RESUMO
BACKGROUND: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate. RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71. CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.
Assuntos
Bacillus amyloliquefaciens , Celulase , Clonagem Molecular , Simulação de Acoplamento Molecular , Oryza , Celulase/genética , Celulase/biossíntese , Celulase/metabolismo , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Oryza/microbiologia , Fermentação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/químicaRESUMO
Halophiles are one of the classes of extremophilic microorganisms that can flourish in environments with very high salt concentrations. In this study, fifteen bacterial strains isolated from various crop rhizospheric soils of agricultural fields along the Southwest coastline of Saurashtra, Gujarat, and identified by 16S rRNA gene sequencing as Halomonas pacifica, H. stenophila, H. salifodinae, H. binhaiensis, Oceanobacillus oncorhynchi, and Bacillus paralicheniformis were investigated for their potentiality to produce extremozymes and compatible solute. The isolates showed the production of halophilic protease, cellulase, and chitinase enzymes ranging from 6.90 to 35.38, 0.004-0.042, and 0.097-0.550 U ml-1, respectively. The production of ectoine-compatible solute ranged from 0.01 to 3.17 mg l-1. Furthermore, the investigation of the ectoine-compatible solute production at the molecular level by PCR showed the presence of the ectoine synthase gene responsible for its biosynthesis in the isolates. Besides, it also showed the presence of glycine betaine biosynthetic gene betaine aldehyde dehydrogenase in the isolates. The compatible solute production by these isolates may be linked to their ability to produce extremozymes under saline conditions, which could protect them from salt-induced denaturation, potentially enhancing their stability and activity. This correlation warrants further investigation.
Assuntos
RNA Ribossômico 16S , Rizosfera , Microbiologia do Solo , RNA Ribossômico 16S/genética , Diamino Aminoácidos/biossíntese , Diamino Aminoácidos/metabolismo , Índia , Produtos Agrícolas/microbiologia , Celulase/metabolismo , Celulase/genética , Celulase/biossíntese , Quitinases/metabolismo , Quitinases/genética , Tolerância ao Sal/genética , Filogenia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genética , Bactérias/genética , Bactérias/metabolismo , Bactérias/isolamento & purificação , Bactérias/classificação , Bacillus/genética , Bacillus/metabolismo , Bacillus/isolamento & purificaçãoRESUMO
To harness the diverse industrial applications of cellulase, including its use in the food, pulp, textile, agriculture, and biofuel sectors, this study focused on the high-yield production of a bioactive insect-derived endoglucanase, Monochamus saltuarius glycoside hydrolase family 5 (MsGHF5). MsGHF5 was introduced into the genome of Kluyveromyces lactis to maintain expression stability, and mass production of the enzyme was induced using fed-batch fermentation. After 40 h of cultivation, recombinant MsGHF5 was successfully produced in the culture broth, with a yield of 29,000 U/L, upon galactose induction. The optimal conditions for the activity of purified MsGHF5 were determined to be a pH of 5 and a temperature of 35 °C, with the presence of ferrous ions enhancing the enzymatic activity by up to 1.5-fold. Notably, the activity of MsGHF5 produced in K. lactis was significantly higher than that produced in Escherichia coli, suggesting that glycosylation is crucial for the functional performance of the enzyme. This study highlights the potential use of K. lactis as a host for the production of bioactive MsGHF5, thus paving the way for its application in various industrial sectors.
