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Vermicomposting is a promising method for corn stover management to achieve bioresource recovery and environmental protection. Most β-glucosidases, which limit the cellulose degradation rate during vermicomposting of corn stover, belong to glycoside hydrolase family 1 (GH 1 ). This study was conducted with different earthworm densities to quantify the GH 1 gene abundance and investigate the evolution of GH 1 cellulase-producing microbial communities using qPCR and pyrosequencing. The results showed that β-glucosidase activity, GH 1 gene abundance, TOC, and microbial communities carrying the GH 1 gene were affected by processing time and earthworm density. After introducing earthworms, β-glucosidase activity increased to 1.90-2.13 U/g from 0.54 U/g. The GH 1 gene abundance of treatments with earthworms (5.82E+09-6.70E+09 copies/g) was significantly higher than that of treatments without earthworms (2.48E+09 copies/g) on Day 45. Earthworms increased the richness of microbial communities. The relative abundances of Sphingobium and Dyadobacter, which are dominant genera harboring the GH 1 gene, were increased by earthworms to peak values of 23.90% and 11.20%, respectively. Correlation analysis showed that Sphingobium, Dyadobacter, Trichoderma, and Starkeya were positively associated with β-glucosidases. This work sheds new light on the mechanism of cellulose degradation during vermicomposting at the molecular level., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Second-generation feedstock, especially nonfood lignocellulosic biomass is a potential source for biofuel production. Cost-intensive physical, chemical, biological pretreatment operations and slow enzymatic hydrolysis make the overall process of lignocellulosic conversion into biofuels less economical than available fossil fuels. Lignocellulose conversions carried out at ≤ 50 °C have several limitations. Therefore, this review focuses on the importance of thermophilic bacteria and thermostable enzymes to overcome the limitations of existing lignocellulosic biomass conversion processes. The influence of high temperatures on various existing lignocellulose conversion processes and those that are under development, including separate hydrolysis and fermentation, simultaneous saccharification and fermentation, and extremophilic consolidated bioprocess are also discussed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A recently discovered thermophilic isolate, Geobacillus sp. R7, was shown to produce a thermostable cellulase with a high hydrolytic potential when grown on extrusion-pretreated agricultural residues such corn stover and prairie cord grass. At 70°C and 15-20% solids, the thermostable cellulase was able to partially liquefy solid biomass only after 36 h of hydrolysis time. The hydrolytic capabilities of Geobacillus sp. R7 cellulase were comparable to those of a commercial cellulase. Fermentation of the enzymatic hydrolyzates with Saccharomyces cerevisiae ATCC 24860 produced ethanol yields of 0.45-0.50 g ethanol/g glucose with more than 99% glucose utilization. It was further demonstrated that Geobacillus sp. R7 can ferment the lignocellulosic substrates to ethanol in a single step that could facilitate the development of a consolidated bioprocessing as an alternative approach for bioethanol production with outstanding potential for cost reductions.

The composition of thermophilic (60 degrees C) mixed cellulose-degrading enrichment culture initiated from compost samples was examined by constructing a 16S rRNA gene clone library and the presence of sequences related to Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria were identified. Eight isolates capable of degrading cellulose, carboxymethyl cellulose (CMC), or ponderosa pine sawdust were identified as belonging to the genera Geobacillus, Thermobacillus, Cohnella, and Thermus. A compost isolate WSUCF1 (Geobacillus sp.) was selected based on its higher growth rate and cellulase activity compared to others in liquid minimal medium containing cellulose as a source of carbon and energy. Strain WSUCF1 and a previously isolated thermophilic cellulose-degrading deep gold mine strain DUSELR13 (Bacillus sp.) were examined for their enzyme properties and kinetics. The optimal pH for carboxymethyl cellulase (CMCase) activity was 5.0 for both isolates. The optimum temperatures for CMCase of WSUCFI and DUSELR13 were 70 and 75 degrees C, respectively. For CMC, the DUSELR13 and WSUCF1 CMCases had K(m) values of 3.11 and 1.08mg/ml, respectively. Most remarkably, WSUCF1 and DUSELR13 retained 89% and 78% of the initial CMCase activities, respectively, after incubation at 70 degrees C for 1day. These thermostable enzymes would facilitate development of more efficient and cost-effective forms of the simultaneous saccharification and fermentation process to convert lignocellulosic biomass into biofuels.

