Hydrolysis Of Cellulose Research Articles

Termite Microbial Symbiosis as a Model for Innovative Design of Lignocellulosic Future Biorefinery: Current Paradigms and Future Perspectives

The hunt for renewable and alternative fuels has driven research towards the biological conversion of lignocellulosic biomass (LCB) into biofuels, including bioethanol and biohydrogen. Among the natural biomass utilization systems (NBUS), termites represent a unique and easy-to-access model system to study host–microbe interactions towards lignocellulose bioconversion/valorization. Termites have gained significant interest due to their highly efficient lignocellulolytic systems. The wood-feeding termites apply a unique and stepwise process for the hydrolysis of lignin, hemicellulose, and cellulose via biocatalytic processes; therefore, mimicking their digestive metabolism and physiochemical gut environments might lay the foundation for an innovative design of nature-inspired biotechnology. This review highlights the gut system of termites, particularly the wood-feeding species, as a unique model for future biorefinery. The gut system of termites is a treasure-trove for prospecting novel microbial species, including protists, bacteria, and fungi, having higher biocatalytic efficiencies and biotechnological potentials. The significance of potential bacteria and fungi for harnessing the enzymes appropriate for lignocellulosic biorefinery is also discussed. Termite digestomes are rich sources of lignocellulases and related enzymes that could be utilized in various industrial processes and biomass-related applications. Consideration of the host and symbiont as a single functioning unit will be one of the most crucial strategies to expedite developments in termite-modeled biotechnology in the future.

Reversible Deacidification and Preventive Conservation of Paper-Based Cultural Relics by Mineralized Bacterial Cellulose.

Paper-based cultural relics experience irreversible aging and deterioration during long-term preservation. The most common process of paper degradation is the acid-catalyzed hydrolysis of cellulose. Nowadays, deacidification has been considered as a practical way to protect acidified literature; however, two important criteria of minimal intervention and reversibility should be considered. Inspired by the superior properties of bacterial cellulose (BC) and its structural similarity to paper, herein, the mineralized BC membranes are applied to deacidification and conservation of paper-based materials for the first time. Based on the enzyme-induced mineralization process, the homogeneous and high-loaded calcifications of hydroxyapatite (HAP) and calcium carbonate (CaCO3) nanoparticles onto the nanofibers of BC networks have been achieved, respectively. The size, morphology, structure of minerals, as well as the alkalinity and alkali reserve of BC membranes are well controlled by regulating enzyme concentration and mineralization time. Compared with HAP/CaCO3-immersed method, HAP/CaCO3-BC membranes show more efficient and sustained deacidification performance on paper. The weak alkalinity of mineralized BC membranes avoids the negative effect of alkali on paper, and the high alkali reserve implies a good sustained-release effect of alkali to neutralize the future generated acid. The multiscale nanochannels of the BC membrane provide ion exchange and acid/alkali neutralization channels between paper and the BC membrane, and the final pH of protected paper can be well stabilized in a certain range. Most importantly, this BC-deacidified method is reversible since the BC membrane can be removed without causing any damage to paper and the original structure and fiber morphology of paper are well preserved. In addition, the mineralized BC membrane provides excellent flame-retardant performance on paper thanks to its unique organic-inorganic composite structure. All of these advantages of the mineralized BC membrane indicate its potential use as an effective protection material for the reversible deacidification and preventive conservation of paper-based cultural relics.

