Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 383
Filtrar
Mais filtros










Filtros aplicados
Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 11(1): 3290, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620929

RESUMO

In mitochondria, ß-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold ß-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane ß-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 ß-barrel opens a lateral gate to accommodate its substrates.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Microscopia Crioeletrônica , Detergentes/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Sordariales/genética , Sordariales/metabolismo
3.
J Biosci Bioeng ; 129(1): 16-22, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31400994

RESUMO

The economical production of pectin oligosaccharides with a specific degree of polymerization and structure from agro-food waste is an industrially important process. This study identified a novel pectate lyase gene (plhy1) from the thermophilic cellulolytic fungus H. insolens Y1 and tested its ability to produce pectin oligosaccharides. The recombinant PLHY1 produced in Pichia pastoris was superior to other similar enzymes due to its high thermal and pH stability. PLHY1 demonstrated optimal enzymatic activity at 55°C and pH 10.0 in the presence of 0.4 mM Ca2+, and preferred methyl esterified substrates for digestion. High performance anion exchange chromatography-pulsed amperometric detector and ultra high performance liquid chromatography in combination with electrospray ionization tandem mass spectrometry analysis showed that galacturonic acid-oligosaccharides with a small degree of polymerization (4-6) were the major hydrolysates produced by the degradation of apple peel pectin by PLHY1. The properties of PLHY1 make it valuable for application in the agro-food industry for the production of pectin oligosaccharides.


Assuntos
Proteínas Fúngicas/química , Oligossacarídeos/metabolismo , Pectinas/química , Polissacarídeo-Liase/química , Sordariales/enzimologia , Biocatálise , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ácidos Hexurônicos/química , Ácidos Hexurônicos/metabolismo , Pectinas/metabolismo , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Sordariales/química , Sordariales/genética
4.
J Ind Microbiol Biotechnol ; 47(1): 133-144, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31786675

RESUMO

Trehalase catalyzes the hydrolysis of the non-reducing disaccharide trehalose. The highly active trehalase MthT from Myceliophthora thermophila was screened from the trehalase genes of six species of filamentous fungi. An ingenious multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool and medium optimization were used to improve MthT production in Aspergillus niger, up to 1698.83 U/mL. The protein background was dramatically abated due to insertion. The recombinant MthT showed optimal activity at pH 5.5 and 60 °C, and exhibited prominent thermal stability between 50 and 60 °C under acid conditions (pH 4.5-6.5). The ethanol conversion rate (ethanol yield/total glucose) was significantly improved by addition of MthT (51.88%) compared with MthT absence (34.38%), using 30% starch saccharification liquid. The results of this study provided an effective strategy, established a convenient platform for heterologous expression in A. niger and showed a potential strategy to decrease production costs in industrial ethanol production.


Assuntos
Aspergillus niger/metabolismo , Etanol/metabolismo , Sordariales/metabolismo , Trealase/metabolismo , Aspergillus niger/genética , Sistemas CRISPR-Cas , Estabilidade Enzimática , Fermentação , Temperatura Alta , Sordariales/genética , Trealase/genética
5.
Biochim Biophys Acta Gen Subj ; 1864(1): 129434, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31525408

RESUMO

Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C12E8). Thus SDS converts ß-lactoglobulin, which has mainly ß-sheet secondary structure, into a state rich in α-helicality, while addition of C12E8 leads to refolding and recovery of the original ß-sheet structure. Here we extend these studies to the large ß-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C12E8 were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3-4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C12E8 recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C12E8 was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.


Assuntos
Celulase/química , Desdobramento de Proteína/efeitos dos fármacos , Dodecilsulfato de Sódio/farmacologia , Tensoativos/farmacologia , Calorimetria , Celulase/efeitos dos fármacos , Dicroísmo Circular , Cinética , Polietilenoglicóis/farmacologia , Conformação Proteica/efeitos dos fármacos , Conformação Proteica em alfa-Hélice/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Espalhamento a Baixo Ângulo , Sordariales/enzimologia , Tensoativos/química , Difração de Raios X
6.
J Comp Pathol ; 172: 22-26, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31690410

RESUMO

Stillbirth and perinatal mortality with neurological signs and lesions were diagnosed in two calves following ingestion by their dams of corn infected with Stenocarpella maydis during the third trimester of gestation. Grossly, the brain and spinal cord were unremarkable. Microscopically, diffuse severe status spongiosis of the white matter was detected in the cerebral hemispheres, brainstem, spinal cord and cerebellum. To the best of our knowledge this is the first pathological description of congenital disease in calves associated with the consumption of S. maydis-infected corn; the findings resemble those reported for the naturally occurring and experimentally induced disease in lambs.


