore the integrity of structure of recombinant mutant endoglucanase B which corresponded to its optimal temperature. Hydrogen bonds analysis showed more stability of recombinant mutant endoglucanase B as compared to native enzyme. Both native and mutant endoglucanase B genes were expressed in pET 28a+ and purified with nickel affinity chromatography. Theoretical masses determined through ExPaSy Protparam were found 38.7 and 38.5 kDa for native and mutant enzymes, respectively. The optimal pH and temperature values for the mutant were 5.0 and 50°C while for native these were found 4.0 and 35°C, respectively. On reacting with carboxy methyl cellulose (CMC) as substrate, the mutant enzyme exhibited less Km (0.452 mg/mL) and more Vmax (50.25 µmol/ml/min) as compared to native having 0.534 mg/mL as Km and 38.76 µmol/ml/min as Vmax. Among metal ions, Mg2+ showed maximum inducing effect (200%) on cellulase activity at 50 mM concentration followed by Ca2+ (140%) at 100 mM concentration. Hence, expression of a recombinant mutant cellulase from A. niger significantly enhanced its cellulytic potential which could be employed for further industrial applications at pilot scale.

Sustainable wheat straw pretreatment process by self-produced and cyclical crude lactic acid.

The purpose of this study was to investigate an environmentally friendly and recyclable pretreatment approach that would enhance the enzymatic digestibility of wheat straw. Wheat straw was pretreated using self-produced crude lactic acid obtained from enzymatic hydrolysate fermentation by Bacillus coagulans. Experimentally, crude lactic acid at low concentration could achieve a pretreatment effect comparable to that of commercial lactic acid. After pretreatment at 180 °C for 60 min with 2.0 % crude lactic acid, hemicellulose could be effectively separated and high recovery of cellulose was ensured, achieving cellulose recovery rate of 95.5 % and hemicellulose removal rate of 92.7 %. Excellent enzymatic hydrolysis was accomplished with a glucose yield of 99.7 %. Moreover, the crude lactic acid demonstrated acceptable pretreatment and enzymatic hydrolysis performance even after three repeated cycles. This not only effectively utilizes the pretreatment solution, but also offers insights into biomass pretreatment using other fermentable acids.

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

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

Ionic liquid-assisted pretreatment of lignocellulosic biomass using purified Streptomyces MS2A cellulase for bioethanol production.

In recent years, the process of producing bioethanol from lignocellulosic biomass through biorefining has become increasingly important. However, to obtain a high yield of ethanol, the complex structures in the feedstock must be broken down into simple sugars. A cost-effective and innovative method for achieving this is ionic liquid pre-treatment, which is widely used to efficiently hydrolyze the lignocellulosic material. The study aims to produce a significant profusion of bioethanol via catalytic hydrolysis of ionic liquid-treated lignocellulose biomass. The current study reports the purification of Streptomyces sp. MS2A cellulase via ultrafiltration and gel permeation chromatography. The kinetic parameters and the biochemical nature of the purified cellulase were analyzed for the effective breakdown of the EMIM[OAC] treated lignocellulose chain. The two-step cellulase purification resulted in 6.28 and 12.44 purification folds. The purified cellulase shows a Km value of 0.82 ± 0.21 mM, and a Vmax value of 85.59 ± 8.87 µmol min-1 mg-1 with the catalytic efficiency of 1.027 S-1. The thermodynamic parameters like ΔH, ΔS, and ΔG of the system were studied along with the thermal deactivation kinetics of cellulase. The optimal temperature and pH of the purified cellulase enzyme for hydrolysis was found to be 40 °C and 7. The rice husk and wheat husk used in this study were pretreated with the EMIM [OAC] ionic liquid and the change in the structure of lignocellulosic biomass was observed via HRSEM. The ionic liquid treated biomass showed the highest catalytic hydrolysis yield of 106.66 ± 0.19 mol/ml on the third day. The obtained glucose was fermented with Saccharomyces cerevisiae to yield 23.43 g of ethanol/l of glucose from the rice husk (RH) and 24.28 g of ethanol/l of glucose from the wheat husk (WH).

Synergism of jet milling and deep eutectic solvent pretreatment on grapevine lignin fractionation and enhancing enzymatic hydrolysis.

