RESUMO
Although studies on non-tuberculous mycobacteria have increased in recent years because they cause a considerable proportion of infections, their cellulolytic system is still poorly studied. This study presents a characterization of the cellulolytic activities of environmental mycobacterial isolates derived from soil and water samples from the central region of Argentina, aimed to evaluate the conservation of the mechanism for the degradation of cellulose in this group of bacteria. The molecular and genomic identification revealed identity with Mycolicibacterium septicum. The endoglucanase and total cellulase activities were assessed both qualitatively and quantitatively and the optimal enzymatic conditions were characterized. A specific protein of around 56 kDa with cellulolytic activity was detected in a zymogram. Protein sequences possibly arising from a cellulase were identified by mass spectrometry-based shotgun proteomics. Results showed that M. septicum encodes for cellulose- and hemicellulose-related degrading enzymes, including at least an active ß-1,4 endoglucanase enzyme that could be useful to improve its survival in the environment. Given the important health issues related to mycobacteria, the results of the present study may contribute to the knowledge of their cellulolytic system, which could be important for their ability to survive in many different types of environments.
Assuntos
Proteínas de Bactérias , Celulase , Celulose , Microbiologia do Solo , Celulose/metabolismo , Celulase/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Argentina , Microbiologia da Água , Proteômica/métodos , Mycobacteriaceae/genética , Mycobacteriaceae/enzimologiaRESUMO
The paper industry is a composite one constituting different types of mills, processes, and products. The paper industries consume large amounts of resources, like wood and water. These industries also create huge amounts of waste that have to be treated. In our study, 23 endophytic bacteria were isolated from Argemone mexicana, and 16 endophytic bacteria were isolated from Papaver rhoeas. Seventeen and 15 bacterial endophytes from A. mexicana and P. rhoeas, respectively, showed cellulose-degrading activity. The biochemical and molecular characterization were done for endophytic bacteria with cellulolytic activity. The consortium of cellulose-degrading endophytic bacteria from A. mexicana showed endoglucanase activity (0.462 IU/ml) and FPCase enzyme activity (0.269 IU/ml) and from P. rhoeas gave endoglucanase activity (0.439 IU/ml) and FPCase enzyme activity (0.253 IU/ml). Degraded carboxy methylcellulose and filter paper were further treated by Saccharomyces cerevisiae and bioethanol was produced. Cellulose-degrading endophytic bacteria were also tested for auxin, siderophore production, and phosphate solubilization activities. Individual cellulose-degrading endophytic bacteria with plant growth-promoting activities were used as biofertilizers, tested for plant growth-promoting activities using Basmati Pusa 1121 rice, and plant growth parameters were recorded. The degraded paper enhances the growth of rice plants. Selected bacterial endophytes and their consortia from A. mexicana and P. rhoeas were powerful cellulose degraders, which can be further employed for ethanol production and as significant biofertilizers in agriculture.
RESUMO
Potato soft rot caused by Pectobacterium spp. are devastating diseases of potato which cause severe economic losses worldwide. Pectobacterium brasiliense is considered as one of the most virulent species. However, the virulence mechanisms and pathogenicity factors of this strain have not been fully elucidated. Here, through pathogenicity screening, we identified two Pectobacterium brasiliense isolates, SM and DQ, with distinct pathogenicity levels. SM exhibits higher virulence compared to DQ in inducing aerial stem rot, blackleg and tuber soft rot. Our genomic and transcriptomic analyses revealed that SM encodes strain specific genes with regard to plant cell wall degradation and express higher level of genes associated with bacterial motility and secretion systems. Our plate assays verified higher pectinase, cellulase, and protease activities, as well as fast swimming and swarming motility in SM. Importantly, a unique endoglucanase S specific to SM was identified. Expression of this cellulase in DQ greatly enhances its virulence compared to wild type strain. Our study sheds light on possible determinants causing different pathogenicity of Pectobacterium brasiliense species with close evolutionary distance and provides new insight into the direction of genome evolution in response to host variation and environmental stimuli.
RESUMO
The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by ß-1,4 bonds. The enzyme ß-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A ß-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a ß-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa ß-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated ß-1,4-endoglucanase had higher activity and stability.
