Journal articles: 'Cellulolytic bacteria; Hydrolytic; Cellulases' – Grafiati (2024)

Abstract:

ABSTRACT Four β-1,4-glucanases (cellulases) of the cellulolytic bacteriumCellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.

Abstract:

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteriaCellulomonas fimion various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B andCfCel48A) slide on crystalline cellulose. The direction of processive movement ofCfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungusTrichoderma reesei(TrCel7A), whose movement was first observed by this technique, whileCfCel48A moves in the same direction asTrCel7A. WhenCfCel6B andTrCel7A were mixed on the same substrate, “traffic accidents” were observed, in which the two cellulases blocked each other’s progress. The processivity ofCfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

Abstract:

Eight isolates of cellulose-degrading bacteria (CDB) were isolated from four different invertebrates (termite, snail, caterpillar, and bookworm) by enriching the basal culture medium with filter paper as substrate for cellulose degradation. To indicate the cellulase activity of the organisms, diameter of clear zone around the colony and hydrolytic value on cellulose Congo Red agar media were measured. CDB 8 and CDB 10 exhibited the maximum zone of clearance around the colony with diameter of 45 and 50 mm and with the hydrolytic value of 9 and 9.8, respectively. The enzyme assays for two enzymes, filter paper cellulase (FPC), and cellulase (endoglucanase), were examined by methods recommended by the International Union of Pure and Applied Chemistry (IUPAC). The extracellular cellulase activities ranged from 0.012 to 0.196 IU/mL for FPC and 0.162 to 0.400 IU/mL for endoglucanase assay. All the cultures were also further tested for their capacity to degrade filter paper by gravimetric method. The maximum filter paper degradation percentage was estimated to be 65.7 for CDB 8. Selected bacterial isolates CDB 2, 7, 8, and 10 were co-cultured withSaccharomyces cerevisiaefor simultaneous saccharification and fermentation. Ethanol production was positively tested after five days of incubation with acidified potassium dichromate.

Abstract:

With decades of studies on cellulose bioconversion, cellulases have been playing an important role in producing fermentable sugars from lignocellulosic biomass. Copious microorganisms that are able to degrade cellulose have been isolated and identified. The present study has been undertaken to isolate and screen the cellulase producing bacteria from soils of agrowaste field. Cellulase production has been qualitatively analyzed in carboxy methylcellulose (CMC) agar medium after congo red staining and NaCl treatment by interpretation with zones around the potent colonies. Out of the seven isolates, only two showed cellulase production. The morphogical and molecular characterization revealed its identity as Escherichia coli and Staphylococcus aureus. The potential of organisms for bioethanol production has been investigated using two substrates, namely, paper and leaves by subjecting with a pre-treatment process using acid hydrolysis to remove lignin which acts as physical barrier to cellulolytic enzymes. Ethanolic fermentation was done using Saccharomyces cerevisiae for 24-48 h and then the bioethanol produced was qualitatively proved by iodoform assay. These finding proves that ethanol can be made from the agricultural waste and the process is recommended as a means of generating wealth from waste.

Abstract:

Microbial cellulases have been drawing attention worldwide because of their massive capacity to process the most abundant cellulosic biomass into sustainable biofuels and other valuable products. Profitable biomass conversion processes are highly dependent on the use of efficient enzymes for lignocellulose degradation. Among the cellulose degrading enzymes, β-glucosidases are essential for efficient hydrolysis of cellulosic biomass as they relieve the inhibition of the cellobiohydrolases and endoglucanases by reducing cellobiose accumulation. In this study cellulolytic bacteria with potential β-glucosidases activity were isolated and screened from biogas plant effluent and dairy effluent near Jahangirnagar University campus. From initial screening a total of 16 isolates were found to have cellulolytic activity, among them three isolates (B1, B5, D4) were selected based on their superior results. All the three bacterial isolates were identified as B. subtilis (B1), Bacillus amyloliquefaciens (B5) and B. subtilis (D4) respectively based on their morphological, biochemical and molecular characteristics. The β-glucosidases activity of these three potential cellulolytic bacteria was performed by measuring the release of PNP using pNPG as a substrate and interestingly D4 strain was resulted with β-glucosidases negative where B1 strain was found to have efficient for β-glucosidases activity.

Abstract:

Cellulase has been widely used as biocatalyst in industries. Production of cellulase from microorganismshas many advantages such as short production time and less expense. Our previous study indicated that oneof cellulolytic bacteria from digestive tract of milkfish (Chanos chanos), namely BSA B1, showed the highestcellulase activity. The objective of this study was to determine the phylogenetic of BSA B1 strain using 16SrRNA gene sequence. Furthermore, this study also determine the specific activity of purified cellulase from BSAB1 strain and its potency to hydrolyze Chlorella zofingiensis cellulose. Cellulase was purified using ammoniumsulphate precipitation, dialysis, and ion exchange chromatography. The purified cellulase was used to hydrolyzecellulose of C. zofingiensis. The result demonstrated that BSA B1 strain was closely related with Bacillus aeriusand Bacillus licheniformis. The specific activity of the crude enzyme was 1.543 U mL-1; after dialysis was 4.384 UmL-1; and after chromatography was 7.543 U mL-1. Purified cellulase exhibited activity in hydrolyzed both CMCand C. zofingiensis. Compared to commercial cellulase, purified cellulase had lower activity in hydrolyzed CMCbut higher activity in hydrolyzed C. zofingiensis. Ethanol dehydration could potentially increase the reducingsugar yield in cellulose hydrolysis when used appropriately. Morphology of C. zofingiensis cell has changedafter incubation with cellulases and ethanol dehydration indicated degradation of cell wall.

Abstract:

Lignocellulose is the most abundant biomass on earth. Agricultural, forest, and agroindustrial activities generate tons of lignocellulosic wastes annually, which present readily procurable, economically affordable, and renewable feedstock for various lignocelluloses based applications. Lignocelluloses are the focus of present decade researchers globally, in an attempt to develop technologies based on natural biomass for reducing dependence on expensive and exhaustible substrates. Lignocellulolytic enzymes, that is, cellulases, hemicellulases, and lignolytic enzymes, play very important role in the processing of lignocelluloses which is prerequisite for their utilization in various processes. These enzymes are obtained from microorganisms distributed in both prokaryotic and eukaryotic domains including bacteria, fungi, and actinomycetes. Actinomycetes are an attractive microbial group for production of lignocellulose degrading enzymes. Various studies have evaluated the lignocellulose degrading ability of actinomycetes, which can be potentially implemented in the production of different value added products. This paper is an overview of the diversity of cellulolytic, hemicellulolytic, and lignolytic actinomycetes along with brief discussion of their hydrolytic enzyme systems involved in biomass modification.

Abstract:

Cellulose, a macromolecule of β -D- anhydroglucopyranose units linked by β (1,4)-glycosidic bonds, is the world’s most abundant organic polymer and is the main component of plant biomass that provides stability. Due to its sta-ble fibrous property, it has become one of the most important commercial raw materials for many industries. However, accumulation of waste cellulose due to natural and/or anthropogenic sources is a matter of concern in terms of environmental pollution. Wastes cellulosic substrates can be utilized as sources of energy through controlled hydrolysis using cellulases- a complex group of enzymes capable of degrading all types of cellulosic waste materials. A number of bacteria, fungi and insects are having the capability to degrade cellulose by production of cellulase enzymes. Further, the symbiotic insect-microbe relationships present in the insect gut microbiome for the production of cellulolytic system is of immense importance as this would lead to applications in different fields like biodegradation of cellulosic wastes, pollution reduction, biofuel production, insect/pest control etc. Cel-lulase gene can also be improved by genetic or protein engineering methods using recent technological advances. This review deals with the advances of cellulase enzymes and its utilization for different application.