Herein, we investigated the synergistic effects of jet milling (JM) and deep eutectic solvent (DES) pretreatment on the fractionation of grapevine lignin and the consequent enhancement of enzymatic hydrolysis. Grapevine, a substantial byproduct of the wine industry, was subjected to JM pretreatment to produce finely powdered particles (median diameter D50 = 98.90), which were then further treated with acidic ChCl-LA and alkaline K2CO3-EG DESs. The results revealed that the combined JM + ChCl-LA pretreatment significantly increased the cellulose preservation under optimal conditions (110 °C, 4 h, and 20 % water content), achieving removal rates of 74.18 % xylan and 66.05 % lignin, respectively. The pretreatment temperature and inhibitor production were reduced, resulting in a remarkable threefold increase in glucose yield compared to untreated samples. Moreover, the structural analysis of the pretreated lignin indicated an enrichment of phenolic units, leading to enhanced antioxidant and antibacterial activities, particularly in the JM pretreated samples. These findings underscore the promising potential of the synergistic JM and DES pretreatment in facilitating the efficient utilization of grapevine lignocellulosic biomass for sustainable biorefinery technologies.

Lignocellulolytic Enzymes Production by Four Wild Filamentous Fungi for Olive Stones Valorization: Comparing Three Fermentation Regimens.

Lignocellulolytic enzymes play a crucial role in efficiently converting lignocellulose into valuable platform molecules in various industries. However, they are limited by their production yields, costs, and stability. Consequently, their production by producers adapted to local environments and the choice of low-cost raw materials can address these limitations. Due to the large amounts of olive stones (OS) generated in Morocco which are still undervalued, Penicillium crustosum, Fusarium nygamai, Trichoderma capillare, and Aspergillus calidoustus, are cultivated under different fermentation techniques using this by-product as a local lignocellulosic substrate. Based on a multilevel factorial design, their potential to produce lignocellulolytic enzymes during 15 days of dark incubation was evaluated. The results revealed that P. crustosum expressed a maximum total cellulase activity of 10.9 IU/ml under sequential fermentation (SF) and 3.6 IU/ml of ß-glucosidase activity under submerged fermentation (SmF). F. nygamai recorded the best laccase activity of 9 IU/ml under solid-state fermentation (SSF). Unlike T. capillare, SF was the inducive culture for the former activity with 7.6 IU/ml. A. calidoustus produced, respectively, 1,009 µg/ml of proteins and 11.5 IU/ml of endoglucanase activity as the best results achieved. Optimum cellulase production took place after the 5th day under SF, while ligninases occurred between the 9th and the 11th days under SSF. This study reports for the first time the lignocellulolytic activities of F. nygamai and A. calidoustus. Furthermore, it underlines the potential of the four fungi as biomass decomposers for environmentally-friendly applications, emphasizing the efficiency of OS as an inducing substrate for enzyme production.

Enhanced crystalline cellulose degradation by a novel metagenome-derived cellulase enzyme.

Metagenomics has revolutionized access to genomic information of microorganisms inhabiting the gut of herbivorous animals, circumventing the need for their isolation and cultivation. Exploring these microorganisms for novel hydrolytic enzymes becomes unattainable without utilizing metagenome sequencing. In this study, we harnessed a suite of bioinformatic analyses to discover a novel cellulase-degrading enzyme from the camel rumen metagenome. Among the protein-coding sequences containing cellulase-encoding domains, we identified and subsequently cloned and purified a promising candidate cellulase enzyme, Celcm05-2, to a state of homogeneity. The enzyme belonged to GH5 subfamily 4 and exhibited robust enzymatic activity under acidic pH conditions. It maintained hydrolytic activity under various environmental conditions, including the presence of metal ions, non-ionic surfactant Triton X-100, organic solvents, and varying temperatures. With an optimal temperature of 40 °C, Celcm05-2 showcased remarkable efficiency when deployed on crystalline cellulose (> 3.6 IU/mL), specifically Avicel, thereby positioning it as an attractive candidate for a myriad of biotechnological applications spanning biofuel production, paper and pulp processing, and textile manufacturing. Efficient biodegradation of waste paper pulp residues and the evidence of biopolishing suggested that Celcm05-2 can be used in the bioprocessing of cellulosic craft fabrics in the textile industry. Our findings suggest that the camel rumen microbiome can be mined for novel cellulase enzymes that can find potential applications across diverse biotechnological processes.