Background: Deinking is an important part of paper recycling that involves the removal of ink particles from the paper fibres. This industrial process is important so that the fibres can be recirculated back into paper production, which enables better sustainability as fewer fresh fibres are needed. In this study, we examined five different alternative fibre materials from different agricultural residues and industrial processes for the pilot production of papers. Papers containing fibres from invasive plants (Japanese knotweed), dedicated crops (miscanthus, acacia), agricultural residues (tomato stems), and industrial waste (jute - fibres from coffee bags) were printed with water-based flexo inks and deinked with two separate processes (chemical and enzymes). Mechanical (break and tensile index, breaking length) and optical properties (ISO whiteness, brightness and CIE L*a*b* values) were measured and ink elimination IR700 and deinking efficiency was calculated for the two deinking processes., Results: Enzymatic treatment improved the mechanical properties of deinked pulp in comparison with the classic chemical treatment. Mechanical strength for almost all papers increased slightly (breaking length up to 20% in tomato and jute), and the optical result (brightness) increased similarly for both processes due to the bleaching action of the colour-shaded samples, whereas the deinking efficiency showed mixed results between chemical- and enzyme-type deinking (with chemical achieving better elimination measured at 700 nm) in the typical range of ink elimination values (15-35%) for flexographic inks. This indicates further optimization of the deinking with enzymes is needed due to different alternative fibre compositions and variations of residues in the delignification processes., Conclusion: Using a combination of adjusted enzymatic treatment as a precursor for deinking of paper-based packaging materials sourced from alternative fibres showed promising results regarding mechanical properties, whereas the optical properties need to be improved with cellulase optimization or by using mixes of different enzymes. These kinds of paper materials printed with flexo inks were found to be successfully deinkable with the chemical ISO-based deinking protocol. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Ligninases and hemicellulases are crucial as accessory enzymes to increase the enzymatic hydrolysis of lignocellulose, boosting sugars production from which biofuels and bioproducts could be obtained. In order to find new sources of these accessory enzymes, this study evaluates the potential of laccase and mannanase enzymes from Streptomyces ipomoeae for improving the conventional hydrolysis with commercial cellulases of steam-pretreated softwood. For that, different laccase treatment and mannanase supplementation strategies were performed. S. ipomoeae laccase increased both glucose and xylose production (17.8% and 9.3%, respectively), which was attributed to a removal of phenols of 29%. Moreover, the combination of laccase and alkaline extraction produced a lignin reduction of 16.2%, improving the glucose and xylose production by almost 41.3% and 44.9%, respectively. On the other hand, the supplementation of S. ipomoeae mannanase to the hydrolysis 24 h before the addition of cellulases increased the glucose (18.4%), xylose (12.3%), and mannose (47.2%) production. [ABSTRACT FROM AUTHOR]

Background: The management of the organic waste recycling process determines the interest in the thermophiles microorganisms involved in composting. Although many microbial enzymes have been isolated and studied for their industrial and commercial uses, there is still a continuous search for microorganisms which could synthesize industrially feasible enzymes, especially when the microbial diversity of cow dung itself makes a potential source of biotechnological enzymes. Results: The composting process studied at the Experimental Station of the University of Naples Federico II (Castel Volturno, Caserta, Italy) was characterized by fresh saw dust 40%, bovine manure 58%, and 2% mature compost as raw organic substrates, and its thermophilic phase exceeded a temperature of 55 °C for at least 5 days, thus achieving sanitation. Six microbial strains were isolated and designated as follow: CV1-1, CV1-2, CV2-1, CV2-2, CV2-3 and CV2-4. Based on 16S rRNA gene sequence, HRMAS–NMR spectroscopy, and biochemical investigations, they were ascribed to the genera Geobacillus and Bacillus. All the microbial isolates were qualitatively screened on plates for the presence of hydrolytic activities, and they were quantitatively screened in liquid for glycoside hydrolase enzymes in the extracellular, cell-bound, and cytosolic fractions. Based on these results, strains CV2-1 and CV2-3 were also quantitatively screened for the presence of cellulase and pectinase activities, and pH and temperature optimum plus thermostability of cellulase from CV2-1 were analyzed. Conclusions: The isolation and the identification of these thermophilic microorganisms such as Geobacillus toebii, Geobacillus galactosidasius, Bacillus composti, Bacillus thermophilus and Aeribacillus composti have allowed the study of the biodiversity of compost, with emphasis on their primary metabolome through an innovative and underutilized technique, that is HRMAS–NMR, also highlighting it as a novel approach to bacterial cell analysis. Subsequently, this study has permitted the identification of enzymatic activities able to degrade cellulose and other polymeric substrates, such as the one investigated from strain CV2-1, which could be interesting from an industrial and a biotechnological point of view, furthermore, increasing the knowledge for potential applicability in different industrial fields as an efficient and environmentally friendly technique. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass is considered the raw material of the future to produce ethanol, due to its low cost and its enormous availability. Lignocellulosic enzymes play a crucial role in the costs and sustainability of biorefining processes by promoting the biomass fractionation. In this study, the melon waste was used as inducer source to produce lignocellulolytic enzymes by Aspergillus niger M4 strain under submerged fermentation (SmF). The chemical characterization of the whole fruit showed a high content of sugars, as well as hemicellulose, followed by lignin and cellulose. The dried and crushed melon was used as a substrate in submerged fermentation, to induce the synthesis of lignocellulosic enzymes. The Aspergillus niger M4 strain was able to grow and synthesize lignocellulose degrading enzymes, allowing free-glucose values of 3.7 g/L. The culture broth showed endo-glucanase activity (1.21 U/mL), xylanase activity (11.00 U/mL), and laccase activity (18.23 U/mL). The entire melon (Cucumis melo L.) wastes can be used as a substrate in the lignocellulosic enzyme production by submerged fermentation process for further application in the biomass pretreatment for bioethanol production. [ABSTRACT FROM AUTHOR]

