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Cellulase is essential for cellulose hydrolysis and is used across industries such as food, feed, textiles, biofuel, and biomass pretreatment. However, its utility is restricted by high temperatures and salt concentrations. This study identified a novel cellulase gene (named c5-cel4 ) from Ebinur Salt Lake in Xinjiang, China using metagenomic technology. Its amino acid sequence has a 90.97% similarity to the GH5 family cellulase of Microbulbifer litoralis (WP_250463697.1). The gene was expressed in Escherichia coli , and the recombinant protein, C5-CEL4, was purified via Ni-NTA affinity chromatography. C5-CEL4, secreted extracellularly (0.886 U/mL), revealed a protein size 14 KDa smaller than predicted, with mass spectrometry indicating a truncated C-terminal of 135 amino acid residues. Optimal activity was observed at 50 °C and pH 7.0, maintaining over 80% activity at 40–65 °C and 45–50 °C for 2 h. The enzyme’s half-life was 60 min at 55–60 °C, retaining over 90% activity after 24 h in pH 5.0–12.0 buffers. C5-CEL4 showed activity against CMC-Na, bagasse xylan, and beech xylan, with Kcat values of 98.20 s − 1 and 12.32 s − 1 for CMC-Na and bagasse xylan, respectively. It also hydrolyzed wheat bran and maize stalks into reducing sugars. Remarkably, C5-CEL4 exhibited high salt tolerance, maintaining 100% activity in 0.5 M-5.0 M NaCl and after 9 months in 5.0 M NaCl. It retained over 90% activity in ionic liquids (BMIM-Ac, EMIM-Cl, BMIM-BF4) at 50% concentration and showed resistance to heavy metal ions (Co 2+ , Cu 2+ , Ag + , Mn 2+ , Pb 2+ , and Ni 2+ ) and inhibitors (PMSF, DTT, Tween80, and SDS). In conclusion, C5-CEL4 is a robust cellulase with heat, alkali, salt, ionic liquid, and inhibitor resistance, alongside cellulase and xylanase activity, presenting significant potential for feed, food, and bioenergy applications.

The present study involved a global search of the Aspergillus oryzae genome database. As a result, the gene (gene ID: AO090011000715 ) encoding a putative endo-β-1,4-glucanase of the glycoside hydrolase family 5 possessing a carbohydrate-binding module was identified. Overexpression of this gene in A. oryzae , followed by the purification of the gene product, revealed that the protein exhibits endoglucanase activity with carboxymethyl cellulose as a substrate. Notably, further enzymatic characterization showed that this enzyme could also hydrolyze glucomannan and galactomannan with relatively lower activity than that of endoglucanase, indicating that the enzyme is likely a bifunctional endoglucanase/mannanase. Detailed analyses of the substrate specificity revealed that the enzyme could hydrolyze cello-oligosaccharides larger than cellotetraose, whereas manno-oligosaccharides could not be hydrolyzed unless they were larger than mannopentaose. Therefore, this enzyme has different modes of action toward cello- and manno-oligosaccharides. To the best of our knowledge, this is the first study to purify and characterize a bifunctional endo-β-1,4-glucanase/mannanase from the Aspergillus genus. The ability of the enzyme to degrade both mannan and cellulose could be used for more efficient degradation of lignocellulosic biomass, indicating that the enzyme shows promise for applications in biomass utilization and the food industry. Key points • A novel bifunctional endoglucanase/mannanase, EgmA, is identified from Aspergillus oryzae. • EgmA is the second bifunctional endo-β-1,4-glucanase/mannanase of fungal origin. • EgmA has different mechanisms of action toward cello- and manno-oligosaccharides.

The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.