Abstract:

Family 45 glycoside hydrolases (GH45) are endoglucanases that are integral to cellulolytic secretomes, and their ability to break down cellulose has been successfully exploited in textile and detergent industries. In addition to their industrial relevance, understanding the molecular mechanism of GH45-catalyzed hydrolysis is of fundamental importance because of their structural similarity to cell wall–modifying enzymes such as bacterial lytic transglycosylases (LTs) and expansins present in bacteria, plants, and fungi. Our understanding of the catalytic itinerary of GH45s has been incomplete because a crystal structure with substrate spanning the −1 to +1 subsites is currently lacking. Here we constructed and validated a putative Michaelis complex in silico and used it to elucidate the hydrolytic mechanism in a GH45, Cel45A from the fungus Humicola insolens, via unbiased simulation approaches. These molecular simulations revealed that the solvent-exposed active-site architecture results in lack of coordination for the hydroxymethyl group of the substrate at the −1 subsite. This lack of coordination imparted mobility to the hydroxymethyl group and enabled a crucial hydrogen bond with the catalytic acid during and after the reaction. This suggests the possibility of a nonhydrolytic reaction mechanism when the catalytic base aspartic acid is missing, as is the case in some LTs (murein transglycosylase A) and expansins. We calculated reaction free energies and demonstrate the thermodynamic feasibility of the hydrolytic and nonhydrolytic reaction mechanisms. Our results provide molecular insights into the hydrolysis mechanism in HiCel45A, with possible implications for elucidating the elusive catalytic mechanism in LTs and expansins.

Abstract:

Silage inoculants consisting of primarily Lactobacillus plantarum, are widely used to ensure that lactic acid bacteria dominate the fermentation of water soluble carbohydrates (WSC) during the ensilage process. Previous studies have shown that the supplementation of ensiled forage crops with cellulases can also improve the quality of silage through i) increasing the generation of WSC, and therefore ensuring an adequate supply of substrate for L. plantanim; ii) Partial hydrolysis of the plant cell wall increasing the rate of cellulose hydrolysis within the rumen. From the above discussion it is apparent that the use of an L.plantarum strain, with the capacity to hydrolyse cellulose, could be beneficial in the ensiling process. No celluloytic lactic bacterium has been isolated from microbial ecosystems. However, the advent of recombinant DNA technology affords us the possibility of engineering a cellulolytic derivative of L. plantarum. This report describes progress towards this objective.

Abstract:

Actinobactérias são bactérias Gram-positivas encontradas principalmente no solo e que se destacam pela capacidade de produção de enzimas hidrolíticas, como amilase e celulase. Essas enzimas degradam, respectivamente, o amido e a celulose, substratos abundantes no solo, mas que por sua complexidade química são indisponíveis para nutrição de outros organismos. Essas bactérias também crescem em condições extremas, como as prevalentes no Semiárido nordestino, onde ainda são raros estudos sobre esse grupo microbiano. Assim, tendo em vista o crescente interesse sobre o funcionamento biológico do solo, aliado ao aspecto biotecnológico, esse trabalho teve por objetivo avaliar a atividade enzimática de vinte e oito cepas de actinobactérias dos gêneros Streptomyces, Terrabacter, Nocardia e Micromonospora isoladas de amostras de solo do Parque Nacional de Ubajara, no Estado do Ceará. As atividades amilolíticas e celulolíticas foram determinadas pelo índice enzimático correspondente à relação entre o diâmetro em milímetros do halo de hidrólise e o diâmetro em milímetros da colônia de cada cepa. A atividade celulolítica foi detectada em 75% e a amilolítica em 82% das cepas avaliadas. Uma das cepas avaliadas destacou-se com um índice enzimático da celulase igual a 6,90 enquanto o maior índice para a amilase foi 6,23. Os resultados indicaram o potencial ecológico e biotecnológico das actinobactérias do solo da região analisada. Actinobacterias are Gram-positive bacterias found in soil and are distinguished by their production of hydrolytic enzymes, such as amylase and cellulase. These enzymes degrade starch and cellulose, respectively, which are abundant substrates in soil, but that are unavailable for use by other organisms due to their chemical complexity. These bacteria grow in extreme conditions, such as those prevalent in the Brazilian semi-arid northeast, where there are also few studies about this microbial group. Thus, due to the growing interest in the biological functioning of the soil, together with the biotechnology aspect, this work aimed to evaluate the enzymatic activity of twenty eight strains of actinobacteria of the genus Streptomyces, Terrabacter, Nocardia, and Micromonospora obtained from the soil samples of the Ubajara National Park in the state of Ceará. The amylolytic and cellulolytic activities were determined by the enzymatic index corresponding to the relation between the diameter of hydrolysis halo, in millimeters, and the diameter in millimeters of the colony of each strain. The cellulolytic and amylolytic indexes were detected in 75% and 85% of the evaluated strains, respectively. One of the strains evaluated stood out with an enzyme cellulase index equal to 6.90 while the highest rate for amylase was 6.23. The results indicated the ecological and biotechnological potential of actinobacteria from the soil of analyzed region. Keywords: enzymes, bacteria, caatinga, Northeast

Abstract:

Bacteria of the genus Bacillus make up a significant (8–12%) part of the soil microbiome. Manifestation of their biological activity, in particular, the antagonistic and lytic activity against other microorganisms directly depends on their exometabolites. According to the literature, such properties of soil bacteria of the genus Bacillus thus can be related to their various lytic exoenzymes. Aim. To evaluate the role of lytic exoenzymes of the studied soil bacteria strains of the genus Bacillus in the manifestation of their biological (antagonistic, lysing) activity. Methods. The antagonistic activity of bacteria strains of the genus Bacillus against phytopathogenic micromycetes was determined by the method of double culture in Petri dishes on potato-glucose agar. For qualitative analysis of the presence of extracellular enzymes, strains of bacteria of the genus Bacillus were plated on Petri dishes with solid mineral-salt medium and a suitable substrate inducer. The ratio of the diameter of substrate hydrolysis zone to the diameter of the colony was taken as the relative enzymatic activity of the culture. Bacteriolytic activity of the studied strains was determined by the change in optical density of living cells of phytopathogenic bacteria suspension at 540 nm. Results. Six strains of bacteria of the genus Bacillus were selected by the results of preliminary screening, with at least five types of lytic activity, namely proteolytic, chitinase, amylolytic, cellulase, and xylanase of different levels (low, average, high). Analysis of the antagonistic activity of the selected strains of bacteria of the genus Bacillus to the main groups of phytopathogenic bacteria (six test cultures) singled out the strain Bacillus sp. 41 for a careful study of the nature and spectrum of its antagonism. Analysis of the level of antagonistic activity of the selected Bacillus strains against the phytopathogenic micromycetes showed that the minimum decrease of antagonism (the decrease of growth inhibition zones) during the observation period (at the 3rd and 7th days) was in Bacillus sp. 41 strain. Therefore, only this strain showed a stable and relatively wide range of antagonistic activity against phytopathogens of bacterial and fungal etiology. The nature of this antagonism is probably complex and conditioned by the participation of various biochemical mechanisms, in particular, the synthesis of a complex of lytic exoenzymes. To assess the lysing activity of Bacillus strains, three strains with the highest proteolytic and cellulolytic activity of exoenzymes were taken from the six previously chosen. Only Bacillus sp.1913 strain showed high (70%) lytic activity against gram-negative polyphagous phytopathogen Pseudomonas syringae pv. syringae UCM B-1027T. Such activity of the strain did not manifest against the rest of the phytopathogenic test cultures. The high lytic activity of Bacillus sp. 1913 strain may be associated with high activity of exogenous proteases and cellulases of the lytic complex, which is quite consistent with the literature data on the lytic activity of bacteria of the genus Bacillus. Conclusions. The spectrum and activity of lytic exoenzymes of strains of the studied soil bacteria of the genus Bacillus indicate the indirect participation of these enzymes in the manifestation of biological activity (antagonistic and lytic).