Green ecofriendly enhancement of cellulase productivity using agricultural wastes by Aspergillus terreus MN901491: statistical designs and detergent ability on cotton fabrics.

BACKGROUND: Cellulase is considered a group member of the hydrolytic enzymes, responsible for catalyzing the hydrolysis of cellulose and has various industrial applications. Agricultural wastes are used as an inexpensive source for several utilizable products throughout the world. So, searching for cellulase enzymes from fungal strains capable of utilizing agricultural wastes to increase productivity, reduce costs and overcome waste accumulation in the environment is very important to evaluate its potency as a bio-additive to detergent agents. RESULTS: In the current study, the previously identified fungal strain Aspergillus terreus MN901491 was screened and selected for cellulase production. Medium parameters were optimized using one-factor-at-a-time (OFAT) and multi-factorial (Plackett-Burman and Box-Behnken) design methods. OFAT showed the ability of the fungal strain to utilize agricultural wastes (corn cob and rice straw) as a substrate. Also, yeast extract was the best nitrogen source for enhancing cellulase productivity. The most significant variables were determined by Plackett-Burman Design (PBD) and their concentrations were optimized by Response Surface Methodology (RSM) using Box-Behnken Design (BBD). Among eleven independent variables screened by PBD, malt extract, (NH4)2SO4, and KCl were the most significant ones followed by rice straw which affected cellulase production positively. The ANOVA results particularly the R2-value of PBD (0.9879) and BBD (0.9883) confirmed the model efficiency and provided a good interpretation of the experiments. PBD and BBD improved cellulase productivity by 6.1-fold greater than that obtained from OFAT. Medium optimization using OFAT and statistical models increased cellulase production from A. terreus MN901491 by 9.3-fold compared to the non-optimized medium. Moreover, the efficiency of cellulase activity on cotton fabrics as a bio-additive detergent was evaluated and estimated using whiteness and scanning electron microscope (SEM) that affirmed its potential effect and remarkable detergent ability to improve whiteness by 200% in comparison with non-washed fabric and by 190% in comparison with fabric washed by water. CONCLUSION: The presented work was stabilized as a multi-efficiency in which wastes were used to produce cellulase enzyme from the fungal strain, Aspergillus terreus MN901491 as a bio-additive to detergent applications that involved ecofriendly and green processes.

A trimeric glycosylated GH45 cellulase from the red abalone (Haliotis rufescens) exhibits endo and exoactivity.

The red abalone (Haliotis rufescens) represents North America's most important aquaculture species. Its hepatopancreas is rich in cellulases and other polysaccharide-degrading enzymes, which provide it the remarkable ability to digest cellulose-rich macroalgae; nevertheless, its cellulolytic systems are poorly explored. This manuscript describes some functional and structural properties of an endogenous trimeric glycosylated endoglucanase from H. rufescens. The purified enzyme showed a molecular mass of 23.4 kDa determined by MALDI-TOF mass spectrometry, which behaved as a homotrimer in gel filtration chromatography and zymograms. According to the periodic acid-Schiff reagent staining, detecting sugar moieties in SDS-PAGE gel confirmed that abalone cellulase is a glycoprotein. Hydrolysis of cello-oligosaccharides and p-nitrophenyl-ß-D-glucopyranosides confirmed its endo/exoactivity. A maximum enzyme activity toward 0.5% (w/v) carboxymethylcellulose of 53.9 ± 1.0 U/mg was achieved at 45°C and pH 6.0. We elucidated the abalone cellulase primary structure using proteases and mass spectrometry methods. Based on these results and using a bioinformatic approach, we identified the gene encoding this enzyme and deduced its full-length amino acid sequence; the mature protein comprised 177 residues with a calculated molecular mass of 19.1 kDa and, according to sequence similarity, it was classified into the glycosyl-hydrolase family 45 subfamily B. An AlphaFold theoretical model and docking simulations with cellopentaose confirmed that abalone cellulase is a ß-sheet rich protein, as also observed by circular dichroism experiments, with conserved catalytic residues: Asp26, Asn109, and Asp134. Interestingly, the AlphaFold-Multimer analysis indicated a trimeric assembly for abalone cellulase, which supported our experimental findings. The discovery and characterization of these enzymes may contribute to developing efficient cellulose bioconversion processes for biofuels and sustainable bioproducts.

