Optimization, gene cloning, expression, and molecular docking insights for enhanced cellulase enzyme production by Bacillus amyloliquefaciens strain elh1.

BACKGROUND: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate. RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71. CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.

Extremozymes and compatible solute production potential of halophilic and halotolerant bacteria isolated from crop rhizospheric soils of Southwest Saurashtra Gujarat.

Halophiles are one of the classes of extremophilic microorganisms that can flourish in environments with very high salt concentrations. In this study, fifteen bacterial strains isolated from various crop rhizospheric soils of agricultural fields along the Southwest coastline of Saurashtra, Gujarat, and identified by 16S rRNA gene sequencing as Halomonas pacifica, H. stenophila, H. salifodinae, H. binhaiensis, Oceanobacillus oncorhynchi, and Bacillus paralicheniformis were investigated for their potentiality to produce extremozymes and compatible solute. The isolates showed the production of halophilic protease, cellulase, and chitinase enzymes ranging from 6.90 to 35.38, 0.004-0.042, and 0.097-0.550 U ml-1, respectively. The production of ectoine-compatible solute ranged from 0.01 to 3.17 mg l-1. Furthermore, the investigation of the ectoine-compatible solute production at the molecular level by PCR showed the presence of the ectoine synthase gene responsible for its biosynthesis in the isolates. Besides, it also showed the presence of glycine betaine biosynthetic gene betaine aldehyde dehydrogenase in the isolates. The compatible solute production by these isolates may be linked to their ability to produce extremozymes under saline conditions, which could protect them from salt-induced denaturation, potentially enhancing their stability and activity. This correlation warrants further investigation.

Metataxonomic Studies to Evaluate the Beneficial Effect of Enzymatic Pretreatment on the Anaerobic Digestion of Waste Generated in Turkey Farming.

Turkey litter waste is lignocellulosic and keratinous, requiring prior enzymatic treatment to facilitate fiber hydrolysis and utilization by microorganisms in anaerobic digestion (AD) process. The understanding of the performance of microorganisms in AD can be facilitated through molecular biology and bioinformatics tools. This study aimed to determine the taxonomic profile and functional prediction of microbial communities in the AD of turkey litter waste subjected to enzymatic pretreatment and correlate it with operational parameters. The tests involved the use of turkey litter (T) at 25 g L-1 of volatile solids, a granular inoculum (S) (10% m/v), and the addition of cellulase (C), and pectinase (P) enzymes at four concentrations. The use of enzymes increased methane production by 19% (turkey litter, inoculum, and cellulase-TSC4) and 15% (turkey litter, inoculum, and enzymatic pectinase-TSP4) compared to the control (turkey litter and inoculum-TS), being more effective in TSC4 (667.52 mLCH4), where there was consumption of acetic, butyric, and propionic acids. The pectinase assay (TSP4) showed a methane production of 648 mLCH4 and there was the accumulation of metabolites. Cellulolytic microorganisms Bacteroides, Ruminofilibacter, Lachnospiraceae, Ruminococcaceae, and Methanosaeta were favored in TSC4. In TSP4, the predominant genus was Macellibacteroides and Methanosarcina, and genes involved in methylotrophic methanogenesis were also found (mtaB, mtmB, and mtbB). Enzymes involved in hydrogenotrophic methanogenesis were identified in both assays (TSC4 and TSP4). Molecular tools helped to understand the metabolic routes involved in AD with enzymatic treatment, allowing the elaboration of strategies to improve the sustainable degradation of turkey litter waste.

Structure elucidation of a multi-modular recombinant endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus ATCC 27405 and its substrate binding analysis.