Abstract:

Cellulose, a major polysaccharide found in agricultural residues and industrial and municipal wastes. In the present study thermophilic cellulolytic microorganisms were isolated. The isolates were tested for their cellulytic activity. The enzyme production from potent isolates was optimised using cellulose basal broth medium. Activity of partially purified enzyme was determined. The most potent thermophilic cellulolytic isolates were identified as Bacillus subtilis. The crude cellulase enzyme concentrated at 80-85% ammonium sulfate produced highest zone of hydrolysis. The enzymatic degradation of cellulose waste has been suggested as a feasible alternative for the conversion of lignocelluloses substrate into fermentable sugars and application for biofuel production. Scientific World, Vol. 10, No. 10, July 2012 p43-46 DOI: http://dx.doi.org/10.3126/sw.v10i10.6861

Abstract:

ABSTRACT A new cellulosomal protein from Clostridium cellulolyticum Cel9M was characterized. The protein contains a catalytic domain belonging to family 9 and a dockerin domain. Cel9M is active on carboxymethyl cellulose, and the hydrolysis of this substrate is accompanied by a decrease in viscosity. Cel9M has a slight, albeit significant, activity on both Avicel and bacterial microcrystalline cellulose, and the main soluble sugar released is cellotetraose. Saccharification of bacterial microcrystalline cellulose by Cel9M in association with two other family 9 enzymes from C. cellulolyticum, namely, Cel9E and Cel9G, was measured, and it was found that Cel9M acts synergistically with Cel9E. Complexation of Cel9M with the mini-CipC1 containing the cellulose binding domain, the X2 domain, and the first cohesin domain of the scaffoldin CipC of the bacterium did not significantly increase the hydrolysis of Avicel and bacterial microcrystalline cellulose.

Abstract:

Mesostructured silica nanoparticles offer a unique opportunity in the field of biocatalysis thanks to their outstanding properties. The tunable pore size in the range of mesopores allows for immobilizing bulky enzyme molecules. The large surface area improves the catalytic efficiency by increasing enzyme loading and finely dispersing the biocatalyst molecules. The easily tunable pore morphology allows for creating a proper environment to host an enzyme. The confining effect of mesopores can improve the enzyme stability and its resistance to extreme pH and temperatures. Benefits also arise from other peculiarities of nanoparticles such as Brownian motion and easy dispersion. Fossil fuel depletion and environmental pollution have led to the need for alternative sustainable and renewable energy sources such as biofuels. In this context, lignocellulosic biomass has been considered as a strategic fuel source. Cellulases are a class of hydrolytic enzymes that convert cellulose into fermentable sugars. This review is intended to survey the immobilization of cellulolytic enzymes (cellulases and β-glucosidase) onto mesoporous silica nanoparticles and their catalytic performance, with the aim to give a contribution to the urgent action required against climate change and its impacts, by biorefineries’ development.

Abstract:

Actinobacteria, conventionally known as actinomycetes are the most unique microorganisms revealing a link between bacteria and fungi. They are highly adaptable to extreme environmental condition and also exhibit a high diversity in metabolic activities. Biochemical, physiological and genetic features are mainly responsible for their higher adoptability to harsh conditions and extra cellular synthesis of wider secondary metabolites in general and enzymes and antibiotics in particular. The limestone quarry and lime powder dwellings are the harsh habitats prevailing in the northern region of Karnataka. These are the typical habitats left behind after the exploration of limestone and lime powder for highly commercial industrial activities such as production of cement and petroleum refining process respectively. In the present investigation, efforts were made to detect cellulolytic actinobacteria from lime powder dwellings. Actinobacteria confirmed by the basic colony characters, microscopic features, biochemical and physiological properties were screened for the potential cellulolytic activity. In all 54 isolates of actinobacteria were detected and screened to obtain three best cellulolytic actinobacteria, namely DSA22, DSA38 and DSA39. The maximum zone of hydrolysis on carboxymethylcellulose medium was an important criterion to screen the best cellulolytic isolates of actinobacteria. Further, the three best isolates of cellulolytic actinobacteria were screened for maximum production of extra cellular cellulase. The isolate DSA22 with higher enzyme activity (12 IU) was subjected to molecular characterization. Based on 16s rRNA analysis (BioEra Laboratory, Pune, Maharashtra) an isolate DSA 22 was identified as Streptomyces enissocaesiles.

Abstract:

The intestinal flagellates of termites produce cellulases that contribute to cellulose digestion of their host termites. However, 75% of all termite species do not harbour the cellulolytic flagellates; the endogenous cellulase secreted from the midgut tissue has been considered a sole source of cellulases in these termites. Using the xylophagous flagellate-free termites Nasutitermes takasagoensis and Nasutitermes walkeri , we successfully solubilized cellulases present in the hindgut pellets. Zymograms showed that the hindguts of these termites possessed several cellulases and contained up to 59% cellulase activity against crystalline cellulose when compared with the midgut. Antibiotic treatment administered to N. takasagoensis significantly reduced cellulase activity in the hindgut, suggesting that these cellulases were produced by symbiotic bacteria.

Abstract:

The production of cellulases of Aspergillus niger ATCC 10864 was carried out by fermentation in biofilms (FB). The extracts of cellulases were used to hydrolyze the cellulose of Capsicum baccatum “escabeche chili” fruit, for this the chili fruits were dehydrated and ground to a particle size < 0.045 mm. Then dry chili: cellulase was mixed and hydrolyzed under conditions of agitation, controlled time and temperature. The medium was filtered and the supernatant of the hydrolyzed chili cake was separated, the latter was dried at 10% humidity and leached with a mixture of hexane:acetone:ethanol to extract the carotenoids and capsaicinoids, which were quantified by HPLC. The extraction yield of oleoresin was five times higher compared to the conventional method; likewise in the T8 and T5 treatments, greater extraction of carotenoids and total capsaicinoids was achieved compared to the other treatments. The hydrolytic action of the cellulases, on the molecular structures of the cellulose of the red drop chili fruit, favored greater release of the carotenoids and total capsaicinoids compared to conventional methods.

Abstract:

Biogas production is one of the means to manage the cassava pulp waste obtained from the cassava processing plants. The success of the process is determined by the hydrolysis in an anaerobic digester. When the digester failure is found, the new microbial consortium inoculum is introduced to the system with the long period of set up time. This research aimed to construct the endemic microbial consortium by re-cultivating the cellulolytic microbial consortia obtained from cassava pulp and digester wastewater with the expected shorter set up time. Modifications of enrichment and re-cultivation methods by varying the nutrients, pH and temperature improved the enzymatic hydrolysis yields, as reducing sugars, of CMC, rice straw and cassava pulp substrates approximately 9, 3, and 13 times, respectively. To analyze the enzymatic activities of the selected microbial consortia, the cellulase enzyme was extracted, partially purified and analyzed on CMC-zymogram. The ~130 kDa-sized cellulase enzyme was identified with endocellulase activity, and it was considered as a relatively large molecular size molecule compared to most bacterial endocellulases. The selected microbial consortia were tested for their biomass degradation capacities, and the optimal operational condition was obtained at pH 7.0 and 30 °C. This optimal condition showed the proof of the concept that this re-cultivated consortium could be applied in on-site digester with high efficiency.

Abstract:

The aim of this study was to compare the hydrolytic potential of the cellulase produced byTrichoderma pseudokoningiiand byTrichoderma koningiiin the enzymatic hydrolysis of furfural residues with different cellulose content. The results showed that the glucose yield was improved by increasing the cellulose of the substrates. The maximum glucose yield of 83.7% was obtained when the substrates with cellulose content of 94.92%, which was hydrolyzed by the enzymes produced byTrichoderma pseudokoningii.The cellulolytic enzyme fromTrichoderma pseudokoningiiwas found to have a high hydrolysis capability to the furfural residues and it possessed more efficient adsorption and desorption on the substrates. There was the nonproductive cellulase adsorption onto lignin in the enzymatic hydrolysis of the furfural residues by the cellulase preparations fromT.strains.