Assuntos
Celulase , Kluyveromyces , Proteínas Recombinantes , Animais , Kluyveromyces/genética , Kluyveromyces/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Celulase/genética , Celulase/química , Celulase/biossíntese , Celulase/isolamento & purificação , Celulase/metabolismo , Besouros/enzimologia , Besouros/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Proteínas de Insetos/genética , Proteínas de Insetos/química , Proteínas de Insetos/biossíntese , Proteínas de Insetos/metabolismo , Proteínas de Insetos/isolamento & purificação , Concentração de Íons de HidrogênioRESUMO
Lignocellulolytic enzymes play a crucial role in efficiently converting lignocellulose into valuable platform molecules in various industries. However, they are limited by their production yields, costs, and stability. Consequently, their production by producers adapted to local environments and the choice of low-cost raw materials can address these limitations. Due to the large amounts of olive stones (OS) generated in Morocco which are still undervalued, Penicillium crustosum, Fusarium nygamai, Trichoderma capillare, and Aspergillus calidoustus, are cultivated under different fermentation techniques using this by-product as a local lignocellulosic substrate. Based on a multilevel factorial design, their potential to produce lignocellulolytic enzymes during 15 days of dark incubation was evaluated. The results revealed that P. crustosum expressed a maximum total cellulase activity of 10.9 IU/ml under sequential fermentation (SF) and 3.6 IU/ml of ß-glucosidase activity under submerged fermentation (SmF). F. nygamai recorded the best laccase activity of 9 IU/ml under solid-state fermentation (SSF). Unlike T. capillare, SF was the inducive culture for the former activity with 7.6 IU/ml. A. calidoustus produced, respectively, 1,009 µg/ml of proteins and 11.5 IU/ml of endoglucanase activity as the best results achieved. Optimum cellulase production took place after the 5th day under SF, while ligninases occurred between the 9th and the 11th days under SSF. This study reports for the first time the lignocellulolytic activities of F. nygamai and A. calidoustus. Furthermore, it underlines the potential of the four fungi as biomass decomposers for environmentally-friendly applications, emphasizing the efficiency of OS as an inducing substrate for enzyme production.
Assuntos
Fermentação , Lignina , Olea , Lignina/metabolismo , Olea/microbiologia , Aspergillus/enzimologia , Aspergillus/metabolismo , Celulase/metabolismo , Celulase/biossíntese , Lacase/metabolismo , Lacase/biossíntese , Penicillium/enzimologia , Penicillium/metabolismo , beta-Glucosidase/metabolismo , beta-Glucosidase/biossíntese , Fusarium/enzimologia , Fusarium/metabolismo , Trichoderma/enzimologia , Trichoderma/metabolismo , Fungos/enzimologia , Fungos/metabolismo , Marrocos , Proteínas Fúngicas/metabolismoRESUMO
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
Endophytic microbial communities colonize plants growing under various abiotic stress conditions. Candelilla (Euphorbia antisyphilitica Zucc.) is a shrub that develops functionally in arid and semi-arid zones of Mexico; these conditions generate an association between the plant and the microorganisms, contributing to the production of enzymes as a defense mechanism for resistance to abiotic stress. The objective of this research was to isolate and identify endophyte fungi of candelilla and bioprospection of these endophytic fungi for enzyme production using candelilla by-products. Fungi were isolated and identified using ITS1/ITS4 sequencing. Their potency index (PI) was evaluated in producing endoglucanase, xylanase, amylase, and laccase. Fermentation was carried out at 30°C for 8 days at 200 rpm, with measurements every 2 days, using candelilla by-products as substrate. All fungi exhibited higher cellulase, amylase, and laccase activities on the 2nd, 6th, and 8th day of fermentation, respectively, of fermentation. The fungus Aspergillus niger ITD-IN4.1 showed the highest amylase activity (246.84 U/mg), the genus Neurospora showed the highest cellulase activity, reaching up to 13.45 FPU/mg, and the strain Neurospora sp. ITD-IN5.2 showed the highest laccase activity (3.46 U/mg). This work provides the first report on the endophytic diversity of E. antisyphilitica and its potential role in enzyme production.