Isolation and characterization of cellulase producing bacteria from forest, cow dung, Dashen brewery and agro-industrial waste.

The continuous accumulation of waste, particularly from industries, often ends up in landfills. However, this waste can be transformed into a valuable resource through innovative methods. This process not only reduces environmental pollution but also generates additional useful products. This study aims to screen novel high-efficiency cellulose-degrading bacteria from cow dung, forest soil, brewery waste, and agro-industrial waste in the Debre Berhan area for the treatment of cellulose-rich agricultural waste. The serial dilution and pour plate method was used to screen for cellulolytic bacteria and further characterized using morphological and biochemical methods. From eleven isolates cow dung 1 (CD1), cow dung 6 (CD6) and cow dung (CD3) which produced the largest cellulolytic index (3.1, 2.9 and 2.87) were selected. Samples from forest soil, and spent grain didn't form a zone of clearance, and effluent treatment and industrial waste (IW9) shows the smallest cellulolytic index. Three potential isolates were then tested for cellulolytic activity, with cow dung 1 (CD1) displaying promising cellulase activity. These bacterial isolates were then identified as Bacillus species, which were isolated from cow dung 1 (CD1) with maximum cellulase production. Cow dung waste is a rich source of cellulase-producing bacteria, which can be valuable and innovative enzymes for converting lignocellulosic waste.

Optimizing microbioreactor cultivation strategies for Trichoderma reesei: from batch to fed-batch operations.

BACKGROUND: Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. RESULTS: This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) - 1 , the lowest rate of 0.3 g (l h) - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in ß -glucosidase activity compared to batch processes using the same type and amount of carbon sources. CONCLUSION: We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology.

Screening of cellulose-degrading bacteria and optimization of cellulase production from Bacillus cereus A49 through response surface methodology.

Cellulose-degrading microorganisms hold immense significance in utilizing cellulose resources efficiently. The screening of natural cellulase bacteria and the optimization of fermentation conditions are the hot spots of research. This study meticulously screened cellulose-degrading bacteria from mixed soil samples adopting a multi-step approach, encompassing preliminary culture medium screening, Congo red medium-based re-screening, and quantification of cellulase activity across various strains. Particularly, three robust cellulase-producing strains were identified: A24 (MT740356.1 Brevibacillus borstelensis), A49 (MT740358.1 Bacillus cereus), and A61 (MT740357.1 Paenibacillus sp.). For subsequent cultivation experiments, the growth curves of the three obtained isolates were monitored diligently. Additionally, optimal CMCase production conditions were determined, keeping CMCase activity as a key metric, through a series of single-factor experiments: agitation speed, cultivation temperature, unit medium concentration, and inoculum volume. Maximum CMCase production was observed at 150 rpm/37 °C, doubling the unit medium addition, and a 5 mL inoculation volume. Further optimization was conducted using the selected isolate A49 employing response surface methodology. The software model recommended a 2.21fold unit medium addition, 36.11 °C temperature, and 4.91 mL inoculant volume for optimal CMCase production. Consequently, three parallel experiments were conducted based on predicted conditions consistently yielding an average CMCase production activity of 15.63 U/mL, closely aligning with the predicted value of 16.41 U/mL. These findings validated the reliability of the model and demonstrated the effectiveness of optimized CMCase production conditions for isolate A49.

Endoglucanase H from Aspergillus westerdijkiae Plays an Important Role in the Virulence on Pear Fruits.

Aspergillus westerdijkiae can infect many agricultural products including cereals, grapes, and pear. Pathogenic fungi secrete diverse effectors as invasive weapons for successful invasion the host plant. During the pathogen-host interaction, 4486 differentially expressed genes were observed in A. westerdijkiae with 2773 up-regulated and 1713 down-regulated, whereas 8456 differentially expressed genes were detected in pear fruits with 4777 up-regulated and 3679 down-regulated. A total of 309 effector candidate genes were identified from the up-regulated genes in A. westerdijkiae. Endoglucanase H (AwEGH) was significantly induced during the pathogen-host interaction. Deletion of AwEGH resulted in altered fungal growth and morphology and reduced conidia production and germination compared to the wild-type. Further experiments demonstrated that AwEGH plays a role in cell wall integrity. Importantly, disruption of AwEGH significantly reduced the fungal virulence on pear fruits, and this defect can be partly explained by the impaired ability of A. westerdijkiae to penetrate host plants.