Alkaline pulping catalyzed ordered conversion of bark in supercritical water

The hydrophilicity of lignocellulosic biomass hinders the application of a high concentration slurry for supercritical water gasification, thereby reducing the gasification efficiency and energy self-sufficiency. A pretreatment method of alkaline pulping with KOH and NaOH was proposed to achieve the high feed concentration for continuous reactor and macromolecular simplification for barrier reduction of conversion. In this study, bark as an example of lignocellulosic biomass, whose 30 wt% and 40 wt% slurries with favorable rheology and stability, was prepared by alkaline pulping successfully. The minimum alkali consumption of NaOH and KOH pulping for 30 wt% and 40 wt% were 11%–14%, 16%–20%, and 16%–21%, 23%–28%, respectively. The most economical conditions were 180 °C and 24% KOH addition. All slurries prepared by NaOH and KOH at 180 and 200 °C could satisfy the industrial standard of coal-water slurry, and only 3–5 times higher than water viscosity. The bark macromolecules were restructured due to the hydrolysis of cellulose and lignin by breaking down the hydrogen bond and ether bond with trivial hydrocarbon matrix loss. The scattered crystal growth facilitated the uniform premixing of bark and water. Meanwhile, a surface particle charges potential increase leading to a heightened repulsive force by alkaline pulping furtherly enhanced the premixing effect. Additionally, metal ions could be deposited onto the bark surface during alkaline pulping. The chemical modification leading by alkaline pulping achieved the premixing of feedstock and water to form bark slurry. The conversion barrier was decreased to fulfill the ordered conversion, and 95.66% gasification efficiency for 40 wt% bark slurry was displayed at 600 °C with a preheated water of 6. Phenolic compounds and hydrocarbons were the most dominant components in liquid products. Polycyclic aromatic hydrocarbons in liquid products were detected at 620 °C with a preheated water of 4. The functional groups of organic matter in the slurry were significantly converted into the gas phase, and lattice oxygen was produced in the solid product. The alkali recovery was 65% at the optimal gasification condition, and achieved the maximum of 80% at 580 °C. The successful completion of this research affirms the dominance of the pulping-alkali recovery-gasification method for expanding the application of SCWG of lignocellulosic biomass in large engineering projects.

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.

Screening the Rumen of Balochi Camel (Camelus dromedarius) and Cashmere Goat (Capra hircus) to Isolate Enzyme-Producing Bacteria as Potential Additives for Animal Feed.

Rumen microbiology has made a significant contribution to the discovery of biodegradation processes, which convert nutrients into energy for ruminants. Therefore, understanding the enzymatic potential in the rumen of different animal species is essential for developing efficient microbial feed additives. The aim of this study was to isolate enzyme-producing bacteria (EPBs) from the rumen of the Balochi camel (Camelus dromedarius) and Cashmere goat (Capra hircus) as potential additives for animal feed. The EPBs were screened based on the hydrolysis of carboxyl methyl cellulose, tannin, starch, and bovine serum albumin. The isolates were then subjected to enzyme activity assays and molecular characterization. Additionally, they were evaluated for their antagonistic effects, antibiotic susceptibility, and growth in acidic, bile, and saline media. Thirteen enzyme-producing strains were identified in the rumen of the camels and goats, belonging to the genera Klebsiella, Escherichia, Raoultella, Enterobacter and Pectobacterium. The highest and lowest tannase activities were recorded for Escherichia coli GHMGHE41 (10.46Um/l-1) and Raoultella planticola GHMGHE15 (1.83Um/l-1), respectively. Enterobacter cloacae GHMGHE18 (2.03U/ml) was the most effective cellulolytic isolate, compared to Klebsiella strains (1.05Um/l-1). The highest protease producer was Klebsiella pneumoniae GHMGHE13 (3.00U/ml-1), while Escherichia coli GHMGHE17 (1.13U/ml-1) had the lowest activity. Klebsiella pneumoniae GHMGHE13 (1.55U/ml-1) and Enterobacter cloacae GHMGHE19 (1.26U/ml-1) were the highest and lowest producers of amylase, respectively. The strains exhibited mixed responses to antibiotics and remained stable under stressful conditions. These findings indicate that ruminal EPBs have the potential to be used in animal feed, pending further in vivo studies.

Enzymatic hydrolysis of cellulose banana stem (alkaline microwave-assisted pre-treatment)

The banana stem waste holds immense promise as a readily available and abundant source of lignocellulosic biomass, making it a compelling alternative for biofuel and biochemical applications. Therefore, this study focuses on investigating the impact of both time and substrate loading on the enzymatic hydrolysis of banana stem cellulose that has undergone alkaline microwave-assisted pre-treatment. The pre-treatment method involves subjecting the biomass to 5% KOH for 30 min, followed by microwave exposure at 300 W for 5 min, a process aimed at enhancing the accessibility of cellulose. Enzymatic hydrolysis experiments were carried out utilizing cellulase enzymes derived from Aspergillus niger, with variations in hydrolysis times (ranging from 5 to 45 h) and enzyme-to-substrate ratios (ranging from 1:1 to 1:10). The results of this investigation revealed a substantial improvement in hydrolysis efficiency, owing to the synergistic effects of alkaline microwave-assisted pre-treatment, signifying enhanced cellulose accessibility. Notably, the highest concentration of reducing sugars (1.3 mg mL−1) was achieved at a substrate-to-enzyme ratio of 1:1 and a hydrolysis duration of 45 h. These findings provide valuable insights into the conversion of lignocellulosic biomass, emphasizing the potential of integrated pre-treatment strategies for sustainable biorefinery applications. This research contributes to advancing our understanding of lignocellulosic biomass utilization, offering a promising avenue for biofuel and biochemical production from banana stem waste.

