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2.
Appl Biochem Biotechnol ; 190(1): 113-128, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31301011

RESUMO

Raw domestic wastewater was used as a culture medium for cellulase production in a bubble column reactor (6.2 UFP/mL, 64.6 U/L h) using the strain Trichoderma harzianum TRIC03-LPBII. Cellulases presented optimum pH and temperature between 4 and 5 and 50 and 70 °C, respectively. Enzymatic extract was concentrated through ultrafiltration and then a cellulolytic formulation was prepared with the addition of sorbitol (50% w/v) and benzoic acid (0.05% w/v). High cellulase stability of around 100% was reached after 30 days at 4 °C. The concentrated extract was also dried in a spray-dryer with the addition of maltodextrin at 20% (w/v), resulting in powder enzymatic formulation with 85% stability after 60 days. With these characteristics, the liquid and powder cellulase products have potential to be used in different industrial applications.


Assuntos
Reatores Biológicos , Celulase/metabolismo , Pós , Águas Residuárias/química , Meios de Cultura , Estabilidade Enzimática , Fermentação , Concentração de Íons de Hidrogênio , Cinética , Temperatura , Trichoderma/enzimologia , Trichoderma/metabolismo
3.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140248, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31279935

RESUMO

Fungi cellulases are used to degrade cellulose-containing biomass for bioethanol production. Industrial cellulases such as Cel7A from Trichoderma reesei (TrCel7A) are critical in this process. Thus, the understanding of structure and dynamics is crucial for engineering variants with improved cellulolytic activity. This cellulase consists of two domains connected by a flexible and highly glycosylated linker. However, the linker flexibility has hindered the determination of Cel7A complete structure. Herein, based on atomic and sparse data, we applied integrative modelling to build a model of the complete enzyme structure. Next, through simulations, we studied the glycosylation effects on the structure and dynamics of a solubilized TrCel7A. Essential dynamics analysis showed that O-glycosylation in the linker led to the stabilization of protein overall dynamics. O-linked glycans seem to restrict protein dihedral angles distribution in this region, selecting more elongated conformations. Besides the reduced flexibility, functional interdomain motions occurred in a more concerted way in the glycosylated system. In contrast, in the absence of glycosylation, we observed vast conformational plasticity with the functional domains frequently collapsing. We report here evidence that targeting Cel7A linker flexibility by point mutations including modification of glycosylation sites could be a promising design strategy to improve cellulase activity.


Assuntos
Celulase/química , Modelos Moleculares , Trichoderma/enzimologia , Proteínas Fúngicas/química , Glicosilação , Conformação Proteica
4.
Enzyme Microb Technol ; 133: 109465, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874695

RESUMO

Beta-glucosidase (BGL1) is widely used in animal feed industries. However, degradation caused by digestive enzymes in the intestine hampers its application. Improving the resistance of feed enzymes against proteases is crucial in livestock farming. To improve the resistance of beta-glucosidase against pepsin and trypsin, a rational molecular design based on the inhibition of bound-state formation and secondary design was developed. The strategy includes: (1) prediction of the interaction surface of the pepsin-BGL1 complex structure, (2) prediction of key amino acids affecting the formation of the complex, (3) optimization of pepsin-resistant mutants by structural evaluation, (4) secondary molecular design based on pepsin-resistant mutants, and optimization of pepsin and trypsin-resistant mutants. Two BGL1 protein mutants (BGL1Q627C and BGL1Q627C/R543H/R646W) were constructed, and then mutated and wild-type BGL1s were expressed in Pichia pastoris. The half-life of BGL1Q627C and BGL1Q627C/R543H/R646W were 1.36 and 1.51 times that of the wild type upon pepsin exposure, respectively. For trypsin resistance, the half-life were 0.93 and 1.53 times that of the wild type, respectively. Compare to those of the wild type, most of the basic enzymatic properties of both mutants were not significantly changed except for increased Michaelis constants. The rational design method can be used as a guide for modifying other feed enzymes.