Thermal hot springs are present throughout the world and constitute a unique habitat for microbial diversity. The current investigation is conducted to study the bacterial diversity of thermophilic microorganisms in thermal hot springs of the Indian Himalayan Region (IHR). As of today, 110 geothermal hot springs have been explored for microbial diversity. In this study, we observed that the growth of thermophilic bacteria isolated from thermal hot springs of IHR ranges between 40 and 100 °C, and pH of 3.5–8 have been reported in the literature. The major bacterial species reported from the thermal hot springs of IHR are Bacillus spp., Geobacillus spp., Paenibacillus spp., Pseudomonas spp., Anoxybacillus, Paenibacillus, Brevibacillus, Aneurinibacillus, Thermus aquaticus, Aquimonas, Flavobacterium, etc. Furthermore, bacterial isolates from thermal hot springs of IHR have been reported to produce various enzymes and metabolites such as amylase, β-galactosidase, cellulase, nitrate reductase, acetoin, caffeine degradation enzymes, lipase, urease, and laccase. Metagenomic study and the entire genomic shotgun project have established the impact of physicochemical parameters (temperature and pH) on developing the microbiome. We have discussed the discoveries of microbiological data on the hot springs of IHR until the end of year 2021. As a whole, the microbiome adapts themselves as successful inhabitants to extreme environmental conditions and also serves as a diverse resource for potential applications in health, food, and environment. [ABSTRACT FROM AUTHOR]

Cellulolytic enzymes have gained considerable importance as potential candidates for the efficient bioconversion of agricultural biomass into second-generation biofuel. This study aimed to improve cellulase production from a bacterial strain isolated from cow dung. The biochemical and molecular identification was performed, and the capacity of the isolated bacterium to hydrolyze sugarcane bagasse was also determined. Response surface methodology (RSM) and one-variable-at-a-time (OVAT) designs were employed to maximize the cellulase production. The bacterium identified as Bacillus subtilis gave the highest cellulase yield at temperature of 45 °C, pH-7.0, and 72 h of incubation time. Optimization by the Plackett–Burman design consisting of 12 experimental runs at two levels of nine independent variables displayed the significant contribution of galactose (22%) and sodium nitrate (16%) in cellulase production. The responses in the form of contour and 3D plots (generated by Box-Behnken design) predicted maximum cellulase production with an activity of 0.8 U/mL/min in a medium containing 3.0 g/L carboxymethyl cellulose, 3.0 g/L galactose, 0.5 g/L sodium nitrate, 0.75 g/L KH2PO4, and 0.375 g/L MgSO4·7H2O. The experimentally deduced activity value was 0.698 U/mL/min, showing 90% validity of the predicted value and gave a threefold increase in cellulase production. The process adequately represented by the quadratic model (p < 0.0343) for all the responses was significant. Results obtained show that optimization of fermentation medium favored the increased production of cellulase by the bacterium. The bacterial isolate B. subtilis CD001 was found to be a potential candidate to hydrolyze the lignocellulosic feedstock, sugarcane bagasse. [ABSTRACT FROM AUTHOR]