Objectives This study was conducted to isolate, screening and purification of cellulase from bacteria present in sugar industry waste (molasses) and characterization by morphological and biochemical analysis. Results Based on experiments, three bacterial strains produced clear transparent zone into carboxymethyl cellulose (CMC) agar plate were identified as cellulase producing bacteria. Different culture parameters such as pH, temperature, incubation period, substrate concentration and carbon sources were optimized for enzyme production. According to the morphological and biochemical tests, the isolated strains were identified as Paenibacillus sp., Bacillus sp. and Aeromonas sp. The first strain Paenibacillus sp. showed high potentiality for maximum cellulase production (0.9 µmol ml −1  min −1 ) at pH 7.0 after 24 h of incubation at 40 °C in a medium containing 1.0% CMC. Then Paenibacillus sp. was selected for enzyme purification by ammonium sulfate precipitation, DEAE-cellulose and CM-cellulose column chromatography, respectively. In last step of purification, specific activity, recovery and purification fold were 2655 U/mg, 35.7% and 9.7, respectively. The molecular weight of the purified cellulase was found to be 67 kDa by SDS-PAGE, had an optimal pH and temperature at 7.0 and 40 °C. According to substrate specificity, the purified cellulase had high specificity on CMC substrate which indicated it to be an endo-β-1,4-glucanase.

Endoglucanase producing bacteria were isolated from Egyptian soils and the most active bacterial strain was identified as Bacillus subtilis strain Fatma/1. Plackett–Burman statistical design was carried out to assess the effect of seven process variables on endoglucanase production. Carboxymethyl cellulose (CMC), yeast extract and peptone were the most significant variables that enhanced the endoglucanase production and thus were selected for further optimization using face-centered central composite design. The highest yield of endoglucanase (32.37 U/mL) was obtained in run no. 9, using 18 g/L CMC, 8 g/L peptone, 7 g/L yeast extract and 0.1 g/L FeSO 4 .7H 2 O. The optimized medium showed about eightfold increase in endoglucanase production compared to the unoptimized medium. The produced crude enzyme was further purified by ammonium sulfate precipitation, then DEAE-Sepharose CL6B column. The purified enzyme was shown to have a molecular weight of 37 kDa. The enzyme showed maximum activity at pH 8.0, temperature of 50 °C, incubation time of 60 min. The half-life time (T 1/2 ) was 139.53 min at 50 °C, while being 82.67 min at 60 °C. Endoglucanase at concentration of 12 U/mL effectively removed 84.61% of biofilm matrix of Pseudomonas aeruginosa with marked reduction in carbohydrate content of the biofilm from 63.4 to 7.9 μg.

Most fungi and bacteria degrade plant cell walls by secreting free, complementary enzymes that hydrolyze cellulose; however, some bacteria use large enzymatic assemblies called cellulosomes, which recruit complementary enzymes to protein scaffolds. The thermophilic bacterium Caldkellulosiruptor besdi uses an intermediate strategy, secreting many free cellulases that contain multiple catalytic domains. One of these, CelA, comprises a glycoside hydrolase family 9 and a family 48 catalytic domain, as well as three type III cellulose-binding modules. In the saccharification of a common cellulose standard, Avicel, CelA outperforms mixtures of commercially relevant exo-and endoglucanases. From transmission electron microscopy studies of cellulose after incubation with CelA, we report morphological features that suggest that CelA not only exploits the common surface ablation mechanism driven by general cellulase processivity, but also excavates extensive cavities into the surface of the substrate. These results suggest that nature's repertoire of cellulose digestion paradigms remain only partially discovered and understood.

The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. We developed a systems-level approach that integrates transcriptomic sequencing, proteomics, phenotype, and biochemical studies of relatively unexplored basal fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, untreated plant biomass and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite-repressed and are further regulated by a rich landscape of noncoding regulatory RNAs. Additionally, we identified several promising sequence-divergent enzyme candidates for lignocellulosic bioprocessing.