Assuntos
Pepsina A/metabolismo , Trichoderma/enzimologia , Tripsina/metabolismo , beta-Glucosidase/genética , Hidrólise , Trichoderma/genética , beta-Glucosidase/metabolismo
5.
World J Microbiol Biotechnol ; 35(12): 194, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776792

RESUMO

Lignocellulosic plant biomass is the world's most abundant carbon source and has consequently attracted attention as a renewable resource for production of biofuels and commodity chemicals that could replace fossil resources. Due to its recalcitrant nature, it must be pretreated by chemical, physical or biological means prior to hydrolysis, introducing additional costs. In this paper, we tested the hypothesis that fungi which thrive on lignocellulosic material (straw, bark or soil) would be efficient in degrading untreated lignocellulose. Wheat straw was used as a model. We developed a fast and simple screening method for cellulase producers and tested one hundred Trichoderma strains isolated from wheat straw. The most potent strain-UB483FTG2/ TUCIM 4455, was isolated from substrate used for mushroom cultivation and was identified as T. guizhouense. After optimization of growth medium, high cellulase activity was already achieved after 72 h of fermentation on raw wheat straw, while the model cellulase overproducing strain T. reesei QM 9414 took 170 h and reached only 45% of the cellulase activity secreted by T. guizhouense. Maximum production levels were 1.1 U/mL (measured with CMC as cellulase substrate) and 0.7 U/mL (ß-glucosidase assay). The T. guizhouense cellulase cocktail hydrolyzed raw wheat straw within 35 h. Our study shows that screening for fungi that successfully compete for special substrates in nature will lead to the isolation of strains with qualitatively and quantitatively superior enzymes needed for their digestion which could be used for industrial purposes.


Assuntos
Celulase/metabolismo , Trichoderma/enzimologia , Trichoderma/metabolismo , Triticum/microbiologia , Biocombustíveis , Carboximetilcelulose Sódica/metabolismo , DNA Fúngico , Fermentação , Hidrólise , Cinética , Filogenia , Trichoderma/genética , Trichoderma/isolamento & purificação , beta-Glucosidase/metabolismo
6.
World J Microbiol Biotechnol ; 35(11): 171, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31673786

RESUMO

Fungal endo-ß-1,4-xylanases (endo-xylanases) can hydrolyze xylan into xylooligosaccharides (XOS), and have potential biotechnological applications for the exploitation of natural renewable polysaccharides. In the current study, we aimed to screen and characterize an efficient fungal endo-xylanase from 100 natural humus-rich soil samples collected in Guizhou Province, China, using extracted sugarcane bagasse xylan (SBX) as the sole carbon source. Initially, 182 fungal isolates producing xylanases were selected, among which Trichoderma sp. strain TP3-36 was identified as showing the highest xylanase activity of 295 U/mL with xylobiose (X2) as the main product when beechwood xylan was used as substrate. Subsequently, a glycoside hydrolase family 11 endo-xylanase, TXyn11A, was purified from strain TP3-36, and its optimal pH and temperature for activity against beechwood xylan were identified to be 5.0 and 55 °C, respectively. TXyn11A was stable across a broad pH range (3.0-10.0), and exhibited strict substrate specificity, including xylan from beechwood, wheat, rye, and sugarcane bagasse, with Km and Vmax values of 5 mg/mL and 1250 µmol/mg min, respectively, toward beechwood xylan. Intriguingly, the main product obtained from hydrolysis of beechwood xylan by TXyn11A was xylobiose, whereas SBX hydrolysis resulted in both X2 and xylotriose. Overall, these characteristics of the endo-xylanase TXyn11A indicate several potential industrial applications.


Assuntos
Dissacarídeos/metabolismo , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/isolamento & purificação , Trichoderma/enzimologia , Xilanos/metabolismo , Celulose , China , Estabilidade Enzimática , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Saccharum/metabolismo , Microbiologia do Solo , Especificidade por Substrato , Temperatura , Trichoderma/genética , Trichoderma/isolamento & purificação
7.
Proc Natl Acad Sci U S A ; 116(46): 23061-23067, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31666327

RESUMO

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.


Assuntos
Celulases/química , Proteínas Fúngicas/química , Trichoderma/enzimologia , Sítios de Ligação , Domínio Catalítico , Celulases/metabolismo , Celulose/química , Celulose/metabolismo , Proteínas Fúngicas/metabolismo , Cinética , Simulação de Dinâmica Molecular , Trichoderma/química
8.
Molecules ; 24(20)2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31652666

RESUMO

Trichothecene mycotoxins are recognized as highly bioactive compounds that can be used in the design of new useful bioactive molecules. In Trichoderma brevicompactum, the first specific step in trichothecene biosynthesis is carried out by a terpene cyclase, trichodiene synthase, that catalyzes the conversion of farnesyl diphosphate to trichodiene and is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin, a trichothecene-type toxin, which is a valuable tool in preparing new molecules with a trichothecene skeleton. In this work, we developed the hemisynthesis of trichodermin and trichodermol derivatives in order to evaluate their antimicrobial and cytotoxic activities and to study the chemo-modulation of their bioactivity. Some derivatives with a short chain at the C-4 position displayed selective antimicrobial activity against Candida albicans and they showed MIC values similar to those displayed by trichodermin. It is important to highlight the cytotoxic selectivity observed for compounds 9, 13, and 15, which presented average IC50 values of 2 µg/mL and were cytotoxic against tumorigenic cell line MCF-7 (breast carcinoma) and not against Fa2N4 (non-tumoral immortalized human hepatocytes).