Cellulases from GH9 family show endo-, exo- or processive endocellulase activity, but the reason behind the variation is unclear. A GH9 recombinant endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus was structurally characterized for conformation, binding and dynamics assessment. Modeled AtGH9C-CBM3A-CBM3B depicted (α/α)6-barrel structure with Asp98, Asp101 and Glu489 acting as catalytic triad. CD results revealed 25.2 % α-helix, 18.4 % ß-sheet and rest 56.4 % of random coils, corroborating with predictions from PSIPRED and SOPMA. MD simulation of AtGH9C-CBM3A-CBM3B bound cellotetraose showed structural stability and global compactness with lowered RMSD values (1.5 nm) as compared with only AtGH9C-CBM3A-CBM3B (1.8 nm) for 200 ns. Higher fluctuation in RMSF values in far-positioned CBM3B pointed to its redundancy in substrate binding. Docking studies showed maximum binding with cellotetraose (ΔG = -5.05 kcal/mol), with reduced affinity towards ligands with degree of polymerization (DP) lower (DP < 4) or higher than 4 (DP > 4). Processivity index displayed the enzyme to be processive with loop 3 (342-379 aa) possibly blocking the non-reducing end of cellulose chain, resulting in cellotetraose release. SAXS analysis of AtGH9C-CBM3A-CBM3B at 5 mg/mL displayed monodispersed state with fist-and-elbow shape in solution. Negative zeta potential of -24 mV at 5 mg/mL indicated stability and free from aggregation.

Enzymes and biosurfactants of industrial interest produced by culturable fungi present in sediments of Boeckella Lake, Hope Bay, north-east Antarctic Peninsula.

This study characterized cultivable fungi present in sediments obtained from Boeckella Lake, Hope Bay, in the north-east of the Antarctic Peninsula, and evaluated their production of enzymes and biosurfactants of potential industrial interest. A total of 116 fungal isolates were obtained, which were classified into 16 genera within the phyla Ascomycota, Basidiomycota and Mortierellomycota, in rank. The most abundant genera of filamentous fungi included Pseudogymnoascus, Pseudeurotium and Antarctomyces; for yeasts, Thelebolales and Naganishia taxa were dominant. Overall, the lake sediments exhibited high fungal diversity and moderate richness and dominance. The enzymes esterase, cellulase and protease were the most abundantly produced by these fungi. Ramgea cf. ozimecii, Holtermanniella wattica, Leucosporidium creatinivorum, Leucosporidium sp., Mrakia blollopis, Naganishia sp. and Phenoliferia sp. displayed enzymatic index > 2. Fourteen isolates of filamentous fungi demonstrated an Emulsification Index 24% (EI24%) ≥ 50%; among them, three isolates of A. psychrotrophicus showed an EI24% > 80%. Boeckella Lake itself is in the process of drying out due to the impact of regional climate change, and may be lost completely in approaching decades, therefore hosts a threatened community of cultivable fungi that produce important biomolecules with potential application in biotechnological processes.

Co-production of fermentable sugars and highly active lignin from eucalyptus via a mild preprocessing with diethylene glycol and chromic chloride.

Eucalyptus was pretreated with diethylene glycol catalyzed by 0.02 mol/L CrCl3 for 10 min, resulting in 91 % delignification and 98 % cellulose recovery, with trace fermentation inhibitors generated. After the mild pretreatment, the accessibility and affinity of cellulase to eucalyptus was enhanced, especially since enzyme adsorption rate increased by 1.6-fold. Therefore, glucose yield of pretreated eucalyptus was 7.9-fold higher than that of untreated eucalyptus after hydrolyzed 48 h, in which the maximum glucose concentration reached 62 g/L from eucalyptus by adding Tween 80. According to the characterization analysis, the structure of the eucalyptus lignin-carbohydrate complexes structure was destroyed during the pretreatment, while lignin fragments was likely reacted with diethylene glycol to form the stabilized aromatic ethers. Moreover, the extracted Deg-lignin exhibited better performances than commercial alkali lignin such as higher fluorescence intensity, less negative surface charge, and lower particle size. The mild pretreatment method with diethylene glycol and CrCl3 provided a promising approach for co-production of fermentable sugars and high activity lignin from lignocellulosic biomass.

Reversible immobilization of cellulase on gelatin for efficient insoluble cellulose hydrolysis.