Assuntos
Bioprospecção , Celulase , Endófitos , Fermentação , Lacase , Endófitos/isolamento & purificação , Endófitos/enzimologia , Endófitos/metabolismo , Endófitos/genética , Lacase/metabolismo , Lacase/biossíntese , Celulase/metabolismo , Celulase/biossíntese , Amilases/metabolismo , Aspergillus niger/isolamento & purificação , Aspergillus niger/enzimologia , México , Neurospora , Fungos/isolamento & purificação , Fungos/enzimologia , Fungos/classificação , Fungos/genéticaRESUMO
The filamentous fungus Trichoderma reesei is widely used for industrial cellulase production. In T. reesei, cellulase gene expression is tightly controlled by a regulatory network involving multiple transcription factors. Here, we isolated a novel protein, Rce2, using a pull-down assay and mass spectrometry analysis, from a partial carbon catabolite de-repression mutant, T. reesei Rut-C30, cultured under glucose-repressing conditions. Deletion and overexpression of Rce2 in T. reesei wild-type QM6a and mutant Rut-C30 revealed that Rce2 acts as a repressor of cellulase gene expression. DNase I footprinting assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Rce2 was located in the nucleus and bound to the consensus sequences 5'-(T/A)NNNNCCG-3' and 5'-CGGNNNN(T/A)-3' in the promoters of cellulase-related genes to repress their transcription. Additionally, Rce2 antagonized Ace3 binding to the cbh1 promoter to repress its transcription. However, Rce2 was not involved in Cre1-mediated carbon catabolite repression. These results demonstrate the mechanism through which Rce2 represses the expression of cellulase genes and provide novel insights into the regulatory system of cellulases and methods that can be used for the regulation of gene expression in T. reesei.
Assuntos
Celulase/biossíntese , Celulase/genética , Hypocreales/genética , Hypocreales/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Repressão Catabólica , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Microbiologia Industrial , Filogenia , Regiões Promotoras GenéticasRESUMO
The filamentous fungus Trichoderma reesei is an extraordinarily efficient cell factory of industrial cellulase for production of biofuels and other bio-based products because of its excellent potential to secrete cellulolytic enzymes. Engineering the protein secretory pathway may be a powerful means for efficient protein production. However, it is uncertain whether this engineering approach could improve cellulase production in T. reesei. Herein, the endoplasmic reticulum (ER) secretory pathway was engineered for the production of cellulolytic enzymes by multiple strategies, including: (I) overexpression of the key components of protein folding (Pdi1, Ero1 and BiP); (II) overexpression of the glycosylation-related elements (Gpt1 and Gls2); (III) knockout of the ER mannosidase I (Mns1) encoding gene mns1. By utilizing these ER engineering strategies, the secretion of ß-glucosidase was remarkably elevated in the engineered strains, ranging from 29.2 % to 112.5 %. Furthermore, it was found that engineering these components also regulated the ER stress resistance. More importantly, the total cellulase production was increased with varying degrees, which reached a maximum of 149.4 %, using the filter paper assay (FPA) as a characterization method. These results demonstrated that engineering the ER secretory pathway can enhance protein secretion, particularly for cellulase production, which shed light for the development of high-efficient cellulolytic enzymes for economically feasible bioethanol production from lignocellulosic biomass.