Assuntos
Tricodermina/análogos & derivados , Animais , Antibacterianos/síntese química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antifúngicos/síntese química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Linhagem Celular , Feminino , Hepatócitos/efeitos dos fármacos , Humanos , Células MCF-7 , Micotoxinas/farmacologia , Coelhos , Trichoderma/enzimologia , Trichoderma/genética , Trichoderma/metabolismo , Tricodermina/síntese química , Tricodermina/química , Tricodermina/farmacologia
9.
Appl Microbiol Biotechnol ; 103(19): 8087-8103, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31384992

RESUMO

Trichothecenes are sesquiterpene toxins produced by diverse fungi, including some species of Trichoderma that are potential plant disease biocontrol agents. Trichoderma arundinaceum produces the trichothecene harzianum A (HA), which consists of the core trichothecene structure (12,13-epoxytrichothec-9-ene, EPT) with a linear polyketide-derived substituent (octa-2,4,6-trienedioyl) esterified to an oxygen at carbon atom 4. The genes required for biosynthesis of EPT and the eight-carbon polyketide precursor of the octa-2,4,6-trienedioyl substituent, as well as for esterification of the substituent to EPT have been described. However, genes required for conversion of the polyketide (octa-2,4,6-trienoic acid) to octa-2,4,6-trienedioyl-CoA, the immediate precursor of the substituent, have not been described. Here, we identified 91 cytochrome P450 monooxygenase genes in the genome sequence of T. arundinaceum, and provided evidence from gene deletion, complementation, cross-culture feeding, and chemical analyses that one of them (tri23) is required for conversion of octa-2,4,6-trienoic acid to octa-2,4,6-trienedioyl-CoA. The gene was detected in other HA-producing Trichoderma species, but not in species of other fungal genera that produce trichothecenes with an octa-2,4,6-trienoic acid-derived substituent. These findings indicate that tri23 is a trichothecene biosynthetic gene unique to Trichoderma species, which in turn suggests that modification of octa-2,4,6-trienoic acid during trichothecene biosynthesis has evolved independently in some fungi.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Trichoderma/enzimologia , Trichoderma/metabolismo , Tricotecenos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Ácidos Graxos Insaturados/metabolismo , Deleção de Genes , Teste de Complementação Genética , Trichoderma/genética
10.
J Biosci Bioeng ; 128(6): 637-654, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31204199

RESUMO

Cellulolytic enzymes play a key role on conversion of lignocellulosic plant biomass to biofuels and biochemicals in sugar platform biorefineries. In this review, we survey composite carbohydrate-active enzymes (CAZymes) among groups of cellulolytic fungi and bacteria that exist under aerobic and anaerobic conditions. Recent advances in designing effective cellulase mixtures are described, starting from the most complex microbial consortium-based enzyme preparations, to single-origin enzymes derived from intensively studied cellulase producers such as Trichoderma reesei, Talaromyces cellulolyticus, and Penicellium funiculosum, and the simplest minimal enzyme systems comprising selected sets of mono-component enzymes tailor-made for specific lignocellulosic substrates. We provide a comprehensive update on studies in developing high-performance cellulases for biorefineries.


Assuntos
Celulases/metabolismo , Biocombustíveis , Biomassa , Penicillium/enzimologia , Talaromyces/enzimologia , Trichoderma/enzimologia
12.
J Food Sci ; 84(6): 1382-1389, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31141617

