Thermostable Cellulase Research Articles

Production and diversity analysis of cellulases from Anoxybacillus genus

Abstract. Sanjaya RE, Puspaningsih NNT, Rohman A, Rahmasari H, Illias RM, Jantarit N, Fujiyama K, Pratama A, Khairunnisa F. 2024. Production and diversity analysis of cellulases from Anoxybacillus genus. Biodiversitas 25: 2705-2718. Bioconversion of cellulose into sustainable renewable resources of oligosaccharides and glucose requires cellulase action. Genomic sequences of Anoxybacillus cellulases are available in GenBank databases, but no information about their biochemical properties. Anoxybacillus flavithermus TP-01 is a thermophilic, facultatively anaerobic, cellulase-producing bacterium isolated from the mountain of Gunung Pancar hot springs, Bogor, Indonesia. This study aimed to characterize the cellulase produced by Anoxybacillus flavithermus TP-01, a local Indonesian isolate. This cellulase was characterized and the diversity analysis on biochemical properties and structural characteristics was done by in silico approaches. SOPMA, SWISS-MODEL, I-TASSER, ProSA, and SAVES were used for computational analysis on physicochemical characterization, phylogenetic construction, functional analysis, multiple sequence alignment, and secondary-tertiary structure prediction. Physicochemical analysis showed pI<7, indicating acidic property for these proteins. Furthermore, the proteins were thermostable and hydrophilic, as proved by their relatively high aliphatic index and negative GRAVY values. The five sequence motifs harbored a conserved domain of the M42 peptidase/endoglucanase family. Alpha helix was the predominant secondary structure, and the tertiary structure fulfilled the structural quality criteria of QMEAN4, ERRAT, Ramachandran plot, and Z-score. Structural comparison between the template structure and the models revealed significant differences in the loop sections. This study identified a potentially thermostable cellulase of Anoxybacillus flavithermus TP-01, and provided new insights of the physicochemical, functional, and explores the structure-function relationship of cellulase from Anoxybacillus genus based on in silico data.

Genome sequence of thermophilic cellulolytic actinobacterium Thermobifida fusca strain UPMC 901 producing thermostable cellulases.

Thermophilic cellulolytic bacteria producing thermostable cellulase have multiple potential industrial and biotechnological applications. In this study, we sequenced the genome of Thermobifida fusca UPMC 901 by using nanopore sequencing technology with 100 % genome coverage. The represented genome is 3.76 Mb with a G + C content of 67.4 %. The genome is composed of 1 scaffold with 3207 protein-coding genes. The results indicated that the genome of the Thermobifida fusca gene encodes at least 112 genes for carbohydrate-active enzymes. Of these genes, 40 glycoside hydrolases (GHs), 36 glycosyl transferases (GTs), 22 carbohydrate-binding modules (CBMs), 9 carbohydrate esterases (CEs), 3 polysaccharide lyases (PL) and 2 auxiliary activity (AAs) which refer to as lytic polysaccharide monooxygenase (LPMO), gene encoding the copper-dependent enzymes that catalyse oxidative cleavage of recalcitrant crystalline cellulose, were identified. In addition, two new functional genes, amylo-α-1,6 glucosidase and endo-α-1,4-mannosidase were also detected in the T. fusca genome. This study represented the ninth genome of the T. fusca and the cellulase genes identified will facilitate the exploration of potential sources for industrial and agricultural applications.

Characterization of Thermostable Cellulase Enzyme Isolated from a Hot Spring Bacterium: Bacillus sp.