Abstract:

Tonle Sap Lake is the largest freshwater lake in Southeast Asia, and it is regarded as one of the most biodiverse freshwater ecosystems in the world. Studies concerning aquatic cellulolytic bacteria from Tonle Sap Lake remain scarce. Cellulolytic bacteria and their cellulases play a vital role in the biogeochemical cycles of lake environments, and their application in biotechnological industries is likewise an important component of their usage. This study aimed to assess the isolation, genetic identification, bioinformatic analyses, and activity characterization of aquatic cellulolytic bacteria. The cellulolytic bacteria isolated from sedimentary water samples in the littoral zone of the lake belong to the genera Aeromonas, Bacillus, and Exiguobacterium. Several isolated aquatic bacteria were designated as rare cellulolytic microbes. Remarkably, B. mojavensis strain REP303 was initially evidenced by the aquatic cellulolytic bacterium in freshwater lake ecosystems. It was considered a highly active cellulolytic bacterium capable of creating a complete cellulase system involving endoglucanase, exoglucanase, and β-glucosidase. The encoded endoglucanase belongs to the glycosyl hydrolase family 5 (GH5), with a carboxymethylcellulase (CMCase) activity of 3.97 ± 0.05 U/mL. The optimum temperature and pH for CMCase activity were determined to be 50 °C at a pH of 7.0, with a stability range of 25–55 °C at a neutral pH of 7.0–8.0. The CMCase activity was enhanced significantly by Mn2+ and was inhibited considerably by EDTA and ethyl-acetate. In conclusion, this study is the first to report data concerning aquatic cellulolytic bacteria isolated from the littoral zone of Tonle Sap Lake. A novel strain of isolated cellulolytic B. mojavensis could be applied in various cellulose-based industries.

Abstract:

AbstractCold−adapted marine bacteria producing extracellular hydrolytic enzymes are important for their industrial application and play a key role in degradation of particulate organic matter in their natural environment. In this work, members of a previously−obtained protease−producing bacterial collection isolated from different marine sources from Potter Cove (King George Island, South Shetlands) were taxonomically identified and screened for their ability to produce other economically relevant enzymes. Eighty−eight proteolytic bacterial isolates were grouped into 25 phylotypes based on their Amplified Ribosomal DNA Restriction Analysis profiles. The sequencing of the 16S rRNA genes from representative isolates of the phylotypes showed that the predominant culturable protease−producing bacteria belonged to the class Gammaproteobacteria and were affiliated to the genera Pseudomonas, Shewanella, Colwellia, and Pseudoalteromonas, the latter being the predominant group (64% of isolates). In addition, members of the classes Actinobacteria, Bacilli and Flavobacteria were found. Among the 88 isolates screened we detected producers of amylases (21), pectinases (67), cellulases (53), CM−cellulases (68), xylanases (55) and agarases (57). More than 85% of the isolates showed at least one of the extracellular enzymatic activities tested, with some of them producing up to six extracellular enzymes. Our results confirmed that using selective conditions to isolate producers of one extracellular enzyme activity increases the probability of recovering bacteria that will also produce additional extracellular enzymes. This finding establishes a starting point for future programs oriented to the prospecting for biomolecules in Antarctica.

Abstract:

ABSTRACTCellulases containing a family 9 catalytic domain and a family 3c cellulose binding module (CBM3c) are important components of bacterial cellulolytic systems. We measured the temperature dependence of the activities of three hom*ologs:Clostridium cellulolyticumCel9G,Thermobifida fuscaCel9A, andC. thermocellumCel9I. To directly compare their catalytic activities, we constructed six new versions of the enzymes in which the three GH9-CBM3c domains were fused to a dockerin both with and without aT. fuscafibronectin type 3 hom*ology module (Fn3). We studied the activities of these enzymes on crystalline cellulose alone and in complex with a miniscaffoldin containing a cohesin and a CBM3a. The presence of Fn3 had no measurable effect on thermostability or cellulase activity. The GH9-CBM3c domains of Cel9A and Cel9I, however, were more active than the wild type when fused to a dockerin complexed to scaffoldin. The three cellulases in complex have similar activities on crystalline cellulose up to 60°C, butC. thermocellumCel9I, the most thermostable of the three, remains highly active up to 80°C, where its activity is 1.9 times higher than at 60°C. We also compared the temperature-dependent activities of different versions of Cel9I (wild type or in complex with a miniscaffoldin) and found that the thermostable CBM is necessary for activity on crystalline cellulose at high temperatures. These results illustrate the significant benefits of working with thermostable enzymes at high temperatures, as well as the importance of retaining the stability of all modules involved in cellulose degradation.

Abstract:

In lower termite such as Coptotermes gestroi, cellulose and hemicellulose are hydrolysed by cellulases and hemicellulases secreted from bacteria, archaea, protozoa and fungy in the hindgut. In which, majority of the enzymes are contributed by protozoa. From the metagenomic DNA data (125,423 open reading frames -ORFs) of free-living bacteria in the gut of C. gestroi harvested in Southern Vietnam and by MEGA 4.0 software, 100.340 ORFs were classified into 1,368 species, 628 genera, 217 families, 97 orders, 41 classes and 22 phyla (Do et al., 2014). Among these, 2,131 ORFs (2,12%) belong to 24 bacterial species (account 1,75% bacterial species), 11 families, 9 orders, 8 classes and 5 phyla were predicted have ability to produce cellulases; 679 ORFs belong to 18 bacterial species 8 families, 6 orders, 5 classes, 4 phyla were predicted have ability to produce hemicellulase. Majority of cellulase producers were species which of Firmicutes (15/24 species), accumulated in class Clostridia, order Clostridiales. The most abundant cellulase producer was Pseudomonas fluorescens (1,258 ORFs) of order Pseudomonadaceae. Out of the 18 hemicellulase producers, the most abundant species was Clostridium thermocellum (113 ORFs) in the phylum Firmicutes, followed by 3 species belonging to the phylum Bacteroidetes. The species predicted to produce both cellulase, hemicellulase were C. thermocellum, Ruminococcusns flavefaciens and Bacillus subtilis. Our study provides a data of gut cellulose and hemicellulose - degrading bacteria composition of C. gestroi

Abstract:

ABSTRACTDegradation of cellulose for biofuels production holds promise in solving important environmental and economic problems. However, the low activities (and thus high enzyme-to-substrate ratios needed) of hydrolytic cellulase enzymes, which convert cellulose into simple sugars, remain a major barrier. As a potential strategy to stabilize cellulases and enhance their activities, we have embedded cellulases of extremophiles into hyperstable α-helical consensus ankyrin domain scaffolds. We found the catalytic domains CelA (CA, GH8;Clostridium thermocellum) and Cel12A (C12A, GH12;Thermotoga maritima) to be stable in the context of the ankyrin scaffold and to be active against both soluble and insoluble substrates. The ankyrin repeats in each fusion are folded, although it appears that for the C12Acatalyticdomain (CD; where the N and C termini are distant in the crystal structure), the two flanking ankyrin domains are independent, whereas for CA (where termini are close), the flanking ankyrin domains stabilize each other. Although the activity of CA is unchanged in the context of the ankyrin scaffold, the activity of C12A is increased between 2- and 6-fold (for regenerated amorphous cellulose and carboxymethyl cellulose substrates) at high temperatures. For C12A, activity increases with the number of flanking ankyrin repeats. These results showed ankyrin arrays to be a promising scaffold for constructing designer cellulosomes, preserving or enhancing enzymatic activity and retaining thermostability. This modular architecture will make it possible to arrange multiple cellulase domains at a precise spacing within a single polypeptide, allowing us to search for spacings that may optimize reactivity toward the repetitive cellulose lattice.

Abstract:

Lignocellulolytic enzymes constitute a very large group of extracellular proteins secreting by fungi who is ecologically involved in the degradation of a variety of complex materials, a property that is attributed to a battery of enzymes produced by these microorganisms like cellulases and xylanases who are of significant industrial value and relevance. Forty fungal isolated from rich soil in organic matter were screened for lignocellulolytic enzymes production, its organized on the basis of their hydrolytic potential of cellulose and xylan. The isolates strains presented enzymatic activity which was ranked as follows: cellulolytic (56%), xylanolytic (44%). Some selected strains that produce high levels of enzymes (cellulase, xylanase) grown in submerged fermentation (SmF) and were quantitatively evaluated. The fermentation experiments were carried out in shake flasks. The highest CMCase (5,10 IU/ml) and xylanase (98,25 IU/ml) activities were obtained from Trichoderma sp strain Mtr6 isolate. Keywords: Fungi, Trichoderma sp, lignocellulolytic enzymes, soil, screening, organic matter.