Assuntos
Celulase , Retículo Endoplasmático , Trichoderma , Celulase/biossíntese , Retículo Endoplasmático/metabolismo , Hypocreales , Microrganismos Geneticamente Modificados , Via Secretória , Trichoderma/genética , Trichoderma/metabolismoRESUMO
<b>Background and Objective:</b> Organic waste dump is a problem that needs to be solved, one of which is by using microbe. Cellulolytic bacteria's ability to produce cellulase enzymes that can hydrolyze cellulose. Cellulose is the major component of the plant cell walls that are difficult to endure degradation naturally. This study aimed to find cellulolytic bacterial isolates on Biological Education and Research Forest floor Andalas University and characterize the cellulolytic bacteria found. Material and Methods: CMC (Carboxymethyl Cellulose) medium is used as a screening for bacteria isolate and this study used the survey method and also conducted catalase, glucose and lactose test for characterizations. <b>Results:</b> We found 16 bacterial isolates on Biological Education and Research Forest floor where 9 isolates were in a shaded area and 7 isolates were in an unshaded area. There were 12 isolates from 16 isolates that have the positive cellulolytic ability with a variety of clear zone sizes, where there were 8 isolates with the large clear zone and 4 isolates producing very small clear zones. Characteristics of bacterial isolates with the large clear zone obtained were 2 gram-positive coccus isolates with positive catalase test, 3 gram-negative coccus isolates with positive glucose test and 3 gram-negative bacilli isolates with negative fructose test. <b>Conclusion:</b> We identified 2 potential isolates with a cellulolytic index value greater than 2, isolate UCB 4 with a cellulolytic index value of 3.5 and UCB 6 with a cellulolytic index value of 2.2.
Assuntos
Bactérias/isolamento & purificação , Celulase/biossíntese , Florestas , IndonésiaRESUMO
Trichoderma reesei has 11 putative ß-glucosidases in its genome, playing key parts in the induction and production of cellulase. Nevertheless, the reason why the T. reesei genome encodes so many ß-glucosidases and the distinct role each ß-glucosidase plays in cellulase production remain unknown. In the present study, the cellular function and distribution of 10 known ß-glucosidases (CEL3B, CEL3E, CEL3F, CEL3H, CEL3J, CEL1A, CEL3C, CEL1B, CEL3G, and CEL3D) were explored in T. reesei, leaving out BGL1 (CEL3A), which has been well investigated. We found that the overexpression of cel3b or cel3g significantly enhanced extracellular ß-glucosidase production, whereas the overexpression of cel1b severely inhibited cellulase production by cellulose, resulting in nearly no growth of T. reesei Four types of cellular distribution patterns were observed for ß-glucosidases in T. reesei: (i) CEL3B, CEL3E, CEL3F, and CEL3G forming clearly separated protein secretion vesicles in the cytoplasm; (ii) CEL3H and CEL3J diffusing the whole endomembrane as well as the cell membrane with protein aggregation, like a reticular network; (iii) CEL1A and CEL3D in vacuoles; (iv) and CEL3C in the nucleus. ß-glucosidases CEL1A, CEL3B, CEL3E, CEL3F, CEL3G, CEL3H, and CEL3J were identified as extracellular, CEL3C and CEL3D as intracellular, and CEL1B as unknown. The extracellular ß-glucosidases CEL3B, CEL3E, CEL3F, CEL3H, and CEL3G were secreted through a tip-directed conventional secretion pathway, and CEL1A, via a vacuole-mediated pathway that was achieved without any signal peptide, while CEL3J was secreted via an unconventional protein pathway bypassing the endoplasmic reticulum (ER) and Golgi.IMPORTANCE Although ß-glucosidases play an important role in fungal cellulase induction and production, our current understanding does not provide a global perspective on ß-glucosidase function. This work comprehensively studies all the ß-glucosidases regarding their effect on cellulase production and their cellular distribution and secretion. Overexpression of cel3b or cel3g significantly enhanced ß-glucosidase production, whereas overexpression of cel1b severely inhibited cellulase production on cellulose. In addition, overexpression of cel3b, cel3e, cel3f, cel3h, cel3j, cel3c, or cel3g delayed endoglucanase (EG) production. We first identified four cellular distribution patterns of ß-glucosidases in Trichoderma reesei Specially, CEL3C was located in the nucleus. CEL3J was secreted through the nonclassical protein secretion pathway bypassing endoplasmic reticulum (ER) and Golgi. CEL1A was secreted via a vacuole-mediated conventional secretion route without a signal peptide. These findings advance our understanding of ß-glucosidase properties and secretory pathways in filamentous fungi, holding key clues for future study.