Assuntos
Bacillus subtilis , Celulase , Papel , Proteínas Recombinantes , Águas Residuárias , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/isolamento & purificação , Águas Residuárias/microbiologia , Águas Residuárias/química , Celulase/genética , Celulase/química , Celulase/biossíntese , Celulase/isolamento & purificação , Celulase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Clonagem Molecular , Expressão GênicaRESUMO
The lignocellulose bioconversion process is an eco-friendly and green-economy alternative technology that allows the reduction of pollution and global warming, so it is necessary for thermophilic and thermostable hydrolytic enzymes from natural sources. This research aimed to isolate cellulolytic and xylanolytic microbial consortia from Huancarhuaz hot spring (Peru) from sludge or in situ baiting cultured with or without sugarcane bagasse. According to the hydrolytic activities consortium T4 from in situ baiting was selected. It was cultivated in submerged fermentation at 65 °C, pH 6.5 for eight days using LB supplemented with sugar cane bagasse (SCB), pine wood sawdust (PWS), CMC, xylan of birchwood, or micro granular cellulose. Crude extract of culture supplemented with SCB (T4B) showed better endoglucanase and xylanase activities with higher activities at 75 °C and pH 6. In these conditions, cellulase activity was kept up to 57% after 1 h of incubation, while xylanase activity was up to 63% after 72 h. Furthermore, this crude extract released reduced sugars from pretreated SCB and PWS. According to metagenomic analysis of 16S rDNA, Geobacillus was the predominant genus. It was found thermostable genes: a type of endoglucanase (GH5), an endo-xylanase (GH10), and alkali xylanase (GH10) previously reported in Geobacillus sp. strains. Finally, Huancarhuaz hot spring harbors a genetic microbial diversity for lignocellulosic waste bioconversion in high temperatures, and the T4B consortium will be a promising source of novel extreme condition stable enzymes for the saccharification process.
RESUMO
Cellulases from anaerobic fungi are enzymes less-studied biochemically and structurally than cellulases from bacteria and aerobic fungi. Currently, only thirteen GH5 cellulases from anaerobic fungi were biochemically characterized and two crystal structures were reported. In this context, here, we report the functional and biophysical characterization of a novel multi-modular cellulosomal GH5 endoglucanase from the anaerobic gut fungus Piromyces finnis (named here PfGH5). Multiple sequences alignments indicate that PfGH5 is composed of a GH5 catalytic domain and a CBM1 carbohydrate-binding module connected through a CBM10 dockerin module. Our results showed that PfGH5 is an endoglucanase from anaerobic fungus with a large spectrum of activity. PfGH5 exhibited preference for hydrolysis of oat ß-glucan, followed by galactomannan, carboxymethyl cellulose, mannan, lichenan and barley ß-glucan, therefore displaying multi-functionality. For oat ß-glucan, PfGH5 reaches its optimum enzymatic activity at 40 °C and pH 5.5, with Km of 7.1 µM. Ion exchange chromatography analyzes revealed the production of oligosaccharides with a wide degree of polymerization indicated that PfGH5 has endoglucanase activity. The ability to bind and cleave different types of carbohydrates evidence the potential of PfGH5 for use in biotechnology and provide a useful basis for future investigation and application of new anaerobic fungi enzymes.
Assuntos
Celulase , Celulases , Celulase/química , Anaerobiose , FungosRESUMO
In this study, a simple and scalable mechanical pretreatment was evaluated as means of enhancing the accessibility of cellulose fibers, with the objective of improving the efficiency of enzymatic reactions for the production of cellulose nanoparticles (CNs). In addition, the effects of enzyme type (endoglucanase - EG, endoxylanase - EX, and a cellulase preparation - CB), composition ratio (0-200UEG:0-200UEX or EG, EX, and CB alone), and loading (0 U-200 U) were investigated in relation to CN yield, morphology, and properties. The combination of mechanical pretreatment and specific enzymatic hydrolysis conditions substantially improved CN production yield, reaching up to 83 %. The production of rod-like or spherical nanoparticles and their chemical composition were highly influenced by the enzyme type, composition ratio, and loading. However, these enzymatic conditions had minimal impact on the crystallinity index (approximately 80 %) and thermal stability (Tmax within 330-355 °C). Overall, these findings demonstrate that mechanical pretreatment followed by enzymatic hydrolysis under specific conditions is a suitable method to produce nanocellulose with high yield and adjustable properties such as purity, rod-like or spherical forms, high thermal stability, and high crystallinity. Therefore, this production approach shows promise in producing tailored CNs with the potential for superior performance in various advanced applications, including, but not limited to, wound dressings, drug delivery, thermoplastic composites, 3D (bio)printing, and smart packaging.