RESUMO

Evolution of Rhizopus oryzae and Trichoderma reesei biomass in rice bran, their enzyme activity, and the profile of phenolic compounds released from the lignocellulosic matrices were determined and correlated by principal component analysis (PCA). PCA analysis confirms that cultivation of rice bran affected the release of methanol-soluble phenolic compounds (MSPC), ethanol-soluble phenolic compounds (ESPC), and bound phenolic compounds (BPC) positively, due to their enzymatic activity. The release of MSPC was influenced by the activity of cellulase and endoglucanase, which increased 110.6% and 136.3%, respectively, for Rhizopus oryzae and Trichoderma reesei. Gallic acid was the main component in the MSPC and ESPC compound fractions. Ferulic and syringic acids were found in its bound (BPC) form in the biomass. This study showed that bioactive compounds be released from lignocellulosic materials by fungus action and this process can be conducted to obtain specific phenolic compounds. PRACTICAL APPLICATION: Due the demand by natural compounds with biological activity, such as phenolic compounds, it is interesting to purpose alternatives to enhance their yield, like for instance, by fungal fermentation of lignocellulosic material. Therefore, understanding the relations among different phenolic compounds released and the production of fungal hydrolases during growth of Rhizopus oryzae and Trichoderma reesei in solid state cultivation using rice bran as a substrate is fundamental to control the process. This knowledge gets viable scale up to apply the phenolic compounds as preservative in food chain, because this becomes possible directing the process to obtain specific bioactive compounds in less time of cultivation and with low cost.


Assuntos
Celulase/metabolismo , Oryza/química , Fenóis/análise , Rhizopus/enzimologia , Trichoderma/enzimologia , Biomassa , Celulose/metabolismo , Ácidos Cumáricos/análise , Meios de Cultura , Fermentação , Ácido Gálico/análogos & derivados , Ácido Gálico/análise , Rhizopus/crescimento & desenvolvimento , Trichoderma/crescimento & desenvolvimento
13.
Can J Microbiol ; 65(9): 653-667, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31059650

RESUMO

We continued our study of high-molecular-mass proteases (HMMPs) using several strains of the genus Trichoderma, and other filamentous fungi (Botrytis cinerea, Aspergillus niger, Fusarium culmorum, and Penicillium purpurogenum). We found that five Trichoderma strains secreted HMMPs into the media after induction with bovine serum albumin. Botrytis cinerea and F. culmorum secreted proteases in the absence of inducer, while A. niger or P. purpurogenum did not secrete proteolytic activity (PA). The activity of HMMPs secreted by or intracellularly located in Trichoderma spp. represents the predominant part of cellular PA, according to zymogram patterns. This observation allowed the study of HMMPs' physiological role(s) independent from the secretion. In studying conidiation, we found that illumination significantly stimulated PA in Trichoderma strains. In the T. atroviride IMI 206040 strain, we demonstrated that this stimulation is dependent on the BLR1 and BLR2 receptors. No stimulation of PA was observed when mechanical injury was used as an elicitor of conidiation. Compounds used as inhibitors or activators of conidiation exerted no congruent effects on both PA and conidiation. These results do not favour a direct role of HMMPs in conidiation. Probably, HMMP activity may be involved in the process of the activation of metabolism during vegetative growth, differentiation, and aging-related processes.


Assuntos
Peptídeo Hidrolases/metabolismo , Trichoderma/enzimologia , Aspergillus niger/enzimologia , Aspergillus niger/fisiologia , Botrytis/enzimologia , Botrytis/fisiologia , Proteínas Fúngicas/metabolismo , Fusarium/enzimologia , Fusarium/fisiologia , Penicillium/enzimologia , Penicillium/fisiologia , Proteólise , Esporos Fúngicos , Trichoderma/fisiologia
14.
Phys Rev Lett ; 122(9): 098102, 2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-30932525

RESUMO

The microscopic kinetics of enzymes at the single-molecule level often deviate considerably from those expected from bulk biochemical experiments. Here, we propose a coarse-grained-model approach to bridge this gap, focusing on the unexpectedly slow bulk hydrolysis of crystalline cellulose by cellulase, which constitutes a major obstacle to mass production of biofuels and biochemicals. Building on our previous success in tracking the movements of single molecules of cellulase on crystalline cellulose, we develop a mathematical description of the collective motion and function of enzyme molecules hydrolyzing the surface of cellulose. Model simulations robustly explained the experimental findings at both the microscopic and macroscopic levels and revealed a hitherto-unknown mechanism causing a considerable slowdown of the reaction, which we call the crowding-out effect. The size of the cellulase molecule impacted significantly on the collective dynamics, whereas the rate of molecular motion on the surface did not.