Assuntos
Proteínas Fúngicas/metabolismo , Expressão Gênica , Hypocreales/enzimologia , Hypocreales/genética , beta-Glucosidase/metabolismo , Celobiose/metabolismo , Celulase/biossíntese , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Hypocreales/metabolismo , beta-Glucosidase/biossíntese , beta-Glucosidase/classificação , beta-Glucosidase/genéticaRESUMO
Caldicellulosiruptor bescii is the most thermophilic, cellulolytic bacterium known and has the native ability to utilize unpretreated plant biomass. Cellulase A (CelA) is the most abundant enzyme in the exoproteome of C. bescii and is primarily responsible for its cellulolytic ability. CelA contains a family 9 glycoside hydrolase and a family 48 glycoside hydrolase connected by linker regions and three carbohydrate-binding domains. A truncated version of the enzyme (TM1) containing only the endoglucanase domain is thermostable and actively degrades crystalline cellulose. A catalytically active TM1 was successfully produced via the attachment of the PelB signal peptide (P-TM1), which mediates post-translational secretion via the SecB-dependent translocation pathway. We sought to enhance the extracellular secretion of TM1 using an alternative pathway, the signal recognition particle (SRP)-dependent translocation pathway. The co-translational extracellular secretion of TM1 via the SRP pathway (D-TM1) resulted in a specific activity that was 4.9 times higher than that associated with P-TM1 overexpression. In batch fermentations, the recombinant Escherichia coli overexpressing D-TM1 produced 1.86 ± 0.06 U/ml of TM1 in the culture medium, showing a specific activity of 1.25 ± 0.05 U/mg cell, 2.7- and 3.7-fold higher than the corresponding values of the strain overexpressing P-TM1. We suggest that the TM1 secretion system developed in this study can be applied to enhance the capacity of E. coli as a microbial cell factory for the extracellular secretion of this as well as a variety proteins important for commercial production.
Assuntos
Celulase/biossíntese , Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Via Secretória , Partícula de Reconhecimento de Sinal/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caldicellulosiruptor/enzimologia , Caldicellulosiruptor/genética , Carboxipeptidases/genética , Celulase/genética , Celulose/metabolismo , DNA Bacteriano , Escherichia coli/genética , Fermentação , Glicosídeo Hidrolases , Microbiologia Industrial , Mutação , Peptidoglicano/genética , Domínios Proteicos , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes/biossínteseRESUMO
Hyphal morphology is considered to have a close relationship with the production level of secreted proteins by filamentous fungi. In this study, the gul1 gene, which encodes a putative mRNA-binding protein, was disrupted in cellulase-producing fungus Trichoderma reesei. The hyphae of Δgul1 strain produced more lateral branches than the parent strain. Under the condition for cellulase production, disruption of gul1 resulted in smaller mycelial clumps and significantly lower viscosity of fermentation broth. In addition, cellulase production was improved by 22% relative to the parent strain. Transcriptome analysis revealed that a set of genes encoding cell wall remodeling enzymes as well as hydrophobins were differentially expressed in the Δgul1 strain. The results suggest that the regulatory role of gul1 in cell morphogenesis is likely conserved in filamentous fungi. To our knowledge, this is the first report on the engineering of gul1 in an industrially important fungus.