Immobilized enzymes are one of the most common tools used in enzyme engineering, as they can substantially reduce the cost of enzyme isolation and use. However, efficient catalysis of solid substrates using immobilized enzymes is challenging, hydrolysis of insoluble cellulose by immobilized cellulases is a typical example of this problem. In this study, inspired by bees and honeycombs, we prepared gelatin-modified cellulase (BEE) and gelatin hydrogels (HONEYCOMB) to achieve reversible recycling versus release of cellulase through temperature-responsive changes in the triple-stranded helix-like interactions between BEE and HONEYCOMB. At elevated temperatures, BEE was released from HONEYCOMB and participated in hydrolytic saccharification. After 24 h, the glucose yields of both the free enzyme and BEE reached the same level. When the temperature was decreased, BEE recombined with HONEYCOMB to facilitate the effective separation and recycling of BEE from the system. The enzymatic system retained >70 % activity after four reuse cycles. In addition, this system showed good biocompatibility and environmental safety. This method increases the mass transfer capacity and enables easy recovery of immobilized cellulase, thereby serving as a valuable strategy for the immobilization of other enzymes.

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.

Enhancing soil amendment for salt stress using pretreated rice straw and cellulolytic fungi.

Rice straw breakdown is sluggish, which makes agricultural waste management difficult, however pretreatment procedures and cellulolytic fungi can address this issue. Through ITS sequencing, Chaetomium globosum C1, Aspergillus sp. F2, and Ascomycota sp. SM2 were identified from diverse sources. Ascomycota sp. SM2 exhibited the highest carboxymethyl cellulase (CMCase) activity (0.86 IU/mL) and filter-paper cellulase (FPase) activity (1.054 FPU/mL), while Aspergillus sp. F2 showed the highest CMCase activity (0.185 IU/mL) after various pretreatments of rice straw. These fungi thrived across a wide pH range, with Ascomycota sp. SM2 from pH 4 to 9, Aspergillus sp. F2, and Chaetomium globosum C1 thriving in alkaline conditions (pH 9). FTIR spectroscopy revealed significant structural changes in rice straw after enzymatic hydrolysis and solid-state fermentation, indicating lignin, cellulose, and hemicellulose degradation. Soil amendments with pretreated rice straw, cow manure, biochar, and these fungi increased root growth and soil nutrient availability, even under severe salt stress (up to 9.3 dS/m). The study emphasizes the need for a better understanding of Ascomycota sp. degradation capabilities and proposes that using cellulolytic fungus and pretreatment rice straw into soil amendments could mitigate salt-related difficulties and improve nutrient availability in salty soils.

Association of enzymatic and optimized ultrasound-assisted aqueous extraction of flavonoid glycosides from dried Hippophae rhamnoides L. (Sea Buckthorn) berries.

The main purpose of the present study was to determine the effect of associating an optimized ultrasound-assisted extraction (UAE) protocol with enzyme-assisted extraction (EAE) in aqueous media, using the dried berries of Hippophae rhamnoides L. (sea buckthorn) as plant material. A specialized software was used for the determination of potential optimal extraction parameters, leading to the development of four optimized extracts with different characteristics (UAE ± EAE). For these extracts, buffered or non-buffered solutions have been used, with the aim to determine the influence of adjustable pH on extractability. As enzymatic solution, a pectinase, cellulase, and hemicellulase mix (2:1:1) has been applied, acting as pre-treatment for the optimized protocol. The highest extractive yields have been identified for non-buffered extracts, and the E-UAE combination obtained extracts with the highest overall in vitro antioxidant activity. The HPLC-MSn analysis demonstrated a rich composition in different types of isorhamnetin-O-glycosides, as well as some quercetin-O-glycosides, showing a high recovery of specific flavonol-type polyphenolic species. Moreover, we have tentatively identified two flavanols (i.e., catechin and epigallocatechin) and one flavone derivative (i.e., luteolin).

Contribution of Aerobic Cellulolytic Gut Bacteria to Cellulose Digestion in Fifteen Coastal Grapsoid Crabs Underpins Potential for Mineralization of Mangrove Production.