Cellulase is a complex of enzymes which consists of β-1,4-endoglucanase, cellobiohydrolase, and β-glucosidase. Cellulase contributes a significant share to the world enzyme market and is used in number of industries viz; paper and pulp, food and beverage, bioethanol, detergent and textile. The harsh industrial conditions such as high temperature, extreme pH levels and high substrate concentration etc. which is used in such industries adversely effect on structure and activity of enzymes. Therefore, huge amount of chemical catalysts which cause chemical wastes are used in industrial settings instead of enzymes. Since the chemical wastes adversely affect the ecosystem, isolation and characterization of thermostable cellulase enzyme-producing bacteria and exploring the industrial perspective of thermostable enzymes is a better approach towards green chemistry. The present study focused on characterization of cellulase enzyme produced by Bacillus sp., which shows optimum activity at 60 C under neutral conditions, isolated from Gomarankadavala hot spring in Sri Lanka. The effect of different Carbon sources: glucose and lactose, Nitrogen sources: peptone, tryptone, yeast extract and urea on enzyme production and different Carboxymethylcellulose (CMC) concentrations; 0.5%, 1.5% and 2%, on enzyme activity were measured using the DNS method. Under the optimum conditions, cellulase enzyme activity on different substrates: corn cob, rice bran and raw leaves were measured. The highest enzyme production was recorded in the culture medium which added tryptone as the nitrogen source and adding carbon sources to the culture medium showed an increase of cellulase enzyme production. The optimum CMC concentration for the enzyme activity was recorded as 1% and from all the optimized parameters, the substrate; raw leaves showed the highest enzyme activity of 11.305 U/ml. Further, a considerable amount of enzyme activity was recorded on corn cobs and rice bran as well. Thus, the thermostable cellulase enzyme produced by bacterium identified as Bacillus cereus isolated from Gomarankadavala hot spring could be successfully used in industrial settings such as kitchen/industrial waste management, bioethanol production, paper and pulp industry and textile industry which use high temperatures and cellulosic substrates as raw materials. 
 Keywords: Thermostable enzymes, Cellulase, Industrial applications, Hot springs

Enhancing Broiler Chick Growth with Locally Produced Thermostable Cellulase in Fibrous Diets

Enhancing Broiler Chick Growth with Locally Produced Thermostable Cellulase in Fibrous Diets

Enhancement effect of AgO nanoparticles on fermentative cellulase activity from thermophilic Bacillus subtilis Ag-PQ

BackgroundCellulase is an important bioprocessing enzyme used in various industries. This study was conducted with the aim of improving the biodegradation activity of cellulase obtained from the Bacillus subtilis AG-PQ strain. For this purpose, AgO and FeO NPs were fabricated using AgNO3 and FeSO4·7H2O salt respectively through a hydro-thermal method based on five major steps; selection of research-grade materials, optimization of temperature, pH, centrifuge, sample washed with distilled water, dry completely in the oven at the optimized temperature and finally ground for characterization. The synthesized NPs were characterized by scanning electron microscope (SEM), energy dispersive X-ray (EDX), and X-ray diffraction (XRD) to confirm the morphology, elemental composition, and structure of the sample respectively. The diameter of the NPs was recorded through SEM which lay in the range of 70–95 nm. ResultsCultural parameters were optimized to achieve better cellulase production, where incubation time of 56 h, inoculum size of 5%, 1% coconut cake, 0.43% ammonium nitrate, pH 8, and 37 °C temperature were found optimal. The enhancing effect of AgO NPs was observed on cellulase activity (57.804 U/ml/min) at 50 ppm concentration while FeO NPs exhibited an inhibitory effect on cellulase activity at all concentrations. Molecular docking analysis was also performed to understand the underlying mechanism of improved enzymatic activity by nanocatalysts. ConclusionThis study authenticates AgO NPs as better nanocatalysts for improved thermostable cellulase biodegradation activity with the extraordinary capability to be potentially utilized in bioethanol production. Graphical Abstract▪

Isolation, Identification and Assessment of Efficient Cellulase Producing Bacteria from the Termite Guts

The present study is concerned with the screening the cellulase-producing bacteria from termite gut, assessed potential cellulase-producing bacteria and partial characterization (optimum parameters) of cellulase from isolated bacteria. The result showed that 15 out of 48 isolated strains was positive for degrading the carboxy methyl cellulose (CMC) in agar by congo-red method. After screening by DNS assay, three selected bacteria exhibited high cellulase activity that were identified as Citrobacter amalonaticus CM 1-3, Bacillus cereus CM 5-1 and Streptococcus salivarius CE 5-1 using 16S rRNA sequence analysis. All bacterial strains utilized CMC and showed the highest cellulase activity. Cellulase characterization of C. amalonaticus CM 1-3 and S. salivarius CE 5-1 was revealed optimum activity at 35°C, pH 7.0 and for 48 h. Bacillus cereus CM 5-1 represented its potential use in industrial processes due to thermostable cellulase production. The crude cellulase of this strain was purified by (NH4)2SO4 precipitation with 1.58 purification fold and 74.38% overall recovery. The optimal temperature and pH for cellulase activity of B. cereus CM 5-1 were at 40°C and pH 7.0. Thus, this study provided additional information about the diversity and partial characteristic cellulase of cellulolytic bacteria from termite gut for future industrial applications.