The biological pretreatment for the enzymatic hydrolysis of lignocellulosic biomasses depends exclusively on the effective pretreatment process. Herein, we report a significant enhancement of enzymatic saccharification obtained with corn stover using a bacterial strain Bacillus sp. P3. The hemicellulose removal from corn stover by the strain Bacillus sp. P3 was evaluated for enhancing subsequent enzymatic hydrolysis. Therefore, our study revealed that an alkaline-resistant xylanase as well as other enzymes produced by Bacillus sp. P3 in fermentation broth led to a substantially enhanced hemicellulose removal rate from corn stover within pH 9.36–9.68. However, after a 20-day pretreatment of corn stover by the strain P3, the glucan content was increased by 51% and the xylan content was decreased by 35%. After 72 h of saccharification using 20 U/g of commercial cellulase, the yield of reducing sugar released from 20-day pretreated corn stover was increased by 56% in comparison to the untreated corn stover. Therefore, the use of the strain P3 could be a promising approach to pretreat corn stover for enhancing the enzymatic hydrolysis process of industrial bioenergy productions. [ABSTRACT FROM AUTHOR]

In the present study, twenty seven cellulose-degrading bacteria (CDB) were isolated from various organic manures and their cellulolytic activities were determined. The bacterial isolate CDB-26 showed the highest cellulolytic index, released 0.507 ± 0.025 mg/ml glucose and produced 0.196 ± 0.014 IU/ml cellulase enzyme under in vitro conditions. Biochemically, all the 27 isolates showed difference in the 6 biochemical tests performed. Further, all the 27 CDB isolates were subjected to various plant growth-promoting activities, and all CDB strains were positive for IAA production, GA3 production and siderophore production, whereas 19 strains were positive for ACC deaminase activity, 21 strains showed NH3 production and 19 strains were positive for HCN production. Out of 27 CDB isolates, 18 isolates were able to solubilize phosphate, 21 isolates were able to solubilize potash and 10 CDB isolates were found positive for silica solubilization. The molecular diversity among different CDB isolates was studied through ARDRA and demonstrated very high genetic diversity among these bacteria. The in vitro cellulose-degradation potential of these CDB isolates using vegetable waste as substrate were also assessed, and the 3 CDB isolates viz. Serratia surfactantfaciens (CDB-26), Stenotrophomonas rhizophila (CDB-16) and Pseudomonas fragi (CDB-5) showed the highest cellulose-degrading potential under in vitro conditions. Hence, the cellulolytic microbes isolated in the present study could be used for effective bioconversion of plant biomasses into enriched compost. [ABSTRACT FROM AUTHOR]

Fungi are plentiful in nature and they are found growing on wastes of wood materials. These wastes are equally found in our environment with no usefulness. The aim of this study was to exploit the probability of the isolated fungi from fruits to produce cellulase from wastes of lignocellulosic materials. Cellulase- producing fungi were isolated from fruits (tomato, banana, plantain). The organisms were screened for cellulase production. Culture conditions were optimized with pH, temperature and carbon. Cellulase was produced using lignocellulosic wastes; sawdust, corn cob, sugarcane bagasse. Six cellulase producers were isolated, four of which were selected for synthesis and quantification of the cellulase. The fungi were identified as Saccharomyces cerevisiae4, Trichoderma species1 and Scopulariopsis brevicaulis1. Of all the tested substrates used in this study, pretreated sugarcane bagasse at 3% w/v concentration with Scopulariopsis brevicaulis gave highest cellulase production 18.18 U/mL at 40°C, 5day incubation time and pH 5, followed by Trichoderma with 12.39 U/mL. These fungi are good potentials cellulase producers that can be considered at industrial level. [ABSTRACT FROM AUTHOR]

To date, the genus Parvularcula consists of 6 species and no potential application of this genus was reported. Current study presents the genome sequence of Parvularcula flava strain NH6-79 T and its cellulolytic enzyme analysis. The assembled draft genome of strain NH6-79 T consists of 9 contigs and 7 scaffolds with 3.68 Mbp in size and GC content of 59.87%. From a total of 3,465 genes predicted, 96 of them are annotated as glycoside hydrolases (GHs). Within these GHs, 20 encoded genes are related to cellulosic biomass degradation, including 12 endoglucanases (5 GH10, 4 GH5, and 3 GH51), 2 exoglucanases (GH9) and 6 β-glucosidases (GH3). In addition, highest relative enzyme activities (endoglucanase, exoglucanase, and β-glucosidase) were observed at 27th hour when the strain was cultured in the carboxymethyl cellulose/Avicel®-containing medium for 45 h. The combination of genome analysis with experimental studies indicated the ability of strain NH6-79 T to produce extracellular endoglucanase, exoglucanase, and β-glucosidase. These findings suggest the potential of Parvularcula flava strain NH6-79 T in cellulose-containing biomass degradation and that the strain could be used in cellulosic biorefining process. [ABSTRACT FROM AUTHOR]