Grapsoid crabs (Decapoda: Grapsoidea) inhabiting along the land-sea transition provided various amounts and quality of vascular plant carbon (e.g., fresh mangrove leaf, leaf litter, and mangrove-derived organic carbon) and perform differing levels of herbivory. Other than endogenous cellulase, symbiotic cellulolytic bacteria could also contribute to the crabs' vascular plant carbon assimilation and mineralization. In this study, we isolated culturable cellulolytic bacteria from three gut regions (i.e., stomach, midgut, and hindgut) of 15 species of grapsoid crabs that inhabit in various coastal habitats (i.e., land margin, mangrove forest, tidal flat, and subtidal area). Bacillus, which was isolated from 11 out of the 15 grapsoid crabs, was the most common genus of culturable prominently cellulolytic bacteria among the target species. Seventy to ninety nine percent of culturable cellulolytic bacteria were removed, and the endoglucanase activity of five species was significantly reduced by 14.4-27.7% after antibiotic treatment. These results suggest that cellulolytic bacteria play a role in assisting mangrove carbon utilization in coastal grapsoid crabs, especially those inhabiting mangrove, mudflat, and subtidal areas. The significantly higher abundance of cellulolytic bacteria and the generally higher hydrolytic capacity of the bacteria in mangrove crab species suggest that they receive more contribution from symbionts for mangrove carbon utilization, while semi-terrestrial crabs seem to depend little on symbiotic cellulase due to the lower abundances.

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.

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.

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.

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.

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).

Enhancing cellulose hydrolysis via cellulase immobilization on zeolitic imidazolate frameworks using physical adsorption.

This study investigates the immobilization of cellulase on zeolitic imidazolate frameworks (ZIFs) by physical adsorption, specifically the ZIF-8-NH2 and Fe3O4@ZIF-8-NH2, to enhance enzymatic hydrolysis efficiency. The immobilization process was thoroughly analyzed, including optimization of conditions and characterization of ZIF carriers and immobilized enzymes. The impacts on the catalytic activity of cellulase under various temperatures, pH levels, and storage conditions were examined. Additionally, the reusability of the immobilized enzyme was assessed. Results showed the cellulase immobilized on Fe3O4@ZIF-8-NH2 exhibited a high loading capacity of 339.64 mg/g, surpassing previous studies. Its relative enzymatic activity was found to be 71.39%. Additionally, this immobilized enzyme system demonstrates robust reusability, retaining 68.42% of its initial activity even after 10 cycles. These findings underscore the potential of Fe3O4@ZIF-8-NH2 as a highly efficient platform for cellulase immobilization, with promising implications for lignocellulosic biorefinery.

Bioprospecting of endophytic fungi from semidesert candelilla (Euphorbia antisyphilitica Zucc): Potential for extracellular enzyme production.

Endophytic microbial communities colonize plants growing under various abiotic stress conditions. Candelilla (Euphorbia antisyphilitica Zucc.) is a shrub that develops functionally in arid and semi-arid zones of Mexico; these conditions generate an association between the plant and the microorganisms, contributing to the production of enzymes as a defense mechanism for resistance to abiotic stress. The objective of this research was to isolate and identify endophyte fungi of candelilla and bioprospection of these endophytic fungi for enzyme production using candelilla by-products. Fungi were isolated and identified using ITS1/ITS4 sequencing. Their potency index (PI) was evaluated in producing endoglucanase, xylanase, amylase, and laccase. Fermentation was carried out at 30°C for 8 days at 200 rpm, with measurements every 2 days, using candelilla by-products as substrate. All fungi exhibited higher cellulase, amylase, and laccase activities on the 2nd, 6th, and 8th day of fermentation, respectively, of fermentation. The fungus Aspergillus niger ITD-IN4.1 showed the highest amylase activity (246.84 U/mg), the genus Neurospora showed the highest cellulase activity, reaching up to 13.45 FPU/mg, and the strain Neurospora sp. ITD-IN5.2 showed the highest laccase activity (3.46 U/mg). This work provides the first report on the endophytic diversity of E. antisyphilitica and its potential role in enzyme production.