Abstract:

Biotransformation via solid state fermentation (SSF) mediated by microorganisms is a promising approach to produce useful products from agricultural biomass. Lactic acid bacteria (LAB) that are commonly found in fermented foods have been shown to exhibit extracellular proteolytic, β-glucosidase, β-mannosidase, and β-mannanase activities. Therefore, extracellular proteolytic, cellulolytic, and hemicellulolytic enzyme activities of seven Lactobacillus plantarum strains (a prominent species of LAB) isolated from Malaysian foods were compared in this study. The biotransformation of palm kernel cake (PKC) biomass mediated by selected L. plantarum strains was subsequently conducted. The results obtained in this study exhibited the studied L. plantarum strains produced versatile multi extracellular hydrolytic enzyme activities that were active from acidic to alkaline pH conditions. The highest total score of extracellular hydrolytic enzyme activities were recorded by L. plantarum RI11, L. plantarum RG11, and L. plantarum RG14. Therefore, they were selected for the subsequent biotransformation of PKC biomass via SSF. The hydrolytic enzyme activities of treated PKC extract were compared for each sampling interval. The scanning electron microscopy analyses revealed the formation of extracellular matrices around L. plantarum strains attached to the surface of PKC biomass during SSF, inferring that the investigated L. plantarum strains have the capability to grow on PKC biomass and perform synergistic secretions of various extracellular proteolytic, cellulolytic, and hemicellulolytic enzymes that were essential for the effective biodegradation of PKC. The substantial growth of selected L. plamtraum strains on PKC during SSF revealed the promising application of selected L. plantarum strains as a biotransformation agent for cellulosic biomass.

Abstract:

Bacteria in the genus Cellulomonas are well known as secretors of a variety of mesophilic carbohydrate degrading enzymes (e.g., cellulases and hemicellulases), active against plant cell wall polysaccharides. Recent proteomic analysis of the mesophilic bacterium Cellulomonas fimi ATCC484 revealed uncharacterized enzymes for the hydrolysis of plant cell wall biomass. Celf_1230 (CfCel6C), a secreted protein of Cellulomonas fimi ATCC484, is a novel member of the GH6 family of cellulases that could be successfully expressed in Escherichia coli. This enzyme displayed very little enzymatic/hydrolytic activity at 30 °C, but showed an optimal activity around 65 °C, and exhibited a thermal denaturation temperature of 74 °C. In addition, it also strongly bound to filter paper despite having no recognizable carbohydrate binding module. Our experiments show that CfCel6C is a thermostable endoglucanase with activity on a variety of β-glucans produced by an organism that struggles to grow above 30 °C.

Abstract:

We previously demonstrated the existence of bacteria degrading carboxy-methyl-cellulose in the gut of the phytophagous snail Helix aspersa and found Enterobacteriaceae predominating in the intestine of snails dissected in aerobic conditions. Here, we investigated the effects of several nutritional treatments on the snail's microflora. Food sterilization led to increased snail growth and reduced cellulase activity in the crop, suggesting a noxious effect of microbial exogenous cellulases. A second aim of this study was to look for anaerobic bacteria. No strict anaerobic cellulolytic, hom*oacetogenic, or methanogenic bacteria were enriched from the gut. However, a motile Gram-positive hom*olactic coccobacillus, grown in anaerobic conditions, dominated in the snail's intestine (1.57 x 109± 0.10 x 109cells.g-1intestine). It was identified as Enterococcus casseliflavus. Its occurrence in the intestine of H. aspersa is discussed with regard to the snail's digestive processes and the presence of a fecal mucous ribbon. A possible snail-bacterium synergistic action is suggested.Key words: snail, Helix aspersa, gut, Enterococcus casseliflavus, fermentative hom*olactic bacterium, antibiotics.

Abstract:

Microbial Cellulases have an escalating demand in many industries and constitute a major group of the industrial enzymes. It has attracted the attention of many researchers because of its tremendous industrial applications including textile industry, pulp, and paper industry, laundry and detergent industry, food and animal feed industry. The present study pursues to unfold a novel cellulase that can overcome existing challenges in biorefineries as well as to reduce biofuel production cost. Therefore, soil from a dairy farm was screened for potent cellulase producers on carboxymethylcellulose agar. Out of 68 isolates, 31 expressed cellulase activity. The best isolate so far had an extracellular crude enzyme activity of 0.167 U/ml and specific activity of 0.333 U/mg. The cell morphology, cultural characteristics, and biochemical tests presumptively identified it to belong to the genus Bacillus. Molecular analysis using 16S rRNA gene of the isolate indicated it to be Bacillus subtilis. The optimum pH and temperature for the activity of the crude enzyme were determined to be 5 and 65°C respectively. Bangladesh J Microbiol, Volume 33, Number 1-2, June-Dec 2016, pp 17-22

Abstract:

The cellulase proteins have a great importance in the enzymatic hydrolysis of woody biomass. Despite of costs being a major concern, it has been a stimulus to study basidiomycetes biochemical properties which degrade lignocellulosic material and have prompted the processes' study for obtaining cellulolytic enzymes in fungi. The objective of this research was to evaluate the effects of the initial nitrogen content on (ammonium sulfate) and on sugar cane bagasse, which hereby, acts as an inducer of hydrolytic enzymes to produce cellulases and xylanases, using three Lentinula edodes (Berk.) Pegler strains as a transformation agent. A factorial design with 22 replications in the central point was conducted, varying concentrations of ammonium sulfate and sugar cane bagasse. The submerged cultures carried out in synthetic culture medium and incubated at 25°C for 7 days on an orbital shaker at 150 rpm. The total protein and cellulase activity as endoglucanase, exoglucanase and β-glucosidase and the xylanase was also determined. The results showed that the production of hydrolytic enzymes was stimulated by the presence of high concentrations of sugar cane bagasse (30g/L), characterizing it as an inducer due to the demonstrated proportional relationship. Thus, ammonium sulfate acted as a reducing agent in the synthesis of enzymes, being the low concentrations (0.1g/L) indicated for the enzyme production system under study. Among the studied strains, the EF52 showed higher activity for xylanase, endoglucanases, β-glucosidase and also protein.

Abstract:

ABSTRACT Lactose (1,4- O -β- d -galacto-pyranosyl- d -glucose) induces cellulolytic enzymes in Trichoderma reesei and is in fact one of the most important soluble carbon sources used to produce cellulases on an industrial level. The mechanism underlying the induction is, however, not fully understood. In this study, we investigated the cellular functions of the intracellular β-glucosidases CEL1a and CEL1b in the induction of cellulase genes by lactose in T. reesei . We demonstrated that while CEL1a and CEL1b were functionally equivalent in mediating the induction, the simultaneous absence of these intracellular β-glucosidases abolished cbh1 gene expression on lactose. d -Galactose restored the efficient cellulase gene induction in the Δ cel1a strain independently of its reductive metabolism, but not in the Δ cel1a Δ cel1b strain. A further comparison of the transcriptional responses of the Δ cel1a Δ cel1b strain complemented with wild-type CEL1a or a catalytically inactive CEL1a version and the Δ cel1a strain constitutively expressing CEL1a or the Kluyveromyces lactis β-galactosidase LAC4 showed that both the CEL1a protein and its glycoside hydrolytic activity were indispensable for cellulase induction by lactose. We also present evidence that intracellular β-glucosidase-mediated lactose induction is further conveyed to XYR1 to ensure the efficiently induced expression of cellulase genes.

Abstract:

Bioconversion of lignocellulose into renewable energy and commodity products faces a major obstacle of inefficient saccharification due to its recalcitrant structure. In nature, lignocellulose is efficiently degraded by some insects, including termites and beetles, potentially due to the contribution from symbiotic gut bacteria. To this end, the presented investigation reports the isolation and characterization of cellulolytic bacteria from the gut system of red flour beetle, Tribolium castaneum. Out of the 15 isolated bacteria, strain RSP75 showed the highest cellulolytic activities by forming a clearance zone of 28 mm in diameter with a hydrolytic capacity of ~4.7. The MALDI-TOF biotyping and 16S rRNA gene sequencing revealed that the strain RSP75 belongs to Bacillus altitudinis. Among the tested enzymes, B. altitudinis RSP75 showed maximum activity of 63.2 IU/mL extract for xylanase followed by β-glucosidase (47.1 ± 3 IU/mL extract) which were manifold higher than previously reported activities. The highest substrate degradation was achieved with wheat husk and corn cob powder which accounted for 69.2% and 54.5%, respectively. The scanning electron microscopy showed adhesion of the bacterial cells with the substrate which was further substantiated by FTIR analysis that depicted the absence of the characteristic cellulose bands at wave numbers 1247, 1375, and 1735 cm−1 due to hydrolysis by the bacterium. Furthermore, B. altitudinis RSP75 showed co-culturing competence with Saccharomyces cerevisiae for bioethanol production from lignocellulose as revealed by GC-MS analysis. The overall observations signify the gut of T. castaneum as a unique and impressive reservoir to prospect for lignocellulose-degrading bacteria that can have many biotechnological applications, including biofuels and biorefinery.