Marine-derived enzymes have recently gained attention particularly for industrial applications. Cellulose-degrading enzymes are among leading biocatalysts with potential utility in biorefineries. This review presents an account of the cellulase production by marine sources from microorganisms including bacteria, yeasts, and molds to marine invertebrates such as protist, rotifer, mollusks, arthropods, and echinoderms. Cellulose-degrading ability of marine invertebrates is attributed to the production of endogenous cellulases and activities by the symbionts. Specialized environments in marine including estuaries and mangroves are rich in lignocellulosic biomass and hence provide a feeding ground for cellulose digesters. Since cellulosic biomass is considered chemical and energy feedstock, therefore, cellulases with the ability to work under extreme environment are much needed to fulfill the demand of modern biotechnological industries. The review also discusses physicochemical parameters of marine-derived cellulases. Key Points: • Cellulolytic ability is widely distributed amongst marine organisms, yet very few have been studied for their biotechnological potential • Cellulase from marine organisms has been demonstrated as a successful agent in degradation of seaweed processing waste to low molecular fragments • Marine derived cellulases can find their application in green processes • Cellulases from marine sources exhibit high specific activity, thermostability, and other important biochemical properties and hence can contend well with the enzymes from terrestrial sources. [ABSTRACT FROM AUTHOR]

Background: Enzymatic hydrolysis is a key step in the conversion of lignocellulosic polysaccharides to fermentable sugars for the production of biofuels and high-value chemicals. However, current enzyme preparations from mesophilic fungi are deficient in their thermostability and biomass-hydrolyzing efficiency at high temperatures. Thermophilic fungi represent promising sources of thermostable and highly active enzymes for improving the biomass-to-sugar conversion process. Here we present a comprehensive study on the lignocellulosic biomass-degrading ability and enzyme system of thermophilic fungus Malbranchea cinnamomea N12 and the application of its enzymes in the synergistic hydrolysis of lignocellulosic biomass. Results: Malbranchea cinnamomea N12 was capable of utilizing untreated wheat straw to produce high levels of xylanases and efficiently degrading lignocellulose under thermophilic conditions. Temporal analysis of the wheat straw-induced secretome revealed that M. cinnamomea N12 successively degraded the lignocellulosic polysaccharides through sequential secretion of enzymes targeting xylan and cellulose. Xylanase-enriched cocktail from M. cinnamomea N12 was more active on native and alkali‑pretreated wheat straw than the commercial xylanases from Trichoderma reesei over temperatures ranging from 40 to 75 °C. Integration of M. cinnamomea N12 enzymes with the commercial cellulase preparation increased the glucose and xylose yields of alkali‑pretreated wheat straw by 32 and 166%, respectively, with pronounced effects at elevated temperature. Conclusions: This study demonstrated the remarkable xylanase-producing ability and strategy of sequential lignocellulose breakdown of M. cinnamomea N12. A new process for the hydrolysis of lignocellulosic biomass was proposed, comprising thermophilic enzymolysis by enzymes of M. cinnamomea N12 followed with mesophilic enzymolysis by commercial cellulases. Developing M. cinnamomea N12 as platforms for thermophilic enzyme mixture production will provide new perspectives for improved conversion yields for current biomass saccharification schemes. [ABSTRACT FROM AUTHOR]

Thermophilic microorganisms and their enzymes have been utilized in various industrial applications. In this work, we isolated and characterized thermophilic anaerobic bacteria with the cellulose and hemicellulose degrading activities from a tropical dry deciduous forest in northern Thailand. Out of 502 isolated thermophilic anaerobic soil bacteria, 6 isolates, identified as Thermoanaerobacterium sp., displayed an ability to utilize a wide range of oligosaccharides and lignocellulosic substrates. The isolates exhibited significant cellulase and xylanase activities at high temperature (65°C). Among all isolates, Thermoanaerobacterium sp. strain R63 exhibited remarkable hydrolytic properties with the highest cellulase and xylanase activities at 1.15 U/mg and 6.17 U/mg, respectively. Extracellular extract of Thermoanaerobacterium sp. strain R63 was thermostable with an optimal temperature at 65°C and could exhibit enzymatic activities on pH range 5.0–9.0. Our findings suggest promising applications of these thermoanaerobic bacteria and their potent enzymes for industrial purposes. [ABSTRACT FROM AUTHOR]