The present study focuses on the isolation and characterization of a bacterium from Lake Van adapted to an alkaline pH environment with significant cellulase production potential. The identified isolate, Bacillus pumilus VLC7 (GenBank Acc No: OR415888.1), exhibited the highest cellulase activity among other alkaliphilic isolates. The purification employing protein precipitation by ammonium sulfate, ultrafiltration, and ion exchange chromatography resulted in 20-fold purification with a specific activity of 16 U/mg protein. Cellulase had a molecular weight of 76 kDa, 3.13 mM K m and 0.160 U/mg V max values. The enzyme displayed maximum activity at pH 9.0 and 40 °C and retained at least 80% of its activity at temperatures of 25–60 °C for 90 min and pH 4–12 for one hour. Stability tests also revealed the enzyme’s resilience to various reagents, metal ions, organic solvents, and detergents. Furthermore, the biotechnological applications of cellulase were explored, demonstrating its effectiveness in fabric biopolishing (removing pilling), as well as in the removal of fabric dyes. The research findings underscore the potential of B. pumilus VLC7 as a valuable source for eco-friendly industrial biotechnology applications. Graphical Abstract

Background The rising concerns about the scarcity of fossil fuels, the emission of green house gasses and air pollution by incomplete combustion of fossil fuel have also resulted in an increasing focus on the use of cellulases to perform enzymatic hydrolysis of the lignocellulosic materials for the generation of bioethanol. The aim of this study was to isolate a potential thermo-solvent tolerant cellulase producing bacterium from natural resources, and then applied for purification and characterization. The purified enzyme was to be accessible for the bioethanol production as well as industrial exploitation (discuss in our next study). Results It is the first instance when thermo-solvent tolerant cellulase producing bacterium was isolated from soil sample. The culture was identified as Bacillus vallismortis RG-07 by 16S rDNA sequence analysis. Bacillus vallismortis RG-07 reported maximum cellulase production from sugarcane baggase (4105 U ml −1 ) used as agro-waste carbon source. The cellulase enzyme produced by the Bacillus sp. was purified by (NH 4 ) 2 SO 4 precipitation, ion exchange and gel filtration chromatography, with overall recovery of 28.8%. The molecular weight of purified cellulase was 80 kDa as revealed by SDS-PAGE and activity gel analysis. The optimum temperature and pH for enzyme activity was determined as 65°C and 7.0 and it retained 95 and 75% of activity even at 95°C, and 9.0 respectively. The enzyme activity was enhanced in the presence of organic solvents (30%) n-dodecane, iso-octane, n-decane, xylene, toluene, n-haxane, n-butanol, and cyclohexane, after prolonged incubation (7 days). The enzyme activity was also stimulated by Ca 2+ , mercaptoethanol, Tween-60, and Sodium hypochloride whereas strongly inhibited by Hg. Kinetic analysis of purified enzyme showed the K m and V max to be 1.923 mg ml −1 and 769.230 μg ml −1  min −1 , respectively. Conclusion The unique property of solvent-thermostable-alkalophilic, nature proves the potential candidature of this isolate for current mainstream biomass conversion into fuel and other industrial process.

A themophilic cellulase-producing bacterium was isolated from a hot spring district and identified as Geobacillus sp. HTA426. The cellulase enzyme produced by the Geobacillus sp. HTA426 was purified through ammonium sulfate precipitation and ion exchange chromatography, with the recovery yield and fold purification of 10.14% and 5.12, respectively. The purified cellulase has a molecular weight of 40 kDa. The optimum temperature and pH for carboxymethyl cellulase (CMCase) activity of the purified cellulase were 60°C and pH 7.0, respectively. The enzyme was also stable over a wide temperature range of 50°C to 70°C after 5 h of incubation. Moreover, the strain HTA426 was able to grow and produce cellulase on alkali-treated sugarcane bagasse, rice straw and water hyacinth as carbon sources. Enzymatic hydrolysis of sugarcane bagasse, which was regarded as the most effective carbon source for cellulase production (CMCase activity = 103.67 U/mL), followed by rice straw (74.70 U/mL) and water hyacinth (51.10 U/mL). This strain producing an efficient thermostable cellulose is a potential candidate for developing a more efficient and cost-effective process for converting lignocellulosic biomass into biofuel and other industrial process.