Assuntos
Celulase/biossíntese , Proteínas Fúngicas/metabolismo , Hifas/metabolismo , Hypocreales/metabolismo , Parede Celular/metabolismo , Fermentação , Proteínas Fúngicas/genética , Hypocreales/genética , ViscosidadeRESUMO
Endoglucanase (EC 3.2.1.4) catalysing the hydrolysis of ß-1.4-glycosidic linkage of cellulose molecules is an enzyme of tremendous industrial importance. The present study describes a response surface methodology based predicted model to deduce a set of fermentation conditions for optimum growth and activity of recombinant endoglucanase in E. coli BL21 (DE3). Numerous significant parameters including fermentation media composition, temperature (Celsius), pH and agitation rate (rpm) were analysed systemically by employing central composite design. This effort reports highly efficient recombinant endoglucanase overproduction (6.9 gl-1 of biomass) with 30% expression by E. coli in modified M9NG media incubated at 37 °C and pH 7 agitated at 200 rpm. Addition of 3 mM glucose and 24 mM glycerol in the M9NG media has shown positive effect on the enzyme yield and activity. The CMCase activity experimentally estimated was found to be 1185 U/mg with the optimized parameters. The outcomes of both the responses by the predicted quadratic model were found in consensus with the obtained values. Our results well depicted the favourable conditions to further scale-up the volumetric yield of other relevant recombinant enzymes and proteins.
Assuntos
Técnicas de Cultura de Células , Celulase/biossíntese , Clostridium thermocellum/genética , Modelos Estatísticos , Celulase/genética , Escherichia coli , Fermentação , Proteínas Recombinantes/biossínteseRESUMO
Some ingredients used in poultry feed formulation contain carbohydrate polymers which are difficult to digest and thus hinder nutritional feed value. Toward overcoming this limitation, exogenous enzymes have been added to poultry feed to improve its nutritive value. The present study was designed to provide first enzymatic characterization of endoglucanase (BsEgl) from the genome of B. sonorensis BD92 expressed in Pichia pastoris. Further, we tested its impact alone and in combination with a ß-glucosidase (Bteqßgluc) on growth in commercial broilers as feed additive. The expressed enzyme displayed features of GH5 family and had optimum activity against carboxymethyl cellulose at pH 5 and 50 °C. The BsEgl was stable at a range of pH from 4 to 8 for 60 min and at 50 °C for 180 min. Supplementing broilers diet with BsEgl alone or in combination with Bteqßgluc resulted in better feed conversion ratio among treatments during a five weeks testing period. Moreover, meat percentage was also highest for this treatment, and all treatments with recombinant enzymes increased intestinal length in birds compared to treatment control group. Blood parameters and serum biochemistry profile showed non-significant difference among groups. These results support that recombinant cellulolytic enzymes supplement high fiber diets improve their nutritional performance.
Assuntos
Ração Animal , Bacillus/genética , Proteínas de Bactérias , Celulase , Saccharomycetales , Animais , Bacillus/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Celulase/biossíntese , Celulase/genética , Celulase/isolamento & purificação , Celulase/farmacologia , Galinhas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Saccharomycetales/enzimologia , Saccharomycetales/genéticaRESUMO
Trichoderma orientalis (T. orientalis) EU7-22 has a complete cellulase system and shows a remarkable enzyme activity with high potential in the industry. Ace2 is an important transcriptional factor for cellulase and hemicellulase expression in Trichoderma reesei (T. reesei). However, the ace2 gene cannot be found in the genome of T. orientalis. Researches show that the mechanism of cellulase transcriptional regulation in T. orientalis keeps high similarity with T. reesei up till now. So, in this study, the ace2 of Trichoderma reesei QM9414 was heterologous expressed in T. orientalis EU7-22. As a result, xylanase activity and ß-glucosidase activity of ace2 heterogeneous expression strains are improved and total cellulase activity is decreased. The result of qPCR is in accordance with enzyme activities. This study provides a reference for an in-depth study on transcriptional regulation mechanisms of T. orientalis.