Formation mechanism of dynamic liquid membranes for cellulose hydrolysis

Formation mechanism of dynamic liquid membranes for cellulose hydrolysis

Cellulolytic Aerobic Bacteria Isolated from Agricultural and Forest Soils: An Overview.

This review provides insights into cellulolytic bacteria present in global forest and agricultural soils over a period of 11 years. It delves into the study of soil-dwelling cellulolytic bacteria and the enzymes they produce, cellulases, which are crucial in both soil formation and the carbon cycle. Forests and agricultural activities are significant contributors to the production of lignocellulosic biomass. Forest ecosystems, which are key carbon sinks, contain 20-30% cellulose in their leaf litter. Concurrently, the agricultural sector generates approximately 998 million tons of lignocellulosic waste annually. Predominant genera include Bacillus, Pseudomonas, Stenotrophomonas, and Streptomyces in forests and Bacillus, Streptomyces, Pseudomonas, and Arthrobacter in agricultural soils. Selection of cellulolytic bacteria is based on their hydrolysis ability, using artificial cellulose media and dyes like Congo red or iodine for detection. Some studies also measure cellulolytic activity in vitro. Notably, bacterial cellulose hydrolysis capability may not align with their cellulolytic enzyme production. Enzymes such as GH1, GH3, GH5, GH6, GH8, GH9, GH10, GH12, GH26, GH44, GH45, GH48, GH51, GH74, GH124, and GH148 are crucial, particularly GH48 for crystalline cellulose degradation. Conversely, bacteria with GH5 and GH9 often fail to degrade crystalline cellulose. Accurate identification of cellulolytic bacteria necessitates comprehensive genomic analysis, supplemented by additional proteomic and transcriptomic techniques. Cellulases, known for degrading cellulose, are also significant in healthcare, food, textiles, bio-washing, bleaching, paper production, ink removal, and biotechnology, emphasizing the importance of discovering novel cellulolytic strains in soil.

Transcriptional regulation of cellobiose utilization by PRD-domain containing Sigma54-dependent transcriptional activator (CelR) and catabolite control protein A (CcpA) in Bacillus thuringiensis.

Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin resulting from the enzymatic hydrolysis of cellulose. It is a major source of carbon for soil bacteria. In bacteria, the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), encoded by the cel operon, is responsible for the transport and utilization of cellobiose. In this study, we analyzed the transcription and regulation of the cel operon in Bacillus thuringiensis (Bt). The cel operon is composed of five genes forming one transcription unit. β-Galactosidase assays revealed that cel operon transcription is induced by cellobiose, controlled by Sigma54, and positively regulated by CelR. The HTH-AAA+ domain of CelR recognized and specifically bound to three possible binding sites in the celA promoter region. CelR contains two PTS regulation domains (PRD1 and PRD2), which are separated by two PTS-like domains-the mannose transporter enzyme IIA component domain (EIIAMan) and the galactitol transporter enzyme IIB component domain (EIIBGat). Mutations of His-546 on the EIIAMan domain and Cys-682 on the EIIBGat domain resulted in decreased transcription of the cel operon, and mutations of His-839 on PRD2 increased transcription of the cel operon. Glucose repressed the transcription of the cel operon and catabolite control protein A (CcpA) positively regulated this process by binding the cel promoter. In the celABCDE and celR mutants, PTS activities were decreased, and cellobiose utilization was abolished, suggesting that the cel operon is essential for cellobiose utilization. Bt has been widely used as a biological pesticide. The metabolic properties of Bt are critical for fermentation. Nutrient utilization is also essential for the environmental adaptation of Bt. Glucose is the preferred energy source for many bacteria, and the presence of the phosphotransferase system allows bacteria to utilize other sugars in addition to glucose. Cellobiose utilization pathways have been of particular interest owing to their potential for developing alternative energy sources for bacteria. The data presented in this study improve our understanding of the transcription patterns of cel gene clusters. This will further help us to better understand how cellobiose is utilized for bacterial growth.