Assuntos
Celulase/química , Modelos Químicos , Celulose/química , Celulose 1,4-beta-Celobiosidase/química , Hidrólise , Cinética , Trichoderma/enzimologia
15.
Int J Biol Macromol ; 134: 113-121, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31034902

RESUMO

In this study, a chitinase gene, Chit46 from a mycoparasitic fungus Trichoderma harzianum was successfully expressed in Pichia pastoris with a high heterologous chitinase production of 31.4 U/mL, much higher than the previous reports. The active center and substrate binding pocket of the recombinant Chit46 (rChit46) were analyzed and the effects of pH, temperature, metal ions and glycosylation on its activity were tested. rChit46 effectively hydrolyzed colloidal chitin with a high conversion rate of 80.5% in 3 h and the chitin hydrolysates were mainly composed of (GlcNAc)2 (94.8%), which make it a good candidate for the green recycling of chitinous waste. rChit46 could also significantly inhibit growth of the phytopathogenic fungus Botrytis cinerea, which endowed it with the potential as a biocontrol agent.


Assuntos
Quitina/química , Quitinases/genética , Quitinases/metabolismo , Coloides , Trichoderma/enzimologia , Trichoderma/genética , Adsorção , Antifúngicos/química , Antifúngicos/farmacologia , Quitina/metabolismo , Quitinases/química , Cromatografia Líquida de Alta Pressão , Ativação Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Proteínas Recombinantes , Especificidade por Substrato , Temperatura
16.
Proc Natl Acad Sci U S A ; 116(12): 5499-5504, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30842286

RESUMO

Fungi have the potential to produce a large repertoire of bioactive molecules, many of which can affect the growth and development of plants. Genomic survey of sequenced biofertilizer fungi showed many secondary metabolite gene clusters are anchored by iterative polyketide synthases (IPKSs), which are multidomain enzymes noted for generating diverse small molecules. Focusing on the biofertilizer Trichoderma harzianum t-22, we identified and characterized a cryptic IPKS-containing cluster that synthesizes tricholignan A, a redox-active ortho-hydroquinone. Tricholignan A is shown to reduce Fe(III) and may play a role in promoting plant growth under iron-deficient conditions. The construction of tricholignan by a pair of collaborating IPKSs was investigated using heterologous reconstitution and biochemical studies. A regioselective methylation step is shown to be a key step in formation of the ortho-hydroquinone. The responsible methyltransferase (MT) is fused with an N-terminal pseudo-acyl carrier protein (ψACP), in which the apo state of the ACP is essential for methylation of the growing polyketide chain. The ψACP is proposed to bind to the IPKS and enable the trans MT to access the growing polyketide. Our studies show that a genome-driven approach to discovering bioactive natural products from biofertilizer fungi can lead to unique compounds and biosynthetic knowledge.


Assuntos
Arabidopsis/metabolismo , Ferro/metabolismo , Policetídeos/metabolismo , Trichoderma/genética , Arabidopsis/enzimologia , Redes e Vias Metabólicas/genética , Metilação , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Família Multigênica/genética , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Trichoderma/enzimologia , Trichoderma/metabolismo
17.
Biochim Biophys Acta Gen Subj ; 1863(6): 1015-1026, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30898558

RESUMO

Cellulases from glycoside hydrolase family 7 (GH7) play crucial roles in plant lignocellulose deconstruction by fungi, but structural information available for GH7 fungal endoglucanases is limited when compared to the number of known sequences in the family. Here, we report the X-ray structure of the glycosylated catalytic domain (CD) of Trichoderma harzianum endoglucanase, ThCel7B, solved and refined at 2.9 Šresolution. Additionally, our extensive molecular dynamics simulations of this enzyme in complex with a variety of oligosaccharides provide a better understanding of its promiscuous hydrolytic activities on plant cell wall polysaccharides. The simulations demonstrate the importance of the hydrogen bond between substrate O2 hydroxyl in the subsite -1 and a side chain of catalytic Glu196 which renders ThCel7B capable to catalytically cleave cello and xylooligosaccharides, but not mannooligosaccharides. Moreover, detailed structural analyses and MD simulations revealed an additional binding pocket, suitable for accommodation of oligosaccharide decorations and/or substrates with mixed glycoside bonds that abuts onto the binding cleft close to subsite +2.