Assuntos
Celulase/biossíntese , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glicosídeo Hidrolases/biossíntese , Hypocreales/genética , Hypocreales/metabolismo , Fatores de Transcrição/metabolismo , Celulase/genética , Proteínas Fúngicas/genética , Glicosídeo Hidrolases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genéticaRESUMO
The essential transcription factor PoxCxrA is required for cellulase and xylanase gene expression in the filamentous fungus Penicillium oxalicum that is potentially applied in biotechnological industry as a result of the existence of the integrated cellulolytic and xylolytic system. However, the regulatory mechanism of cellulase and xylanase gene expression specifically associated with PoxCxrA regulation in fungi is poorly understood. In this study, the novel regulator PoxCbh (POX06865), containing a centromere protein B-type helix-turn-helix domain, was identified through screening for the PoxCxrA regulon under Avicel induction and genetic analysis. The mutant ∆PoxCbh showed significant reduction in cellulase and xylanase production, ranging from 28.4% to 59.8%. Furthermore, PoxCbh was found to directly regulate the expression of important cellulase and xylanase genes, as well as the known regulatory genes PoxNsdD and POX02484, and its expression was directly controlled by PoxCxrA. The PoxCbh-binding DNA sequence in the promoter region of the cellobiohydrolase 1 gene cbh1 was identified. These results expand our understanding of the diverse roles of centromere protein B-like protein, the regulatory network of cellulase and xylanase gene expression, and regulatory mechanisms in fungi.
Assuntos
Proteína B de Centrômero/genética , Proteínas Cromossômicas não Histona/biossíntese , Regulação Fúngica da Expressão Gênica/genética , Sequências Hélice-Volta-Hélice/genética , Penicillium/genética , Penicillium/metabolismo , Celulase/biossíntese , Celulase/genética , Celulose 1,4-beta-Celobiosidase/genética , Proteína B de Centrômero/biossíntese , Proteínas Cromossômicas não Histona/genética , Endo-1,4-beta-Xilanases/biossíntese , Endo-1,4-beta-Xilanases/genética , Fatores de Transcrição/genéticaRESUMO
Phospholipases play vital roles in immune and inflammatory responses in mammals and plants; however, knowledge of phospholipase functions in fungi is limited. In this study, we investigated the effects of deleting predicted phospholipase genes on cellulase and xylanase production, and morphological phenotype, in Penicillium oxalicum. Individual deletion of nine of the ten predicted phospholipase genes resulted in alteration of cellulase and xylanase production, and the morphological phenotypes, to various degrees. The mutant ∆POX07277 lost 22.5 to 82.8% of cellulase (i.e., filter paper cellulase, carboxymethylcellulase, and p-nitrophenyl-ß-cellobiosidase) and xylanase production, whereas p-nitrophenyl-ß-glucopyranosidase production increased by 5.8-127.8 fold. POX07277 (P. oxalicum gene No. 07277) was predicted to encode phospholipase A2 and was found to negatively affect the sporulation of P. oxalicum. Comparative transcriptomic and quantitative reverse transcription-PCR analysis indicated that POX07277 dynamically affected the expression of cellulase and xylanase genes and the regulatory genes for fungal sporulation, under micro-crystalline cellulose induction. POX07277 was required for the expression of the known regulatory gene PoxCxrB (cellulolytic and xylanolytic regulator B in P. oxalicum), which is involved in cellulase and xylanase gene expression in P. oxalicum. Conversely, POX07277 expression was regulated by PoxCxrB. These findings will aid the understanding of phospholipase functions and provide novel insights into the mechanism of fungal cellulase and xylanase gene expression. KEY POINTS : ⢠The roles of phospholipases were investigated in Penicillium oxalicum. ⢠POX07277 (PLA2) is required for the expression of cellulase and xylanase genes. ⢠PoxCxrB dynamically regulated POX07277 expression.