Investigation of physical, nutritional and sensory properties of wheat bread treated with purified thermostable cellulase and alpha amylase

Investigation of physical, nutritional and sensory properties of wheat bread treated with purified thermostable cellulase and alpha amylase

CRISPR/Cas9 mediated gene editing of transcription factor ACE1 for enhanced cellulase production in thermophilic fungus Rasamsonia emersonii

BackgroundThe filamentous fungus Rasamsonia emersonii has immense potential to produce biorefinery relevant thermostable cellulase and hemicellulase enzymes using lignocellulosic biomass. Previously in our lab, a hyper-cellulase producing strain of R. emersonii was developed through classical breeding and system biology approaches. ACE1, a pivotal transcription factor in fungi, plays a crucial role in negatively regulating the expression of cellulase genes. In order to identify the role of ACE1 in cellulase production and to further improve the lignocellulolytic enzyme production in R. emersonii, CRISPR/Cas9 mediated disruption of ACE1 gene was employed.ResultsA gene-edited ∆ACE1 strain (GN11) was created, that showed 21.97, 20.70 and 24.63, 9.42, 18.12%, improved endoglucanase, cellobiohydrolase (CBHI), β-glucosidase, FPase, and xylanase, activities, respectively, as compared to parental strain M36. The transcriptional profiling showed that the expression of global regulator (XlnR) and different CAZymes genes including endoglucanases, cellobiohydrolase, β-xylosidase, xylanase, β-glucosidase and lytic polysaccharide mono-oxygenases (LPMOs) were significantly enhanced, suggesting critical roles of ACE1 in negatively regulating the expression of various key genes associated with cellulase production in R. emersonii. Whereas, the disruption of ACE1 significantly down-regulated the expression of CreA repressor gene as also evidenced by 2-deoxyglucose (2-DG) resistance phenotype exhibited by edited strain GN11 as well as appreciably higher constitutive production of cellulases in the presence of glucose and mixture of glucose and disaccharide (MGDs) both in batch and flask fed batch mode of culturing. Furthermore, ∆ACE1 strains were evaluated for the hydrolysis of biorefinery relevant steam/acid pretreated unwashed rice straw slurry (Praj Industries Ltd; 15% substrate loading rate) and were found to be significantly superior when compared to the benchmark enzymes produced by parent strain M36 and Cellic Ctec3.ConclusionsCurrent work uncovers the crucial role of ACE1 in regulating the expression of the various cellulase genes and carbon catabolite repression mechanism in R. emersonii. This study represents the first successful report of utilizing CRISPR/Cas9 genome editing technology to disrupt the ACE1 gene in the thermophlic fungus R. emersonii. The improved methodologies presented in this work might be applied to other commercially important fungal strains for which genetic manipulation tools are limited.

Characterization of Thermostable Cellulase from Bacillus licheniformis PANG L Isolated from the Himalayan Soil.

This study aimed to isolate, purify, and characterize a potential thermophilic cellulase-producing bacterium from the Himalayan soil. Eleven thermophilic bacteria were isolated, and the strain PANG L was found to be the most potent cellulolytic producer. Morphological, physiological, biochemical, and molecular characterization identified PANG L as Bacillus licheniformis. This is the first study on the isolation of thermostable cellulase-producing Bacillus licheniformis from the Himalayan soil. This bacterium was processed for the production of cellulase enzyme. The optimum conditions for cellulase production were achieved at 45°C after 48 h of incubation at pH 6.5 in media-containing carboxymethyl cellulose (CMC) and yeast extract as carbon and nitrogen sources, respectively, in a thermo-shaker at 100 rpm. The enzyme was partially purified by 80% ammonium sulphate precipitation followed by dialysis, resulting in a 1.52-fold purification. The optimal activity of partially purified cellulase was observed at a temperature of 60°C and pH 5. The cellulase enzyme was stable within the pH ranges of 3-5 and retained 67% of activity even at 55°C. Cellulase activity was found to be enhanced in the presence of metal ions such as Cd2+, Pb2+, and Ba2+. The enzyme showed the highest activity when CMC was used as a substrate, followed by cellobiose. The Km and Vmax values of the enzyme were 1.8 mg/ml and 10.92 μg/ml/min, respectively. The cellulase enzyme obtained from Bacillus licheniformis PANG L had suitable catalytic properties for use in industrial applications.