Assuntos
Micotoxicose/veterinária , Micotoxinas/toxicidade , Malformações do Sistema Nervoso/veterinária , Sordariales/patogenicidade , Animais , Animais Recém-Nascidos , Bovinos , Feminino , Micotoxicose/embriologia , Micotoxicose/patologia , Malformações do Sistema Nervoso/patologia , Gravidez , Zea mays/microbiologia
7.
Pak J Pharm Sci ; 32(4): 1717-1722, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31608896

RESUMO

Fungi have been used in modern scientific research due to their high potential for different enzymes production based on genomic features. The great proportion of soil mycoflora represented by saprobic fungi plays an important role in decomposition, thus contribute to the global carbon cycle. Sordaria fimicola strains (n= 61) collected from different environments were evaluated for catalase enzyme activity at first stage. Among all 61 isolates of S. fimicola, five strains viz. S1, S2, N7, N6 and SF13 were found to be most efficient in catalase enzyme activity. The complete catalase gene including exons and introns was amplified and sequenced from the most efficient strains of S. fimicola and then submitted in the NCBI data base under accession numbers KM282183, KM282184, KM282186, KM282185 and KM282182 for strains S1, S2, N7, N6 and SF13 respectively. The significant differences in the genes sequences and theoretically translated proteins were observed for all five strains of S. fimicola. As regards catalase enzyme activity, S. fimicola strains were found comparable to the Aspergillus niger strains, therefore being a saprophytic fungus with short life cycle S. fimicola can become a fungus of choice to produce catalase enzyme at large scale.


Assuntos
Catalase/genética , Catalase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Sordariales/enzimologia , Filogenia , Mutação Puntual , Microbiologia do Solo
8.
Molecules ; 24(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470567

RESUMO

BACKGROUND: The development of new antifungal agents has always been a hot research topic in pesticide development. In this study, a series of derivatives of natural compound ß-pinene were prepared, and the antifungal activities of these derivatives were evaluated. The purpose of this work is to develop some novel molecules as promising new fungicides. METHODS: Through a variety of chemical reactions, ß-pinene was transformed into a series of ß-pinene-based derivatives containing amide moieties and acylthiourea moieties. The antifungal activities of these derivatives against five plant pathogens including Colletotrichum gloeosporioides, Fusarium proliferatum, Alternaria kikuchiana, Phomopsis sp. and Phytophthora capsici were tested; preliminary structure-activity relationship was discussed. RESULTS: Some derivatives exhibited moderate or significant antifungal activity due to the fusion of the amide moiety or the acylthiourea moiety with the pinane skeleton. The structure-activity relationship analysis showed that the fluorine atom and the strong electron withdrawing nitro group, or trifluoromethyl group on the benzene ring of the derivatives had a significant effect on the improvement of the antifungal activity against Colletotrichum gloeosporioides, Fusarium proliferatum, Alternaria kikuchiana and Phomopsis sp. Meanwhile, the introduction of an ethyl group at the meta-position on the benzene ring of the derivatives could improve the antifungal activity against Phytophthora capsici. Compounds 4e, 4h, 4q, 4r exhibited broad-spectrum antifungal activity against the tested strains. Compound 4o had significant antifungal activity against Phytophthora capsici (IC50 = 0.18 µmol/L). These derivatives were expected to be used as precursor molecules for novel pesticide development in further research.


Assuntos
Alternaria/efeitos dos fármacos , Monoterpenos Bicíclicos/síntese química , Colletotrichum/efeitos dos fármacos , Fungicidas Industriais/síntese química , Fusarium/efeitos dos fármacos , Phytophthora/efeitos dos fármacos , Sordariales/efeitos dos fármacos , Alternaria/crescimento & desenvolvimento , Amidas/química , Monoterpenos Bicíclicos/farmacologia , Colletotrichum/crescimento & desenvolvimento , Fungicidas Industriais/farmacologia , Fusarium/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Phytophthora/crescimento & desenvolvimento , Doenças das Plantas/microbiologia , Doenças das Plantas/terapia , Plantas/microbiologia , Sordariales/crescimento & desenvolvimento , Relação Estrutura-Atividade , Tioureia/química
9.
J Microbiol ; 57(12): 1048-1055, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31555992