Abstract:

There is a need to explore lignocellulosic materials to select an adequate substrate for lignocellulolytic enzyme production. Utilization of some residues provides an opportunity to produce high yields of lignocellulolytic enzymes in a simple medium. The aim of the present work was to study the effect of wood flour as a carbon source on the cellulolytic and xylanolytic secretion of white-rot fungi native from Misiones. Fungi were incubated with 5 g Pinus sp. wood flour/L and 5 g Eucalyptus sp. wood flour/L as carbon sources in a reciprocal shaker at 80 rpm and 29ºC for 15 days. Total cellulase, endo-1,4-β-glucanase, β-glucosidase, cellobiohydrolase and endo-1,4-β-xylanase activities were determined in culture supernatants. Tested fungi showed high endo-1,4-β-xylanase activity between 6 and 12 days. Total cellulase showed the highest activity between 12-15 culture days. The test did not show differences among Pycnoporus sanguineus LBM 014, P. sanguineus BAFC 2126, Irpex lacteus BAFC 1171, Irpex sp. LBM 032, Irpex sp. LBM 034 and Lenzites elegans BAFC 2127, showing the highest activity for this group regard the others strains. I. lacteus BAFC 1171 was the strain with major endo-1,4-β-glucanase activity at day 9 (847 U/L). In the case of β-glucosidase and cellobiohydrolase, P. sanguineus BAFC 2126 was the strain with the highest activity between 12-15 culture days (18 U/L), and between 9-15 culture days (39 U/L), respectively. Wood flour proved to be a suitable carbon source to produce hydrolytic enzymes. I. lacteus BAFC 1171 and P. sanguineus BAFC 2126 have potential for cellulase production whereas P. sanguineus LBM 008 is a good endo-1,4-β-xylanase producer.

Abstract:

AbstractIn this study, selected heavy metals resistant heterotrophic bacteria isolated from soil samples at the Windmill Islands region, Wilkes Land (East Antarctica), were characterized. Phylogenetic analysis revealed affiliation of isolates to genera Bacillus, Lysinibacillus, Micrococcus and Stenotrophom*onas. The strains were found to be psychrotolerant and halotolerant, able to tolerate up to 10% NaCl in the growth medium. The Minimum Inhibitory Concentration of the seven heavy metals Cr, Cu, Ni, Co, Cd, Zn, and Pb was determined in solid media for each bacterial strain. Gram−positive Vi−2 strain and Gram−negative Vi−4 strain showed highest multiply heavy metals resistance, and Vi−3 and Vi−4 strains showed multi−antibiotic resistance to more than a half of the 13 used antibiotics. Plasmids were detected only in Gram−negative Vi−4 strain. The bacteria were able to produce different hydrolytic enzymes including industrially important proteases, xylanases, cellulases, and β−glucosidases. High heavy metals resistance of the Antarctic bacteria suggests their potential application for wastewater treatment in cold and temperate climates. Highly sensitive to Cd and Co ions Vi−1, Vi−5 and Vi−7 strains would be promising for developing biosensors to detect these most toxic heavy metals in environmental samples.

Abstract:

To test the hypothesis that selective pressure has led to the retention of cellulose-binding domains (CBDs) by hemicellulase enzymes from aerobic bacteria, four new xylanase (xyn) genes from two cellulolytic soil bacteria, Pseudomonas fluorescens subsp. cellulosa and Cellvibrio mixtus, have been isolated and sequenced. Pseudomonas genes xynE and xynF encoded modular xylanases (XYLE and XYLF) with predicted M(r) values of 68,600 and 65000 respectively. XYLE contained a glycosyl hydrolase family 11 catalytic domain at its N-terminus, followed by three other domains; the second of these exhibited sequence identity with NodB from rhizobia. The C-terminal domain (40 residues) exhibited significant sequence identity with a non-catalytic domain of previously unknown function, conserved in all the cellulases and one of the hemicellulases previously characterized from the pseudomonad, and was shown to function as a CBD when fused to the reporter protein glutathione-S-transferase. XYLF contained a C-terminal glycosyl hydrolase family 10 catalytic domain and a novel CBD at its N-terminus. C. mixtus genes xynA and xynB exhibited substantial sequence identity with xynE and xynF respectively, and encoded modular xylanases with the same molecular architecture and, by inference, the same functional properties. In the absence of extensive cross-hybridization between other multiple cel (cellulase) and xyn genes from P. fluorescens subsp. cellulosa and genomic DNA from C. mixtus, similarity between the two pairs of xylanases may indicate a recent transfer of genes between the two bacteria.

Abstract:

AbstractAssigning a functional role to a microorganism has historically relied on cultivation of isolates or detection of environmental genome-based biomarkers using a posteriori knowledge of function. However, the emerging field of function-driven single-cell genomics aims to expand this paradigm by identifying and capturing individual microbes based on their in situ functions or traits. To identify and characterize yet uncultivated microbial taxa involved in cellulose degradation, we developed and benchmarked a function-driven single-cell screen, which we applied to a microbial community inhabiting the Great Boiling Spring (GBS) Geothermal Field, northwest Nevada. Our approach involved recruiting microbes to fluorescently labeled cellulose particles, and then isolating single microbe-bound particles via fluorescence-activated cell sorting. The microbial community profiles prior to sorting were determined via bulk sample 16S rRNA gene amplicon sequencing. The flow-sorted cellulose-bound microbes were subjected to whole genome amplification and shotgun sequencing, followed by phylogenetic placement. Next, putative cellulase genes were identified, expressed and tested for activity against derivatives of cellulose and xylose. Alongside typical cellulose degraders, including members of the Actinobacteria, Bacteroidetes, and Chloroflexi, we found divergent cellulases encoded in the genome of a recently described candidate phylum from the rare biosphere, Goldbacteria, and validated their cellulase activity. As this genome represents a species-level organism with novel and phylogenetically distinct cellulolytic activity, we propose the name Candidatus ‘Cellulosimonas argentiregionis’. We expect that this function-driven single-cell approach can be extended to a broad range of substrates, linking microbial taxonomy directly to in situ function.

Abstract:

ABSTRACT The nucleotide sequence of the Clostridium thermocellumF7 cbhA gene, coding for the cellobiohydrolase CbhA, has been determined. An open reading frame encoding a protein of 1,230 amino acids was identified. Removal of a putative signal peptide yields a mature protein of 1,203 amino acids with a molecular weight of 135,139. Sequence analysis of CbhA reveals a multidomain structure of unusual complexity consisting of an N-terminal cellulose binding domain (CBD) hom*ologous to CBD family IV, an immunoglobulin-like β-barrel domain, a catalytic domain hom*ologous to cellulase family E1, a duplicated domain similar to fibronectin type III (Fn3) modules, a CBD hom*ologous to family III, a highly acidic linker region, and a C-terminal dockerin domain. The cellulosomal localization of CbhA was confirmed by Western blot analysis employing polyclonal antibodies raised against a truncated enzymatically active version of CbhA. CbhA was identified as cellulosomal subunit S3 by partial amino acid sequence analysis. Comparison of the multidomain structures indicates striking similarities between CbhA and a group of cellulases from actinomycetes. Average linkage cluster analysis suggests a coevolution of the N-terminal CBD and the catalytic domain and its spread by horizontal gene transfer among gram-positive cellulolytic bacteria.