Testing and improving the performance of protein thermostability predictors for the engineering of cellulases.

Thermostability of cellulases can be increased through amino acid substitutions and by protein engineering with predictors of protein thermostability. We have carried out a systematic analysis of the performance of 18 predictors for the engineering of cellulases. The predictors were PoPMuSiC, HoTMuSiC, I-Mutant 2.0, I-Mutant Suite, PremPS, Hotspot, Maestroweb, DynaMut, ENCoM ([Formula: see text] and [Formula: see text], mCSM, SDM, DUET, RosettaDesign, Cupsat (thermal and denaturant approaches), ConSurf, and Voronoia. The highest values of accuracy, F-measure, and MCC were obtained for DynaMut, SDM, RosettaDesign, and PremPS.A combination of the predictors provided an improvement in the performance. F-measure and MCC were improved by 14% and 28%, respectively. Accuracy and sensitivity were also improved by 9% and 20%, respectively, compared to the maximal values of single predictors. The reported values of the performance of the predictors and their combination may aid research in the engineering of thermostable cellulases as well as the further development of thermostability predictors.

Biological pretreatment of sugarcane bagasse for the production of fungal laccase and bacterial cellulase.

Sugarcane bagasse (SB) is a promising source of appreciable quantities of fermentable sugars. However, the presence of lignin hinders utilization of these carbohydrates and hence pretreatment to remove lignin is necessarily carried out. Here, a biological pretreatment method was synchronized with the production of a thermostable cellulase using SB as a raw material. Initially, bagasse was fermented by a laccase producing fungus, Trametes pubescens MB 89 under solid state fermentation (SSF) and a titer of 1758 IU mL-1 of laccase was obtained. Investigations of nine factors affecting laccase production through Plackett Burman design improved the titers to 6539 IU mL-1 . Five factors (incubation period, concentration of CuSO4 , temperature, moisture content, and particle size) were found significant which were optimized through Central Composite design leading to an improvement in the titers by ~5 folds (8841 IU mL-1 ). Biologically pretreated SB was fermented by a thermophilic bacterium, Neobacillus sedimentimangrovi UE25, that yielded 8.64 IU mL-1 of cellulase. Delignification and cellulose utilization were affirmed by structural analysis through FTIR and SEM. The synchronized process yielded higher titers of laccase and cellulase under SSF of SB with the minimum use of corrosive chemicals.

Effect of pretreatment strategies on halophyte Atriplex crassifolia to improve saccharification using thermostable cellulases.