Assuntos
Celulase , Nanopartículas , Celulose/química , Hidrólise , Celulase/química , Endo-1,4-beta-Xilanases/química , Nanopartículas/químicaRESUMO
Cellulose nanofibrils (CNFs) have emerged as a potential alternative to synthetic polymers in packaging applications owing to their oxygen and grease barrier performance, as well as their strong mechanical properties. However, the performance of CNF films relies on the inherent characteristics of fibers, which undergo changes during the CNF isolation process. Understanding these variations in characteristics during CNF isolation is crucial for tailoring CNF film properties to achieve optimum performance in packaging applications. In this study, CNFs were isolated by endoglucanase-assisted mechanical ultra-refining. The alterations in the intrinsic characteristics of CNFs and their impact on CNF films were systematically investigated by considering the degree of defibrillation, enzyme loading, and reaction time through a design of experiments. Enzyme loading had a significant influence on the crystallinity index, crystallite size, surface area, and viscosity. Meanwhile, the degree of defibrillation greatly affected the aspect ratio, degree of polymerization, and particle size. CNF films prepared from CNFs isolated under two optimized scenarios (casting and coating applications) exhibited remarkable properties, including high thermal stability (approximately 300 °C), high tensile strength (104 - 113 MPa), excellent oil resistance (kit n°12), and low oxygen transmission rate (1.00 - 3.17 cc·m-2.day-1). Therefore, endoglucanase pretreatment can aid in obtaining CNFs with lower energy consumption, resulting in films that possess higher transmittance, superior barrier performance, and reduced surface wettability compared to control samples without enzymatic pretreatment and other unmodified CNF films reported in the literature, all while maintaining mechanical and thermal performance without significant loss.
Assuntos
Celulase , Nanofibras , Celulose , Embalagem de Produtos , Resistência à Tração , OxigênioRESUMO
Bleached kraft pulps from eucalyptus and pine were subjected to cold caustic extraction (CCE) with NaOH (5, 10, 17.5, and 35%) for hemicelluloses removal and to increase cellulose accessibility. The effect of these changes was evaluated in enzymatic saccharification with the multicomponent Cellic CTec3 enzyme cocktail, and in viscosity reduction of pulps with the monocomponent Trichoderma reesei endoglucanase (EG). After CCE with 10% NaOH (CCE10) and 17.5% NaOH (CCE17.5), hemicellulose content lower than 1% was achieved in eucalyptus and pine pulps, respectively. At these concentrations, cellulose I started to be converted into cellulose II. NaOH concentrations higher than 17.5% decreased the intrinsic viscosity (from 730 to 420 mL/g in eucalyptus and from 510 to 410 mL/g in pine). Cellulose crystallinity was reduced from 60% to 44% in eucalyptus and from 71% to 44% in pine, as the NaOH concentration increased. Enzymatic multicomponent saccharification showed higher glucose yields in all CCE-treated eucalyptus samples (up to 93%) while only CCE17.5 and CCE35 pine pulps achieved 90% after 40 h of incubation. Untreated bleached pulps of both species presented saccharification yields lower than 70%. When monocomponent EG was used to treat the same pulps, depending on enzyme charge and incubation time, a wide range of intrinsic viscosity reduction was obtained (up to 74%). Results showed that eucalyptus pulps are more accessible and easier to hydrolyze by enzymes than pine pulps and that the conversion of cellulose I to cellulose II hydrate only has the effect of increasing saccharification of CCE pine samples. Viscosity reduction of CCE pulps and EG treated pulps were obtained in a wide range indicating that pulps presented characteristics suitable for cellulose derivatives production.
RESUMO
Enzyme-mediated isolation of cellulose nanocrystals (CNCs) is a promising environment friendly method with expected lower capital and operating expenditures compared to traditional processes. However, it is still poorly understood. In this study, an endoxylanase was applied as accessory enzyme to assess its potential to increase the selectivity of an endoglucanase during cellulose hydrolysis to isolate CNCs with improved properties. Only combinations of the enzymes with xylanase activity equal to or higher than the endoglucanase activity resulted in CNCs with improved properties (i.e., crystallinity, thermostability, uniformity, suspension stability and aspect ratio). The beneficial effects of the accessory enzyme are related to its hydrolytic (xylan and cellulose hydrolysis) and non-hydrolytic action (swelling of cellulose fibers and fiber porosity) and on the ratio of the enzymes, which in turn allows to tailor the properties of the CNCs. In conclusion, compared to the traditional sulfuric acid hydrolysis method, accessory enzymes help to isolate cellulose nanomaterials with improved and customized (sizes, aspect ratio and morphology) properties that may allow for new applications.