Mechanochemical Hydrolysis of Polysaccharide Biomass: Scope and Mechanistic Insights.

Mechanical forces can affect chemical reactions in a way that thermal reactions cannot do, which may have a variety of applications. In biomass conversion, the selective conversion of cellulose and chitin is a grand challenge because they are the top two most abundant resources and recalcitrant materials that are insoluble in common solvents. However, recent works have clarified that mechanical forces enable the depolymerization of these polysaccharides, leading to the selective production of corresponding monomers and oligomers. This article reviews the mechanochemical hydrolysis of cellulose and chitin, particularly focusing on the scope and mechanisms to show a landscape of this research field and future subjects. We introduce the background of mechanochemistry and biomass conversion, followed by recent progress on the mechanochemical hydrolysis of the polysaccharides. Afterwards, a considerable space is devoted to the mechanistic consideration on the mechanochemical reactions.

Cloning of AA9 Polysaccharide Monooxygenase gene AN3860 into pEX2B for expression in Aspergillus oryzae

Polysaccharide monooxygenases (PMOs) catalyze oxidative degradation of recalcitrant carbohydrate chains in cellulose, starch, and chitin. In biofuel industry, the conversion of rich lignocellulose source to fermentable sugars by hydrolytic cellulases can be synergistically boosted by cellulose-active PMOs (AA9 PMOs) found in a vast number of fungi that grow on biomass. Aspergillus nidulans, a filamentous fungus, possess a dozen of PMO-encoding genes, but only the AN3860 is expressed at a high level when cultured with wheat straw as the sole carbon source. Bioinformatic analysis indicates that AN3860 belongs to type 3 AA9 PMO subfamily that is capable of hydroxylating both C1 and C4 of the glycosidic linkages. Therefore, AN3860 may be a potential enzyme to improve cellulose hydrolysis efficiency, which has not been characterized. In this study, we describe the AN3860 cloning into Aspergillus oryzae AUT1-PlD. To facilitate the purification of AN3860, we added a CBM20 tag to its C-terminal. The recombinant vector was designed and constructed successfully. Simultaneously, we have obtained the clone of A. oryzae carrying the target gene by the ATMT method. Further expression optimization and characterization of AN3860 by both activity assays and spectroscopic techniques are underway.

Metabolic Remodulation of Chassis and Corn Stover Bioprocessing to Unlock 3-Hydroxypropionic Acid Biosynthesis from Agrowaste-Derived Substrates.

Embracing the principles of sustainable development, the valorization of agrowastes into value-added chemicals has nowadays received significant attention worldwide. Herein, Escherichia coli was metabolically rewired to convert cellulosic hydrolysate of corn stover into a key platform chemical, namely, 3-hydroxypropionic acid (3-HP). First, the heterologous pathways were introduced into E. coli by coexpressing glycerol-3-P dehydrogenase and glycerol-3-P phosphatase in both single and fusion (gpdp12) forms, making the strain capable of synthesizing glycerol from glucose. Subsequently, a glycerol dehydratase (DhaB123-gdrAB) and an aldehyde dehydrogenase (GabD4) were overexpressed to convert glycerol into 3-HP. A fine-tuning between glycerol synthesis and its conversion into 3-HP was successfully established by 5'-untranslated region engineering of gpdp12 and dhaB123-gdrAB. The strain was further metabolically modulated to successfully prevent glycerol flux outside the cell and into the central metabolism. The finally remodulated chassis produced 32.91 g/L 3-HP from the cellulosic hydrolysate of stover during fed-batch fermentation.