Bioethanol is believed to be an influential revolutionary gift of biotechnology, owing to its elevating global demand and massive production. Pakistan is home to a rich diversity of halophytic flora, convertible into bounteous volumes of bioethanol. On the other hand, the accessibility to the cellulosic part of biomass is a major bottleneck in the successful application of biorefinery processes. The most common pre-treatment procedures existent include physicochemical and chemical approaches, which are not environmentally benign. To overcome these problems, biological pre-treatment has gained importance but the drawback is the low yield of the extracted monosaccharides. The current research was aimed at exploring the best pre-treatment method for the bioconversion of halophyte Atriplex crassifolia into saccharides using three thermostable cellulases. Atriplex crassifolia was subjected to acid, alkali and microwave pre-treatments, followed by compositional analysis of the pre-treated substrates. Maximum delignification i.e. 56.6% was observed in the substrate pre-treated using 3% HCl. Enzymatic saccharification using thermostable cellulases also validated the results where the highest saccharification yield i.e. 39.5% was observed for the sample pre-treated using same. Maximum enzymatic hydrolysis of 52.7% was obtained for 0.40g of the pre-treated halophyte Atriplex crassifolia where Endo-1,4- -glucanase (300U), Exo-1,4- -glucanase (400U) and -1,4-glucosidase (1000U) were simultaneously added and incubated for 6h at 75°C. The reducing sugar slurry obtained after optimization of saccharification was utilized as glucose in submerged fermentation for bioethanol production. The fermentation medium was inoculated with Saccharomyces cerevisiae, incubated at 30°C and 180rpm for 96h. Ethanol production was estimated using potassium dichromate method. Maximum production of bioethanol i.e. 16.33% was noted at 72h. It can be concluded from the study that Atriplex crassifolia owing to its high cellulosic content after pre-treatment using dilute acid method, yields substantial amount of reducing sugars and high saccharification rates when subjected to enzymatic hydrolysis using thermostable cellulases, under optimized reaction conditions. Hence, the halophyte Atriplex crassifolia is a beneficial substrate that can be utilized to extract fermentable saccharides for bioethanol production.

Characterization of a novel end product tolerant and thermostable cellulase from Neobacillus sedimentimangrovi UE25

Recent advancements in biorefinery processes necessitate search for cost effective and thermostable cellulases. This study was designed to characterize the cellulase obtained from a thermophilic bacterium, Neobacillus sedimentimangrovi UE25. A combined pretreatment of NaOH and methyltrioctylammonium chloride was given to sugarcane bagasse (SB) for lignin removal and the pretreated SB was utilized as a carbon source for the cellulase production. The thermostable cellulase thus obtained was characterized by adopting central composite design which has not been reported earlier for this purpose. Cellulase showed its maximum activity at pH 7 and temperature 60 ℃ and it remained active in the presence of many salts and detergents. Endoglucanase (EG) was found to be stable for 30 min at 80 ℃. The purification of EG by using DEAE column yielded specific activity and purification fold of 365.866 IU mg−1 and 4.264, respectively. The purified EG had a molecular weight of ∼45 kDa. End product tolerance of EG was also evident, as an activity of 228.57 IU mL−1 was observed in the presence of 60 mM glucose which revealed that it does not lose its activity upon accumulation of end-product when the reaction is prolonged. The purified EG exhibited Vmax and Km of 294 U mL−1 min−1 and 36 µM, respectively, in the presence of 60 mM glucose. This novel thermostable cellulase can finds its applications in industrial sector.

Isolation and characterization of thermostable and alkali-tolerant cellulase from litter endophytic fungus Bartalinia pondoensis.

Endophytic fungi in plant tissues produce a wide range of secondary metabolites and enzymes, which exhibit a variety of biological activities. In the present study, litter endophytic fungi were isolated from a fire-prone forest and screened for thermostable cellulases. Among nine endophytic fungi tested, two isolates, Bartalinia pondoensis and Phoma sp., showed the maximum cellulase activity. Bartalinia pondoensis was further selected for its cellulase production and characterization. Among the carbon and nitrogen sources tested, maximum cellulase production was observed with maltose and yeast extract, and the eucalyptus leaves and rice bran served as the best natural substrates. The cellulase activity increased with increasing temperature, with maximum activity recorded at 100°C. The maximum CMCase activity was observed between pH 6.0 and 7.0 and retained 80% of its activity in the pH range of 8-10. Partially purified cellulase of B. pondoensis retained 50% of its activity after 2h of incubation at 60°C, 80°C and 100°C. These results suggest that litter endophytic fungus B. pondoensis is a potential source for the production of thermostable and alkali-tolerant cellulase.

Sustainable Production of Bioethanol Using Levulinic Acid Pretreated Sawdust.