Abstract:

ABSTRACTThe extremely thermophilic, Gram-positive bacteriaCaldicellulosiruptor besciiandCaldicellulosiruptor obsidiansisefficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses fromC. besciiandC. obsidiansis, label-free quantitative proteomics was used to analyze protein abundance over the course of fermentative growth on crystalline cellulose. Both organisms' secretomes consisted of more than 400 proteins, of which the most abundant were multidomain glycosidases, extracellular solute-binding proteins, flagellin, putative pectate lyases, and uncharacterized proteins with predicted secretion signals. Among the identified proteins, 53 to 57 significantly changed in abundance during cellulose fermentation in favor of glycosidases and extracellular binding proteins. Mass spectrometric characterizations, together with cellulase activity measurements, revealed a substantial abundance increase of a few bifunctional multidomain glycosidases composed of glycosidase (GH) domain family 5, 9, 10, 44, or 48 and family 3 carbohydrate binding (CBM3) modules. In addition to their orthologous cellulases, the organisms expressed unique glycosidases with different domain organizations:C. obsidiansisexpressed the COB47_1671 protein with GH10/5 domains, whileC. besciiexpressed the Athe_1857 (GH10/48) and Athe_1859 (GH5/44) proteins. Glycosidases containing CBM3 domains were selectively enriched via binding to amorphous cellulose. Preparations from both bacteria contained highly thermostable enzymes with optimal cellulase activities at 85°C and pH 5. TheC. obsidiansispreparation, however, had higher cellulase specific activity and greater thermostability. TheC. besciiculture produced more extracellular protein and additional SDS-PAGE bands that demonstrated glycosidase activity.

Abstract:

Wood-feeding insects should have a source of enzymes like cellulases to digest their food. These enzymes can be produced by the insect, or by microbes living in the wood and/or inside the insect gut. The coconut rhinoceros beetle, Oryctes rhinoceros, is a pest whose digestive microbes are of considerable interest. This study describes the compartments of the O. rhinoceros gut and compares their microbiomes using culturing-enriched metabarcoding. Beetle larvae were collected from a coconut grove in southern Taiwan. Gut contents from the midgut and hindgut were plated on nutrient agar and selective carboxymethylcellulose agar plates. DNA was extracted from gut and fat body samples and 16S rDNA metabarcoding performed to identify unculturable bacteria. Cellulase activity tests were performed on gut fluids and microbe isolates. The midgut and hindgut both showed cellulolytic activity. Bacillus cereus, Citrobacter koseri, and the cellulolytic fungus Candida xylanilytica were cultured from both gut sections in most larvae. Metabarcoding did not find Bacillus cereus, and found that either Citrobacter koseri or Paracoccus sp. were the dominant gut microbes in any given larva. No significant differences were found between midgut and hindgut microbiomes. Bacillus cereus and Citrobacter koseri are common animal gut microbes frequently found in Oryctes rhinoceros studies while Candida xylanilytica and the uncultured Paracoccus sp. had not been identified in this insect before. Some or all of these may well have digestive functions for the beetle, and are most likely acquired from the diet, meaning they may be transient commensalists rather than obligate mutualists. Broader collection efforts and tests with antibiotics will resolve ambiguities in the beetle–microbe interactions.

Abstract:

<abstract> <p>The camel is well-adapted to utilize the poor-quality forages in the harsh desert conditions as the camel rumen sustains fibrolytic microorganisms, mainly bacteria that are capable of breaking down the lignocellulosic biomass efficiently. Exploring the composition of the bacterial community in the rumen of the camel and quantifying their cellulolytic and xylanolytic activities could lead to understanding and improving fiber fermentation and discovering novel sources of cellulases and xylanases. In this study, Illumina MiSeq sequencing of the V4 region on 16S rRNA was applied to identify the bacterial and archaeal communities in the rumen of three camels fed wheat straw and broom corn. Furthermore, rumen samples were inoculated into bacterial media enriched with xylan and different cellulose sources, including filter paper (FP), wheat straw (WS), and alfalfa hay (AH) to assess the ability of rumen bacteria to produce endo-cellulase and endo-xylanase at different fermentation intervals. The results revealed that the phylum Bacteroidetes dominated the bacterial community and <italic>Candidatus Methanomethylophilus</italic> dominated the archaeal community. Also, most of the bacterial community has fibrolytic potential and the dominant bacterial genera were <italic>Prevotella</italic>, <italic>RC9_gut_group</italic>, <italic>Butyrivibrio</italic>, <italic>Ruminococcus</italic>, <italic>Fibrobacteres</italic>, and <italic>Treponema</italic>. The highest xylanase production (884.8 mU/mL) was observed at 7 days. The highest cellulase production (1049.5 mU/mL) was observed when rumen samples were incubated with Alfalfa hay for 7 days.</p> </abstract>

Abstract:

Cellulosomes, which are multienzyme complexes from anaerobic bacteria, are considered nature’s finest cellulolytic machinery. Thus, constructing a cellulosome in an industrial yeast has long been a goal pursued by scientists. However, it remains highly challenging due to the size and complexity of cellulosomal genes. Here, we overcame the difficulties by synthesizing the Clostridium thermocellum scaffoldin gene (CipA) and the anchoring protein gene (OlpB) using advanced synthetic biology techniques. The engineered Kluyveromyces marxianus, a probiotic yeast, secreted a mixture of dockerin-fused fungal cellulases, including an endoglucanase (TrEgIII), exoglucanase (CBHII), β-glucosidase (NpaBGS), and cellulase boosters (TaLPMO and MtCDH). The confocal microscopy results confirmed the cell-surface display of OlpB-ScGPI and fluorescence-activated cell sorting analysis results revealed that almost 81% of yeast cells displayed OlpB-ScGPI. We have also demonstrated the cellulosome complex formation using purified and crude cellulosomal proteins. Native polyacrylamide gel electrophoresis and mass spectrometric analysis further confirmed the cellulosome complex formation. Our engineered cellulosome can accommodate up to 63 enzymes, whereas the largest engineered cellulosome reported thus far could accommodate only 12 enzymes and was expressed by a plasmid instead of chromosomal integration. Interestingly, CipA 2B9C (with two cellulose binding modules, CBM) released significantly higher quantities of reducing sugars compared with other CipA variants, thus confirming the importance of cohesin numbers and CBM domain on cellulosome complex. The engineered yeast host efficiently degraded cellulosic substrates and released 3.09 g/L and 8.61 g/L of ethanol from avicel and phosphoric acid-swollen cellulose, respectively, which is higher than any previously constructed yeast cellulosome.

Abstract:

ABSTRACT Thermococcus species are widely distributed in terrestrial and marine hydrothermal areas, as well as in deep subsurface oil reservoirs. Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a well of the never flooded oil-bearing Jurassic horizon of a high-temperature oil reservoir. To obtain insight into the genome of an archaeon inhabiting the oil reservoir, we have determined and annotated the complete 1,845,800-base genome of T. sibiricus. A total of 2,061 protein-coding genes have been identified, 387 of which are absent in other members of the order Thermococcales. Physiological features and genomic data reveal numerous hydrolytic enzymes (e.g., cellulolytic enzymes, agarase, laminarinase, and lipases) and metabolic pathways, support the proposal of the indigenous origin of T. sibiricus in the oil reservoir, and explain its survival over geologic time and its proliferation in this habitat. Indeed, in addition to proteinaceous compounds known previously to be present in oil reservoirs at limiting concentrations, its growth was stimulated by cellulose, agarose, and triacylglycerides, as well as by alkanes. Two polysaccharide degradation loci were probably acquired by T. sibiricus from thermophilic bacteria following lateral gene transfer events. The first, a “saccharolytic gene island” absent in the genomes of other members of the order Thermococcales, contains the complete set of genes responsible for the hydrolysis of cellulose and β-linked polysaccharides. The second harbors genes for maltose and trehalose degradation. Considering that agarose and laminarin are components of algae, the encoded enzymes and the substrate spectrum of T. sibiricus indicate the ability to metabolize the buried organic matter from the original oceanic sediment.