Assuntos
Celulase/biossíntese , Endo-1,4-beta-Xilanases/biossíntese , Penicillium , Fosfolipases/metabolismo , Regulação Fúngica da Expressão Gênica , Penicillium/enzimologia , Penicillium/genéticaRESUMO
The cellulase production by filamentous fungi Aspergillus fumigatus JCM 10253 was carried out using agro-industrial waste ragi husk as a substrate in the microbial fermentation. The effect of the process parameters such as temperature, substrate concentration, pH, and incubation process time and their interdependence was studied using response surface methodology. The optimum cellulase activities were obtained at 50 °C under the conditions with 1-2% of substrate concentration at pH 2-4 for the incubation period of 7-8 days. The maximum carboxymethyl cellulase (CMCase) and ß-glucosidase activities with optimized process variables were 95.2 IU/mL and 0.174 IU/mL, respectively. The morphological characterization of fungus by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) revealed the presence of secondary protein structures. Furthermore, this study demonstrated that the application of ragi husk could be a promising feedstock for value-added industrial products. The thermo-acidophilic nature of isolated strain Aspergillus fumigatus JCM 10253 possessed a significant potential for higher titer of cellulase production that could be further employed for lignocellulosic bioethanol production.
Assuntos
Aspergillus fumigatus/crescimento & desenvolvimento , Celulase/biossíntese , Eleusine/química , Proteínas Fúngicas/biossíntese , Concentração de Íons de HidrogênioRESUMO
One of the reasons hindering large-scale application of sophorolipids (SLs) is high production cost. In this study, six recombinant strains of Starmerella bombicola, sbEG1, sbEG2, sbCBH1, sbCBH1-2, sbBGL1, and sbCBH2 expressing cellulase genes eg1, eg2, cbh, cbh1-2, bgl1, and cbh2 from Penicillium oxalicum were respectively constructed. Four strains showed cellulase activities and were co-cultivated in fermentation media containing 2% glucose, 1% Regenerated Amorphous Cellulose (RAC), 2% glucose, and 1% RAC, respectively. After 7 days' cultivation, concentration of SLs in medium with 1% RAC (g/L) reached 1.879 g/L. When 2% glucose and 1% of RAC were both contained, the titer of SLs increased by 39.5% than that of control strain and increased by 68.8% than that in the medium with only 2% glucose. Results demonstrated that cellulase genes from filamentous fungi in S. bombicola can function to degrade lignocellulosic cellulose to produce SLs.
Assuntos
Biomassa , Celulase , Proteínas Fúngicas , Lignina/metabolismo , Microrganismos Geneticamente Modificados , Ácidos Oleicos/biossíntese , Penicillium/genética , Saccharomycetales , Celulase/biossíntese , Celulase/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Microrganismos Geneticamente Modificados/enzimologia , Microrganismos Geneticamente Modificados/genética , Ácidos Oleicos/genética , Saccharomycetales/enzimologia , Saccharomycetales/genéticaRESUMO
Optimization of cellulase production by Bacillus subtilis subsp. subtilis JJBS300 resulted in maximum cellulase (CMCase 9.7 U/g substrate) using wheat bran and rice straw in 1:1 ratio at substrate to moisture ratio of 1:3 at 35 °C and pH 4.0 after 48 h. Partially purified cellulase of B. subtilis subsp. subtilis showed optimal activity at 50 °C and pH 5.0. Among the metal ions, Na+, Ca2+ and Fe2+ stimulated the cellulase activity. Glutaraldehyde and 1-butanol also enhanced the cellulase activity as compared to other solvents. Bacterial cellulase hydrolyzed ammonia-pretreated rice straw more efficiently as compared to sodium-carbonate pretreated and untreated biomass. Optimization of saccharification of untreated and pretreated (sodium carbonate and ammonia) rice straw by bacterial cellulase resulted in high liberation of reducing sugars with enzyme dose of 100 U/g substrate (221 mg/g substrate) at pH 5.0 (103 mg/g substrate) and 50 °C (142 mg/g substrate) after 6 h in ammonia-pretreated rice straw. Furthermore, liberation of reducing sugars increased with incubation time showing maximum reducing sugars (171 mg/g substrate) after 24 h in ammonia-pretreated rice straw. HPLC analysis of enzymatic hydrolysate of ammonia-pretreated rice straw verified the ability of bacterial cellulase in liberation of various monomeric and oligomeric sugars.