The sustainability and economic viability of the bioethanol production process from lignocellulosic biomass depend on efficient and effective pretreatment of biomass. Traditional pretreatment strategies implicating the use of mineral acids, alkalis, and organic solvents release toxic effluents and the formation of inhibitory compounds posing detrimental effects on the environment and interfering with the enzymatic saccharification process, respectively. Ionic liquids (ILs) as green solvents were used to overcome this issue, but the deep eutectic solvent as an emerging class of ionic liquids performed better in terms of making the process environmentally and economically viable. The green solvent-based pretreatment strategy applied in the current research was levulinic, acid-based natural deep eutectic solvent (NADES). Three different hydrogen bond acceptors (HBAs)—acetamide, betaine, and choline chloride—in combination with levulinic acid as hydrogen bond donor (HBD) in (HBD: HBA) molar ratio 2:1, were screened for biomass pretreatment. The best deep eutectic solvent was levulinic acid: choline chloride in an optimized molar ratio of 1:0.5, resulting in 91% delignification. The physicochemical parametric optimization of saccharification exhibited maximum enzymatic hydrolysis of 25.87% with 125 mg of pretreated sawdust via simultaneous addition of three thermostable cellulases [i.e., endo-1,4-β-D-glucanase (240 U), exo-1,4-β-D-glucanase (180 U), and β-glucosidase (320 U)] for 5 h of incubation at 75°C. The reducing sugar slurry obtained from the saccharified biomass was then added to a fermentation medium for bioethanol production, and a maximum of 11.82% of production was obtained at 30°C, 72 h, and 180 rpm using a 2.5% 24 h old Saccharomyces cerevisiae seed culture. The current study revealed that the levulinic-based deep eutectic solvent exhibited remarkable delignification, which led to the efficient enzymatic hydrolysis of sawdust and hence bioethanol production. Furthermore, it will prospect new avenues in bioethanol production using a deep eutectic solvent. Deep eutectic solvent overcame the issues posed by ionic liquids: toxicity, expensive and complex preparation, and non-biodegradability.

Production Optimization and Biochemical Characterization of Cellulase from Geobacillus sp. KP43 Isolated from Hot Spring Water of Nepal.

This study is aimed at isolating and identifying a thermophilic cellulolytic bacterium from hot spring water and characterizing thermostable cellulase produced by the isolate. Enrichment and culture of water sample was used for isolation of bacterial strains and an isolate with highest cellulase activity was chosen for the production, partial purification, and biochemical characterization of the enzyme. Different staining techniques, enzymatic tests, and 16s ribosomal DNA (16s rDNA) gene sequencing were used for the identification of the isolate. The cellulase producing isolate was Gram positive, motile, and sporulated rod-shaped bacterium growing optimally between 55°C and 65°C. Based on partial 16s rDNA sequence analysis, the isolate was identified as Geobacillus sp. and was designated as Geobacillus sp. KP43. The cellulase enzyme production condition was optimized, and the product was partially purified and biochemically characterized. Optimum cellulase production was observed in 1% carboxymethyl cellulose (CMC) at 55°C. The molecular weight of the enzyme was found to be approximately 66 kDa on 12% SDS-PAGE analysis. Biochemical characterization of partially purified enzyme revealed the temperature optimum of 70°C and activity over a wide pH range. Further, cellulase activity was markedly stimulated by metal ion Fe2+. Apart from cellulases, the isolate also depicted good xylanase, cellobiase, and amylase activities. Thermophilic growth with a variety of extracellular enzyme activities at elevated temperature as well as in a wide pH range showed that the isolated bacteria, Geobacillus sp. KP43, can withstand the harsh environmental condition, which makes this organism suitable for enzyme production for various biotechnological and industrial applications.

Thermostable Cellulases / Xylanases From Thermophilic and Hyperthermophilic Microorganisms: Current Perspective.

The bioconversion of lignocellulose into monosaccharides is critical for ensuring the continual manufacturing of biofuels and value-added bioproducts. Enzymatic degradation, which has a high yield, low energy consumption, and enhanced selectivity, could be the most efficient and environmentally friendly technique for converting complex lignocellulose polymers to fermentable monosaccharides, and it is expected to make cellulases and xylanases the most demanded industrial enzymes. The widespread nature of thermophilic microorganisms allows them to proliferate on a variety of substrates and release substantial quantities of cellulases and xylanases, which makes them a great source of thermostable enzymes. The most significant breakthrough of lignocellulolytic enzymes lies in lignocellulose-deconstruction by enzymatic depolymerization of holocellulose into simple monosaccharides. However, commercially valuable thermostable cellulases and xylanases are challenging to produce in high enough quantities. Thus, the present review aims at giving an overview of the most recent thermostable cellulases and xylanases isolated from thermophilic and hyperthermophilic microbes. The emphasis is on recent advancements in manufacturing these enzymes in other mesophilic host and enhancement of catalytic activity as well as thermostability of thermophilic cellulases and xylanases, using genetic engineering as a promising and efficient technology for its economic production. Additionally, the biotechnological applications of thermostable cellulases and xylanases of thermophiles were also discussed.