RESUMO

A Gram-stain-negative strictly aerobic, marine bacterium, designated GH2-2T, was isolated from a rhizosphere mudflat of a halophyte (Carex scabrifolia) in Gangwha Island, the Republic of Korea. The cells of the organism were oxidase-positive, catalase-positive, flagellated, short rods that grew at 10-40°C, pH 4-10, and 0-13% (w/v) NaCl. The predominant ubiquinone was Q-10. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. The major fatty acid is C18:1. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the novel isolate formed an independent lineage at the base of the radiation encompassing members of the genus Thioclava, except for Thioclava arenosa. The closest relatives were T. nitratireducens (96.03% sequence similarity) and T. dalianensis (95.97%). The genome size and DNA G+C content were 3.77 Mbp and 59.6 mol%, respectively. Phylogenomic analysis supported phylogenetic distinctness based on 16S rRNA gene sequences. Average nucleotide identity values were 73.6-74.0% between the novel strain and members of the genus Thioclava. On the basis of data obtained from a polyphasic approach, the strain GH2-2T (= KCTC 62124T = DSM 105743) represents a novel species of a new genus for which the name Hahyoungchilella caricis gen. nov., sp. nov. is proposed. Moreover, the transfer of Thioclava arenosa Thongphrom et al. 2017 to Pseudothioclava gen. nov. as Pseudothioclava arenosa comb. nov. is also proposed. Finally, Thioclava electrotropha Chang et al. 2018 is proposed to be a later heterosynonym of Thioclava sediminum Liu et al. 2017.


Assuntos
Carex (Planta)/microbiologia , Rizosfera , Rhodobacteraceae/classificação , Rhodobacteraceae/isolamento & purificação , Plantas Tolerantes a Sal/microbiologia , Sordariales/classificação , Sordariales/isolamento & purificação , Composição de Bases , Carex (Planta)/fisiologia , DNA Bacteriano , Ácidos Graxos/análise , Concentração de Íons de Hidrogênio , Ilhas , Técnicas Microbiológicas , Filogenia , RNA Ribossômico 16S/genética , República da Coreia , Rhodobacteraceae/genética , Rhodobacteraceae/fisiologia , Plantas Tolerantes a Sal/fisiologia , Sordariales/genética , Sordariales/fisiologia , Ubiquinona/análogos & derivados , Ubiquinona/análise , Sequenciamento Completo do Exoma
10.
Int J Biol Macromol ; 139: 570-576, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31381927

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) have attracted vast attention because of their unique mechanism of oxidative degradation of carbohydrate polymers and the potential application in biorefineries. This study characterized a novel LPMO from Myceliophthora thermophila, denoted MtLPMO9L. The structure model of the enzyme indicated that it belongs to the C1-oxidizing LPMO, which has neither an extra helix in the L3 loop nor extra loop region in the L2 loop. This was confirmed subsequently by the enzymatic assays since MtLPMO9L only acts on cellulose and generates C1-oxidized cello-oligosaccharides. Moreover, synergetic experiments showed that MtLPMO9L significantly improves the efficiency of cellobiohydrolase (CBH) II. In contrast, the inhibitory rather than synergetic effect was observed when combining used MtLPMO9L and CBHI. Changing the incubation time and concentration ratio of MtLPMO9L and CBHI could attenuate the inhibitory effects. This discovery suggests a different synergy detail between MtLPMO9L and two CBHs, which implies that the composition of cellulase cocktails may need reconsideration.


Assuntos
Celulose 1,4-beta-Celobiosidase/química , Celulose/química , Oxigenases de Função Mista/química , Sordariales/enzimologia , Ativação Enzimática , Hidrólise , Cinética , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Termodinâmica
11.
Int J Mol Sci ; 20(15)2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31357701

RESUMO

The thermophilic fungus Humicola insolens produces cellulolytic enzymes that are of great scientific and commercial interest; however, few reports have focused on its cellulase expression regulation mechanism. In this study, we constructed a creA gene (carbon catabolite repressor gene) disruption mutant strain of H. insolens that exhibited a reduced radial growth rate and stouter hyphae compared to the wild-type (WT) strain. The creA disruption mutant also expressed elevated pNPCase (cellobiohydrolase activities), pNPGase (ß-glucosidase activities), and xylanase levels in non-inducing fermentation with glucose. Unlike other fungi, the H. insolens creA disruption mutant displayed lower FPase (filter paper activity), CMCase (carboxymethyl cellulose activity), pNPCase, and pNPGase activity than observed in the WT strain when fermentation was induced using Avicel, whereas its xylanase activity was higher than that of the parental strain. These results indicate that CreA acts as a crucial regulator of hyphal growth and is part of a unique cellulase expression regulation mechanism in H. insolens. These findings provide a new perspective to improve the understanding of carbon catabolite repression regulation mechanisms in cellulase expression, and enrich the knowledge of metabolism diversity and molecular regulation of carbon metabolism in thermophilic fungi.