Acetic acid as a protic solvent for reducing sulphuric acid concentrations in the production of cellulose nanocrystals alongside transition metal co-catalysts

Cellulose nanocrystals (CNC) conventionally involve highly concentrated sulphuric acid, which typically resulted in the formation of undesirable by-products. Although less corrosive mineral acids have been explored as alternatives, high concentrations are still required. In this study, CNC was successfully isolated from Leucaena leucocephala wood using mild sulphuric acid with acetic acid as protic solvent, and it was further studied with the addition of Lewis acids in the form of multivalent transition metal salts as co-catalyst. Selected divalent and trivalent transition metal salts including (Cr(NO3)3, Fe(NO3)3, Co(NO3)2, and Ni(NO3)2) were investigated. The morphology, chemical structure, particle size, and physicochemical properties of the CNCs were determined. Controlled depolymerization of cellulose was observed using transmission electron microscopy (TEM). Rod-like morphology for all CNCs was obtained during the hydrolysis process with the smallest CNC particles found at an average length of 278.1 ± 35.1 nm and a diameter of 13.4 ± 3.0 nm. The results showed that higher valence state metal ions resulted in better cellulose hydrolysis efficiency. In addition, the use of transition metal salt as a co-catalyst improved production efficiency and minimised carbonization of CNC while maintaining desired crystallinity and thermal properties.

The cellulose hydrolysis into glucose with carbon-based solid acid catalyst via machine learning, life cycle assessment and bibliometric analysis

The cellulose hydrolysis into glucose with carbon-based solid acid catalyst contributes to sustainable and low-carbon development. Determining the quantitative impact of key variables on glucose yield and environmental sustainability is a notoriously time-consuming and labor-intensive process. In this study, a bibliometric analysis was carried out to examine the current research on cellulose hydrolysis into glucose using solid acid catalyst. The application of machine learning (ML) has demonstrated that the random forest (RF) model possesses optimal predictive performance concerning the glucose yield (Training R2 = 0.89, Testing R2 = 0.80, MAE = 6.52, RMSE = 8.29). The SO3H acid densities was emerged as a most crucial influential parameter. The interactions relationship of influenced factors for glucose production was clarified by the partial dependence plot analysis (PDPs). Furthermore, the life cycle assessment (LCA) further indicated that the carbon solid acid catalysts used in cellulose hydrolysis to produce glucose have lower emissions of CO2, NOx, and SO2 compared to traditional acid catalysts, which is 85.5 kg ep of CO2, 0.17 kg ep of NOx, 0.23 kg ep of SO2 for carbon solid acid catalysts, while 91.84 kg ep of CO2, 0.24 kg ep of NOx, 0.61 kg ep of SO2 emission for acid hydrolysis. Thus, carbon solid acid catalysts have a comparably lower negative environmental impact than traditional acid hydrolysis. This study provides crucial insights into improving the prediction accuracy of ML models and reducing the carbon footprint involved in glucose production.

Integrating ball milling assisted enzymatic hydrolysis of bamboo cellulose for controllable production of xylo-oligosaccharides, monosaccharides and cellulose nanofibrils

Green bioconversion technology has been regarded as a fundamental solution to energy challenges and environmental issues. During bioconversion, a combination of mechanical and enzymatic treatment on lignocellulosic biomass can not only conformity with environmental concepts but also enable efficient conversion. In this study, a ball milling assisted enzymatic treatment was applied for full utilization of bamboo cellulose. In addition to the production of soluble saccharides, the residues that remained after enzymatic hydrolysis were collected for producing high-quality nanocellulose. Ball milling efficiently broke the hydrogen bonding interactions between glucan chains thus promoting the following xylanase and cellulase conversion. The highest Xylo-oligosaccharides (XOS) yield reached 21.6%. Meanwhile, xylanase facilitated cellulase hydrolysis by separation of hemicellulose on cellulose. With a low commercial cellulase loading of 5 FPU/g glucan, a maximum yield of 76.6% glucose and 53.3% xylose was reached. Finally, nanocellulose was extracted from the enzymatic hydrolyzate residue with a minimum width of 3.5 nm and an average size of 294.8 nm. The present study proposed an efficient combination of mechanical treatment assisted enzymatic digestion, which is not only environmentally friendly but also allows for complete bioconversion of bamboo cellulose.

In situ bifunctional solid acids bearing B–OH and –COOH groups for efficient hydrolysis of cellulose to sugar in a pure aqueous phase

Pyruvaldehyde as a carbon source is synthesized together with boric acid to produce a carbon-based solid catalyst with in situ bifunctional groups, which is utilized to induce the hydrolysis of cellulose to sugar in a pure aqueous phase.

Spatiotemporal dynamics of cellulose during enzymatic hydrolysis studied by infrared spectromicroscopy

Spatial and temporal resolved mapping of infrared spectra of cellulose during enzymatic hydrolysis towards developing sustainable bioconversion processes to materials, fuels, and chemicals.