Screening, cloning, enzymatic properties of a novel thermostable cellulase enzyme, and its potential application on water hyacinth utilization.

Cellulose is the cheapest, natural, renewable organic substance that is used as a carbon source in various fields. Water hyacinth, an aquatic plant rich in cellulose, is often used as a raw material in fuel production. However, natural cellulase can be hardly used in industrial production on account of its low thermal stability and activity. In this study, a metagenomic library was constructed. Then, a new cellulase gene, cel1029, was screened by Congo red staining and expressed in the prokaryotic system. Enzymatic properties of Cel1029 were explored, including optimum temperature and pH, thermal and pH stability, and tolerance against organic solvents, metal ions, and salt solutions. Finally, its ability of degrading water hyacinth was identified and evaluated. Cel1029 displayed high homology with endoglucanase in the glycoside hydrolase family 5 (GH5) and had high stability across a broad temperature range. More than 86% of its enzymatic activities were retained between 4 and 60 °C after 24 h of incubation. Single-factor analysis and orthogonal design were further conducted to determine the optimal conditions for the highest reducing sugar yield of water hyacinth. Interestingly, Cel1029 efficiently transformed water hyacinth with a reducing sugar yield of 430.39 mg/g in 22 h. These findings may open the door for significant industrial applications of a novel GH5 cellulase (NCBI Reference Sequence: MK051001, Cel1029) and help identify more efficient methods to degrade cellulose-rich plants.

Isolation, Biochemical Characterisation and Identification of Thermotolerant and Cellulolytic Paenibacillus lactis and Bacillus licheniformis.

SUMMARYResearch backgroundCellulose is an ingredient of waste materials that can be converted to other valuable substances. This is possible provided that the polymer molecule is degraded to smaller particles and used as a carbon source by microorganisms. Because of the frequently applied methods of pretreatment of lignocellulosic materials, the cellulases derived from thermophilic microorganisms are particularly desirable.Experimental approachWe were looking for cellulolytic microorganisms able to grow at 50 °C and we described their morphological features and biochemical characteristics based on carboxymethyl cellulase (CMCase) activity and the API® ZYM system. The growth curves during incubation at 50 °C were examined using the BioLector® microbioreactor.Results and conclusionsForty bacterial strains were isolated from fermenting hay, geothermal karst spring, hot spring and geothermal pond at 50 °C. The vast majority of the bacteria were Gram-positive and rod-shaped with the maximum growth temperature of at least 50 °C. We also demonstrated a large diversity of biochemical characteristics among the microorganisms. The CMCase activity was confirmed in 27 strains. Hydrolysis capacities were significant in bacterial strains: BBLN1, BSO6, BSO10, BSO13 and BSO14, and reached 2.74, 1.62, 1.30, 1.38 and 8.02 respectively. Rapid and stable growth was observed, among others, for BBLN1, BSO10, BSO13 and BSO14. The strains fulfilled the selection conditions and were identified based on the 16S rDNA sequences. BBLN1, BSO10, BSO13 were classified as Bacillus licheniformis, whereas BSO14 as Paenibacillus lactis.Novelty and scientific contributionWe described cellulolytic activity and biochemical characteristics of many bacteria isolated from hot environments. We are also the first to report the cellulolytic activity of thermotolerant P. lactis. Described strains can be a source of new thermostable cellulases, which are extremely desirable in various branches of circular bioeconomy.

Erratum to "Thermostable cellulase biosynthesis from Paenibacillus alvei and its utilization in lactic acid production by simultaneous saccharification and fermentation".

[This corrects the article DOI: 10.1515/biol-2020-0019.].