Background: The current production of bioethanol based on lignocellulosic biomass (LCB) highly depends on thermostable enzymes and extremophiles owing to less risk of contamination. Thermophilic bacterial cellulases are preferred over fungi due to their higher growth rate, presence of complex multi-enzymes, stability, and enhanced bioconversion efficiency. Corncob, underutilized biomass, ensures energy conservation due to high lignocellulosic and more fermentable sugar content. In the present study, the thermophilic bacterium Bacillus aerius CMCPS1, isolated from the thermal springs of Manikaran, Himachal Pradesh, India, was characterized in terms of its activity, stability, and hydrolytic capacity. A two-step process comprising: (i) a combined strategy of hydrodynamic cavitation reaction (HCR)-coupled enzymatic (LccH at 6.5 U) pretreatment for delignification and (ii) subsequent hydrolysis of pre-treated (HCR-LccH) corncob biomass (CCB) using a thermostable cocktail of CMCPS1 was adopted to validate the efficiency of the process. Some of the parameters studied include lignin reduction, cellulose increase, and saccharification efficiency. Result: Among the five isolates obtained by in situ enrichment on various substrates, B. aerius CMCPS1, isolated from hot springs, exhibited the maximum hydrolytic activity of 4.11. The GH activity of the CMCPS1 strain under submerged fermentation revealed maximum filter paper activity (FPA) and endoglucanase activity of 4.36 IU mL−1 and 2.98 IU mL−1, respectively, at 44 h. Similarly, the isolate produced exoglucanase and β-glucosidase with an activity of 1.76 IU mL−1 and 1.23 IU mL−1 at 48 h, respectively. More specifically, the enzyme endo-1,4-β-d glucanase E.C.3.2.1.4 (CMCase) produced by B. aerius CMCPS1 displayed wider stability to pH (3–9) and temperature (30–90 °C) than most fungal cellulases. Similarly, the activity of CMCase increased in the presence of organic solvents (118% at 30% acetone v/v). The partially purified CMCase from the culture supernatant of CMCPS1 registered 64% yield with twofold purification. The zymogram and SDS-PAGE analyses further confirmed the CMCase activity with an apparent molecular mass of 70 kDa. The presence of genes specific to cellulases, such as cellulose-binding domain CelB, confirmed the presence of GH family 46 and β-glucosidase activity (GH3). The multifunctional cellulases of CMCPS1 were evaluated for their saccharification efficiency on laccase (LccH, a fungal laccase from Hexagonia hirta MSF2)-pretreated corncob in a HCR. The lignin and hemicelluloses removal efficiency of HCR-LccH was 54.1 and 6.57%, respectively, with an increase in cellulose fraction (42.25%). The saccharification efficiency of 55% was achieved with CMCPS1 multifunctional cellulases at 50 °C and pH 5.0. Conclusion: The multifunctional cellulase complex of B. aerius CMCPS1 is a potential biocatalyst for application in lignocellulosic biomass-based biorefineries. The saccharification ability of HCR-LccH-pretreated corncob at elevated temperatures would be an advantage for biofuel production from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Cellulose-degrading auxiliary activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) are known to be widely distributed among filamentous fungi and participate in the degradation of lignocellulose via the oxidative cleavage of celluloses, cello-oligosaccharides, or hemicelluloses. AA9 LPMOs have been reported to have extensive interactions with not only cellulases but also oxidases. The addition of AA9 LPMOs can greatly reduce the amount of cellulase needed for saccharification and increase the yield of glucose. The discovery of AA9 LPMOs has greatly changed our understanding of how fungi degrade cellulose. In this review, apart from summarizing the recent discoveries related to their catalytic reaction, functional diversity, and practical applications, the stability, expression system, and protein engineering of AA9 LPMOs are reviewed for the first time. This review may provide a reference value to further broaden the substrate range of AA9 LPMOs, expand the scope of their practical applications, and realize their customization for industrial utilization. Key Points • The stability and expression system of AA9 LPMOs are reviewed for the first time. • The protein engineering of AA9 LPMOs is systematically summarized for the first time. • The latest research results on the catalytic mechanism of AA9 LPMOs are summarized. • The application of AA9 LPMOs and their relationship with other enzymes are reviewed. [ABSTRACT FROM AUTHOR]