Assuntos
Parede Celular/química , Celulase/química , Proteínas Fúngicas/química , Simulação de Dinâmica Molecular , Oligossacarídeos/química , Células Vegetais/química , Trichoderma/enzimologia
18.
Ecotoxicol Environ Saf ; 174: 699-705, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30878010

RESUMO

In the present study, we investigated the role of calcite, i.e., microbiologically-induced precipitate by ureolytic Trichoderma sp. MG, in remediation of soils contaminated with arsenic (As) and lead (Pb). The fungus tolerates high concentrations of As (500 mg/L) and Pb (650 mg/L). The effects of three factors, i.e., urea concentration, CaCl2 concentration and pH, on urease production and bio-mineralization of As and Pb were investigated using Box-Behnken design. The maximum urease production (920 U/mL) and metal removal efficiency (68% and 59% for Pb and AS, respectively) were observed in the medium containing urea of 300 mM and CaCl2 of 75 mM at pH 9.0. Fourier transform infrared spectroscopy result revealed the formation of metal carbonates by the isolate MG. Sequential extraction of metals revealed that the carbonate fractions of As and Pb were increased to 46.4% and 42.4% in bioremediated soil, whereas in control they were 35.5% and 32.5%, respectively. The X-ray powder diffraction result further confirmed the role of calcite precipitate in bioremediation of As- and Pb-contaminated soils. The results points out that the microbiologically-induced calcite precipitation is a feasible, eco-friendly technology for the bioremediation of As- and Pb-contaminated sites.


Assuntos
Arsênico/metabolismo , Chumbo/metabolismo , Microbiologia do Solo , Poluentes do Solo/metabolismo , Trichoderma/metabolismo , Arsênico/química , Biodegradação Ambiental , Carbonato de Cálcio/química , Carbonatos/química , Chumbo/química , Solo/química , Trichoderma/enzimologia , Urease/biossíntese
19.
Environ Microbiol ; 21(8): 2644-2658, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30815928

RESUMO

When resources are limited, the hypocrealean fungus Trichoderma guizhouense can overgrow another hypocrealean fungus Fusarium oxysporum, cause sporadic cell death and arrest growth. A transcriptomic analysis of this interaction shows that T. guizhouense undergoes a succession of metabolic stresses while F. oxysporum responded relatively neutrally but used the constitutive expression of several toxin-encoding genes as a protective strategy. Because of these toxins, T. guizhouense cannot approach it is potential host on the substrate surface and attacks F. oxysporum from above. The success of T. guizhouense is secured by the excessive production of hydrogen peroxide (H2 O2 ), which is stored in microscopic bag-like guttation droplets hanging on the contacting hyphae. The deletion of NADPH oxidase nox1 and its regulator, nor1 in T. guizhouense led to a substantial decrease in H2 O2 formation with concomitant loss of antagonistic activity. We envision the role of NOX proteins in the antagonism of T. guizhouense as an example of metabolic exaptation evolved in this fungus because the primary function of these ancient proteins was probably not linked to interfungal relationships. In support of this, F. oxysporum showed almost no transcriptional response to T. guizhouense Δnox1 strain indicating the role of NOX/H2 O2 in signalling and fungal communication.


Assuntos
Fusarium/metabolismo , Peróxido de Hidrogênio/metabolismo , NADPH Oxidases/metabolismo , Trichoderma/metabolismo , Evolução Biológica , Fusarium/crescimento & desenvolvimento , Hifas/crescimento & desenvolvimento , NADPH Oxidases/genética , Oxirredução , Trichoderma/enzimologia , Trichoderma/crescimento & desenvolvimento
20.
J Ind Microbiol Biotechnol ; 46(6): 831-842, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30809754

RESUMO

Filamentous fungi produce a variety of proteases with significant biotechnological potential and show diverse substrate specificities. Proteolytic analysis of the industrial enzyme producer Trichoderma reesei has been sparse. Therefore, we determined the substrate specificity of T. reesei secretome and its main protease Trichodermapepsin (TrAsP) up to P1 position using FRETS-25Xaa-libraries. The role of TrAsP was analyzed using T. reesei QM9414 and the deletant QM∆trasp in Avicel. We observed higher activities of CMCase, Avicelase, and Xylanase in QM∆trasp compared to that of QM9414. Saccharification rate of cellulosic biomass also increased when using secretome of QM∆trasp but the effect was not significant due to the absence of difference in BGL activity compared to QM9414. Higher TrAsP was produced when monosaccharides were used as a carbon source compared to cellulase inducers such as Avicel and α-sophorose. These results elucidate the relationship between TrAsP and cellulase production in T. reesei and suggest a physiological role for TrAsP.


Assuntos
Celulase/metabolismo , Proteínas Fúngicas/metabolismo , Trichoderma/enzimologia , Biomassa , Celulose/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Proteólise
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