Abstract:

Background Lignocellulose is a renewable and enormous biomass resource, which can be degraded efficiently by a range of co*cktails of carbohydrate-active enzymes secreted by termite gut symbiotic bacteria. There is an urgent need to find enzymes with novel characteristics for improving the conversion processes in the production of lignocellulosic-based products. Although various studies dedicated to the genus Cellulosimicrobium as gut symbiont, genetic potential related to plant biomass-acting enzymes and exopolysaccharides production has been fully untapped to date. Methods The cellulolytic bacterial strain MP1 was isolated from termite guts and identified to the species level by phenotypic, phylogenetic, and genomic analysis. To further explore genes related to cellulose and hemicellulose degradation, the draft genome of strain MP1 was obtained by using whole-genome sequencing, assembly, and annotation through the Illumina platform. Lignocellulose degrading enzymes and levan production in the liquid medium were also examined to shed light on bacterial activities. Results Among 65 isolates obtained, the strain MP1 was the most efficient cellulase producer with cellulase activity of 0.65 ± 0.02 IU/ml. The whole genome analysis depicted that strain MP1 consists of a circular chromosome that contained 4,580,223 bp with an average GC content of 73.9%. The genome comprises 23 contigs including 67 rRNA genes, three tRNA genes, a single tmRNA gene, and 4,046 protein-coding sequences. In support of the phenotypic identification, the 16S rRNA gene sequence, average nucleotide identity, and whole-genome-based taxonomic analysis demonstrated that the strain MP1 belongs to the species Cellulosimicrobium cellulans. A total of 30 genes related to the degradation of cellulases and hemicellulases were identified in the C. cellulans MP1 genome. Of note, the presence of sacC1-levB-sacC2-ls operon responsible for levan and levan-type fructooligosaccharides biosynthesis was detected in strain MP1 genome, but not with closely related C. cellulans strains, proving this strain to be a potential candidate for further studies. Endoglucanases, exoglucanases, and xylanase were achieved by using cheaply available agro-residues such as rice bran and sugar cane bagasse. The maximum levan production by C. cellulans MP1 was 14.8 ± 1.2 g/l after 20 h of cultivation in media containing 200 g/l sucrose. To the best of our knowledge, the present study is the first genome-based analysis of a Cellulosimicrobium species which focuses on lignocellulosic enzymes and levan biosynthesis, illustrating that the C. cellulans MP1 has a great potential to be an efficient platform for basic research and industrial exploitation.

Abstract:

Cellulases are inducible enzymes that are synthesized by many microorganisms during their growth on cellulosic materials. Production of cellulase enzyme from bacteria has gained interest for applications in industries owing to their stability, catalytic activity and ease of production. In current study, total 40 cellulolytic bacteria were screened by agar well diffusion assay followed by Congo red stain. Cellulolytic bacteria were evaluated for detergent compatibility and de-staining ability. Bacterial cellulase production was optimized at different environmental conditions. Biochemical testing was done following Bergey’s manual. Ten cellulolytic bacteria selected, were gram positive. Bacteria showed best cellulolytic activity at 40oC to 60oC, at pH 9, lactose as carbon source and peptone as nitrogen source. Best hydrolysis zone shown was 45mm from strain MS22 and least zone was 6mm. The maximum detergent activity for surf excel was observed for G2 while the lowest was observed for MW22. Similarly, maximum activity for ariel and bonus were observed for RS5 and C3. G1 and C3 showed better clearance of ink as compared to other strains. In de-staining, G1, C1and MW17 showed better clearance of edible oil and G1, C2 and MW18 showed better clearance of grease. These promising cellulolytic bacteria can be used for various applications in different industries.

Abstract:

Knowledge in the field of bacterial endophytes associated with the plant Capsicum chinense is negligible. So in order to characterize the endophytic population in the targeted plant, different accessions of C. chinense plant were procured from different agro-climatic zones of India. Bacterial endophytes were isolated by using standard protocols. After isolation of the endophytes, a biochemical identification study was performed using the standard key. Secondary metabolites of these bacterial species were studied for their economic importance. One isolate of cellulose-degrading bacteria (CDB) was isolated from the roots of C. chinense. The cellulase activity of the endophyte, containing cellulose Congo Red agar. Finally, enzyme assays for the cellulase (endoglucanase) and filter paper cellulase or FPC assay was studied. The maximum clearing zone for the isolate was 50mm, and the hydrolytic capacity (HC) was found to be 5.96. The endoglucanase activity of 0.95 IU/mL and the filter paper Filter Paper Cellulase (FPCase) activity was found to be 0.25 IU/mL. The importance of the study is attributed to the fact that this is the first-ever study of the enzymatic activity of endophytic bacteria isolated from C. chinense collected from North-East India.

Abstract:

Research background. Cellulose is an ingredient of waste materials that can be converted to other valuable substances. This is possible provided that, the polymer molecule will be degraded to smaller particles, used as a carbon source by microorganisms. Because of the frequently applied methods of pre-treatment of lignocellulosic materials, the cellulases derived from thermophilic microorganisms are particularly desirable. Experimental approach. We were looking for cellulolytic microorganisms able to grow at 50 °C. We described their morphological features and biochemical characteristics based on CMCase activity and the api®ZYM. The growth curves, during incubation at 50 °C, were examined using the microbioreactor BioLector®. Results and conclusions. 40 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 microorganism. The CMCase activity was confirmed for 27 strains. However, the hydrolysis capacities (HC) 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 presented, among others, by 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 contribution. We 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.s lactis. Described strains can be a source of new thermostable cellulases, which are extremely desirable in various branches of the circular bioeconomy.

Abstract:

ABSTRACTHeterologous display of enzymes on microbial cell surfaces is an extremely desirable approach, since it enables the engineered microbe to interact directly with the plant wall extracellular polysaccharide matrix. In recent years, attempts have been made to endow noncellulolytic microbes with genetically engineered cellulolytic capabilities for improved hydrolysis of lignocellulosic biomass and for advanced probiotics. Thus far, however, owing to the hurdles encountered in secreting and assembling large, intricate complexes on the bacterial cell wall, only free cellulases or relatively simple cellulosome assemblies have been introduced into live bacteria. Here, we employed the “adaptor scaffoldin” strategy to compensate for the low levels of protein displayed on the bacterial cell surface. That strategy mimics natural elaborated cellulosome architectures, thus exploiting the exponential features of their Lego-like combinatorics. Using this approach, we produced several bacterial consortia ofLactobacillus plantarum, a potent gut microbe which provides a very robust genetic framework for lignocellulosic degradation. We successfully engineered surface display of large, fully active self-assembling cellulosomal complexes containing an unprecedented number of catalytic subunits all producedin vivoby the cell consortia. Our results demonstrate that the enzyme stability and performance of the cellulosomal machinery, which are superior to those seen with the equivalent secreted free enzyme system, and the high cellulase-to-xylanase ratios proved beneficial for efficient degradation of wheat straw.IMPORTANCEThe multiple benefits of lactic acid bacteria are well established in health and industry. Here we present an approach designed to extensively increase the cell surface display of proteins via successive assembly of interactive components. Our findings present a stepping stone toward proficient engineering ofLactobacillus plantarum, a widespread, environmentally important bacterium and potent microbiome member, for improved degradation of lignocellulosic biomass and advanced probiotics.

Abstract:

Chaetomium species are saprophytic fungi, havethe ability to degrade cellulose with hydrolytic enzymes. Hydrolytic enzymes are a cluster of enzymes, which have the flexibility of breaking down complicated molecules into smaller molecules. The objective of this study was to clarify the genetic diversity among the isolated Chaetomium population by using PCR-based methods, along with the study of their cellulolytic activities. Chaetomium spp. were isolated from twenty-eight samples of Cuminum cyminum and Pimpinella anisum, and identified morphologically and molecularly by ITS1 and ITS4 primers. The sequencing indicated that all the isolates of Chaetomium had a 99% sequence identity with Chaetomium globosum sequences from GenBank. Molecular techniques with Internal Transcribed Spacer (ITS) region sequencing and specific genes random primers polymerase chain reaction (SGRP-PCR) showed the existence of high DNA polymorphism of Chaetomium globosum. All isolates were tested for their ability of cellulases production. Nine of the thirteen isolates of Chaetomium globosumcould produce cellulases. There are variations between C. globosum isolates and the source of the isolates and their habitat do not correlate to their ability to secrete cellulases.