The present study investigates the cellulolytic potential of three prokaryotic strains belonging to genus Bacillus isolated from the benthic soil of two aquaculture farms in East Kolkata Wetlands. For the purpose of identification and phylogenetic analyses, biochemical characterization of these three strains were performed followed by molecular characterization of 16S rRNA sequences. Maximum cellulolytic potential was found in Bacillus cereus Strain SWA6a for both Exo - β,1-4- glucanase and Endo -β,1-4- glucanase with an activity of 0.08214 ± 0.00412 and 0.11263 ± 0.00478 IU ml-1min-1 respectively. The enzyme activity ranged from 0.06112 to 0.08214 IU ml-1min-1 for Exo - β,1-4- glucanase and 0.10018 to 0.11263 IU ml-1min-1 for Endo - β,1-4- glucanase. Degradation of free insoluble cellulose ranged from 55 to 63% within a time span of 96 hours in pH 6.8 - 7.2 when incubated at 37°C. Overall, the cellulolytic activity of the isolated strains may have strong implications in the field of biological conversion of cellulose-containing municipal wastes, agricultural and forestry wastes. These isolates maintain the equilibrium of the aquaculture pond by maintaining the biogeochemical cycle of carbon and hence has the prospect to be used for bioaugmentation purposes as a step towards a microbial-based aquaculture method. [ABSTRACT FROM AUTHOR]

Cellulases are important in the hydrolysis of lignocellulosic materials and thereby contribute to biomass conversion into fuels and chemicals. A cellulase-producing bacterium was isolated from decayed plant leaf litter in soil of a botanical garden. Based on morphological, biochemical and 16S rRNA gene sequencing, it was identified asEnterobacter cloacaeIP8, with gene bank accession number NR118568.1. The bacterial cellulase was purified in a three-step procedure using lyophilization, ion exchange chromatography (QAE Sephadex A-50) and gel filtration (Biogel P-100). Two isoforms of the enzyme were purified 1.21 and 1.23 folds, respectively, with yields of 30 and 29% for isoforms A and B, respectively. Apparent molecular weights of 36.61 ± 1.40 and 14.1 ± 0.10 kDa were obtained for isoforms A and B, respectively, using gel filtration chromatography. Kinetic parametersKmandVmaxwere 0.13 ± 0.04 mg/ml and 3.84 ± 0.05 U/ml/min, respectively, for isoform A and 0.58 ± 0.06 mg/ml and 13.8 ± 0.10 U/ml/min, respectively, for isoform B. Optimum pH (7.0) and temperature (60 °C) of cellulase activity were determined for both isoforms A and B. Na+and Ca2+enhanced the activities of both isoforms. Mg2+inhibited the enzyme activity at concentrations 4–15 mM but, while it stimulated the activity of isoform A at concentrations 15–200 mM, it inhibited that of isoform B at same concentration range. The strong inhibition of the enzyme by ethylenediaminetetraacetic acid (EDTA) confirmed the enzyme as a metalloenzyme. These results reveal the purified cellulase fromE. cloacaeIP8 as a thermostable, acidic to neutral metalloenzyme, suggesting that it has good potential for biotechnological applications. [ABSTRACT FROM AUTHOR]

The valorization of carbonoclastic materials to economically viable products are imperative to the global economy hence, abundance of lignocellulosic biomass in the biosphere presents an imperative opportunity for use as cheap substrate and/or sources of organisms with novel properties. Consequently, the Bacillus specie isolated from decaying wood shavings was evaluated for cellulose and xylan degradation potentials. Preliminary screening of the bacterial isolate revealed substantial activities with the development of 37 mm and 55.3 mm halo zone diameters, an indication of cellulase and xylanase, respectively. The bacteria were identified through partial 16S ribosomal deoxyribonucleic acids (rDNA) nucleotide sequencing, and Basic local alignment search tool (BLAST) analysis of the gene sequence showed that the bacterial isolate had 99% similarity to Bacillus cereus strain MCCC and the sequences were deposited in the GenBank as Bacillus cereus SAMRC-UFH1 with the accession number KU171369. The optimum culture conditions for cellulase production under submerged fermentation were as follow: pH 6 (103 U/ml), temperature 25∘C (93 U/ml) and agitation speed 150 rpm (126.3 U/ml) for cellulase; and pH 5 (298.5 U/ml), temperature 30∘C (428.5 U/ml), and agitation speed 100 rpm (248.5 U/ml) for xylanase. Under optimized conditions, cellulase was optimally produced (102.7 U/ml) after 84 h of cultivation whereas; xylanase production was peaked (390.5 U/ml) at 72 h. Conclusively, the bacteria exhibit promise as a source of potential cellulase and xylanase that could be useful for degrading lignocellulosic matter into value-added products of economic importance. [ABSTRACT FROM AUTHOR]