Recent advances in the processing of Napier grass (Pennisetum purpureum Schumach) as a potential bioenergy crop for bioethanol production

Pennisetum purpureum is an easily cultivated fast-growing perennial grass with great potential as a bioenergy crop due to its high biomass production. Recent advancements in the processing of P. purpureum for the generation of bioethanol are highlighted in this review paper. Various pretreatment methods, namely, enzymatic, acid, and alkaline pretreatment have been investigated to enhance the efficiency of cellulose hydrolysis, which is a crucial step in bioethanol production. Moreover, genetic engineering approaches for enhancing the biomass yield and reducing the lignin are being discussed in this review. The production of bioethanol can be increased by several fermentation processes, including saccharification, separated hydrolysis, simultaneous saccharification, and fermentation. This comprehensive review explores recent advancements in bioethanol production, encompassing technological innovations, diversification of feedstock sources, genetic engineering methodologies, inventive pretreatment techniques, refinements in enzymatic hydrolysis, and enhancements in fermentation, ultimately highlighting the potential of P. purpureum as a sustainable bioethanol source while addressing the challenges and opportunities in its commercial-scale production.

Cloning and characterization of bgl6111 gene encoding β‐glucosidase from bagasse metagenome

β‐Glucosidase (BGL) is an essential enzyme for the hydrolysis of cellulose in industrial processes, but natural BGL enzymes are poorly understood. Metagenomics is a robust tool for bioprospecting in the search for novel enzymes from the entire community’s genomic DNA present in nature. The metagenomics approach simplifies the process of searching for new BGL enzymes by extracting DNA and retrieving its gene information through a series of bioinformatic analyses. In this study, we report the gene cloning, heterologous expression of the bgl6111 gene (accession number MW221260) in Pichia pastoris KM71, and the biochemical characterization of the recombinant enzyme. We successfully identified the bgl6111 sequence of 2,520 bp and 839 amino acids with a molecular size of 89.4 kDa. The amino acid sequence of the bgl6111 gene showed 67.61% similarity to BGL from an uncultured bacterium (ABB51613.1). The BGL product has the highest activity on the third day at 1.210 U/mL, categorized as low production. The enzymatic activity could enhance up to 539.8% of 7.742 U/mL by using the ultrafiltration method. Our findings provide insightful information that bgl6111 obtained from bagasse metagenome could be an alternative candidate for industrial applications in the future.

Ionic-liquid-tolerant enzymes from an adapted Bacillus paralicheniformis for one-pot conversion of lignocellulosic substrates to ethanol

A newly isolated thermophilic cellulase-producing Bacillus paralicheniformis was subjected to adaptive laboratory evolution in the presence of increasing concentration (from 1 to 5% w w−1) of an otherwise toxic ionic liquid (either 1-ethyl-3-methylimidazolium chloride, or 1-butyl-3-methylimidazolium chloride) over 200-days to obtain strains that produced ionic-liquid-stable enzymes. The ability of the ionic-liquid-adapted strains to grow and produce cellulose hydrolyzing enzymes was proven using several different untreated agroindustrial lignocellulosic substrates. The cellulase of the adapted isolate retained more than 99% of its initial activity after incubation (12 h, ∼25 °C) with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([Emim][Ac]; 50% w w−1 concentration of ionic liquid). In contrast, the wildtype cellulase retained < 9% of its initial activity after an equivalent exposure to a lesser concentration (15% w w−1) of the ionic liquid. Using untreated sugarcane bagasse as the substrate, the optimal conditions for producing the ionic-liquid-stable enzymes (cellulase, xylanase, β-glucosidase) by the adapted bacterium were 48 °C and an initial pH ∼6. The ionic-liquid-adapted crude enzymes were used in a one-pot process (at 50 °C, 48 h) to release glucose from the ionic-liquid-pretreated wheat straw (pretreatment at 90 °C, 1 h, using 10% (w w−1) wheat straw in [Emim][Ac]), in the presence of the residual ionic liquid. At 48 h of enzymolysis, the glucose concentration exceeded 50 g L−1. This glucose mixed with the residual ionic liquid was fermented (24 h, 37 °C) to ethanol using the conventional yeast Saccharomyces cerevisiae. The ethanol yield on glucose was nearly 56% of the theoretical maximum, despite the yeast not having been adapted to withstand the toxic ionic liquid.