Background: The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable ß-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scafoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates. Results: The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scafoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, "thermostable" designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scafoldin proved critical for enhanced enzymatic performance under conditions of high temperatures. Conclusions: Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scafoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars. [ABSTRACT FROM AUTHOR]

Background: In the bioconversion of lignocellulosic substrates, the adsorption behavior of cellulase onto lignin has a negative effect on enzymatic hydrolysis of cellulose, decreasing glucose production during enzymatic hydrolysis, thus decreasing the yield of fermentation and the production of useful products. Understanding the interaction between lignin and cellulase is necessary to optimize the components of cellulase mixture, genetically engineer high-efficiency cellulase, and reduce cost of bioconversion. Most lignin is not removed during liquid hot water (LHW) pretreatment, and the characteristics of lignin in solid substrate are also changed. To understand the interactions between cellulase and lignin, this study investigated the change in the characteristics of lignin obtained from corn stover, as well as the behavior of cellulase adsorption onto lignin, under various severities of LHW pretreatment. Results: LHW pretreatment removed most hemicellulose and some lignin in corn stover, as well as improved enzymatic digestibility of corn stover. After LHW pretreatment, the molecular weight of lignin obviously increased, whereas its polydispersity decreased and became more negative. The hydrophobicity and functional groups in lignin also changed. Adsorption of cellulase from Penicillium oxalicum onto lignin isolated from corn stover was enhanced after LHW pretreatment, and increased under increasing pretreatment severity. Different adsorption behaviors were observed in different lignin samples and components of cellulase mixtures, even in different cellobiohydrolases (CBHs), endo-beta-1, 4-glucanases (EGs). The greatest reduction in enzyme activity caused by lignin was observed in CBH, followed by that in xylanase and then in EG and β-Glucosidase (BGL). The adsorption behavior exerted different effects on subsequent enzymatic hydrolysis of various biomass substrates. Hydrophobic and electrostatic interactions may be important factors affecting different adsorption behaviors between lignin and cellulase. Conclusions: LHW pretreatment changed the characteristics of the remaining lignin in corn stover, thus affected the adsorption behavior of lignin toward cellulase. For different protein components in cellulase solution from P. oxalicum, electrostatic action was a main factor influencing the adsorption of EG and xylanase onto lignin in corn stover, while hydrophobicity affected the adsorption of CBH and BGL onto lignin. [ABSTRACT FROM AUTHOR]

Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 310 helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity. [ABSTRACT FROM AUTHOR]

In this article, the authors focus on a study related to the characterization and phylogenetic diversity of cellulase producing bacterial isolates obtained from extreme environments. They inform that phylogenetic analyses based on 16S ribosomal RNA gene sequences have showed that the isolates are clustered within Actinobacteria and Firmicutes. They mention that isolates identified by researchers include Bacillus, Paenibacillus and Lysinibacillus.

The biomass degrading enzymatic potential of 101 thermophilic bacterial strains isolated from a volcanic environment (Santorini, Aegean Sea, Greece) was assessed. 80 % of the strains showed xylanolytic activity in Congo Red plates, while only eight could simultaneously hydrolyze cellulose. Fifteen isolates were selected on the basis of their increased enzyme production, the majority of which was identified as Geobacilli through 16S rDNA analysis. In addition, the enzymatic profile was evaluated in liquid cultures using various carbon sources, a procedure that revealed lack of correlation on xylanase levels between the two cultivation modes and the inability of solid CMC cultures to fully unravel the cellulose degrading potential of the isolates. Strain SP24, showing more than 99 % 16S DNA similarity with Geobacillus sp. was further studied for its unique ability to simultaneously exhibit cellulase, xylanase, β-glucosidase and β-xylosidase activities. The first two enzymes were produced mainly extracellularly, while the β-glycosidic activities were primarily detected in the cytosol. Maximum enzyme production by this strain was attained using a combination of wheat bran and xylan in the growth medium. Bioreactor cultures showed that aeration was necessary for both enhanced growth and enzyme production. Aeration had a strong positive effect on cellulase production while it negatively affected expression of β-glucosidase. Xylanase and β-xylosidase production was practically unaffected by aeration levels. [ABSTRACT FROM AUTHOR]