Assuntos
Carbono/metabolismo , Repressão Catabólica/genética , Sordariales/enzimologia , Ureo-Hidrolases/genética , Carbono/química , Carboximetilcelulose Sódica/metabolismo , Celulase/química , Celulase/genética , Celulase/metabolismo , Celulose/farmacologia , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Fermentação , Regulação Fúngica da Expressão Gênica/genética , Glucose/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Mutação/genética , Sordariales/metabolismo , Ureo-Hidrolases/química , beta-Glucosidase/química , beta-Glucosidase/metabolismo
12.
PLoS One ; 14(7): e0220319, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31344104

RESUMO

Disease spread of Pseudocercospora fijiensis, causal agent of the black Sigatoka disease of banana, depends on ascospores produced through the sexual reproductive cycle. We used phylogenetic analysis to identify P. fijiensis homologs (PKS8-4 and Hybrid8-3) to the PKS4 polyketide synthases (PKS) from Neurospora crassa and Sordaria macrospora involved in sexual reproduction. These sequences also formed a clade with lovastatin, compactin, and betaenone-producing PKS sequences. Transcriptome analysis showed that both the P. fijiensis Hybrid8-3 and PKS8-4 genes have higher expression in infected leaf tissue compared to in culture. Domain analysis showed that PKS8-4 is more similar than Hybrid8-3 to PKS4. pPKS8-4:GFP transcriptional fusion transformants showed expression of GFP in flask-shaped structures in mycelial cultures as well as in crosses between compatible and incompatible mating types. Confocal microscopy confirmed expression in spermagonia in leaf substomatal cavities, consistent with a role in sexual reproduction. A disruption mutant of pks8-4 retained normal pathogenicity on banana, and no differences were observed in growth, conidial production, and spermagonia production. GC-MS profiling of the mutant and wild type did not identify differences in polyketide metabolites, but did identify changes in saturated fatty acid methyl esters and alkene and alkane derivatives. To our knowledge, this is the first report of a polyketide synthase pathway associated with spermagonia.


Assuntos
Ascomicetos/genética , Família Multigênica , Musa/microbiologia , Policetídeo Sintases/genética , Ascomicetos/enzimologia , Ascomicetos/patogenicidade , Ascomicetos/fisiologia , Proteínas Fúngicas/genética , Neurospora crassa/enzimologia , Neurospora crassa/genética , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Reprodução/genética , Homologia de Sequência , Sordariales/enzimologia , Sordariales/genética
13.
Appl Biochem Biotechnol ; 189(4): 1304-1317, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31254227

RESUMO

Feruloyl esterases (FAEs) have great potential applications in paper and breeding industry. A new thermo-stable feruloyl esterase gene, TtfaeB was identified from the thermophilic fungus Thielavia terrestris h408. Deduced protein sequence shares the identity of 67% with FAEB from Neurospora crassa. The expression vector pPIC9K-TtfaeB was successfully constructed and electro-transformed into GS115 strain of Pichia pastoris. One transformant with high feruloyl esterase yield was obtained through plate screening and named TtFAEB1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TtFAEB1 showed a distinct protein band appearing at the position of about 35-kDa, indicating that TtfaeB gene has been successfully expressed in P. pastoris. The recombinant TtFAEB was purified by affinity chromatography and the specific activity of purified TtFAEB was 6.06 ± 0.72 U/mg. The optimal temperature and pH for purified recombinant TtFAEB was 60 °C and 7.0, respectively. TtFAEB was thermostable, retaining 96.89 and 84.16% of the maximum activity after being treated for 1 h at 50 °C and 60 °C, respectively. Additionally, the enzyme was stable in the pH range 4.5-8.0. The homology model of TtFAEB showed that it consists of a single domain adopting a typical α/ß-hydrolase fold and contains a catalytic triad formed by Ser117, Asp201, and His260. TtFAEB in association with xylanase from Trichoderma reesei could release 77.1% of FA from destarched wheat bran. The present results indicated that the recombinant TtFAEB with excellent enzymatic properties is a promising candidate for potential applications in biomass deconstruction and biorefinery.


Assuntos
Hidrolases de Éster Carboxílico , Clonagem Molecular , Proteínas Fúngicas , Sordariales , Biomassa , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/isolamento & purificação , Estabilidade Enzimática , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sordariales/enzimologia , Sordariales/genética
14.
J Biol Chem ; 294(32): 12157-12166, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31235519

RESUMO

Degradation of polysaccharides is central to numerous biological and industrial processes. Starch-active polysaccharide monooxygenases (AA13 PMOs) oxidatively degrade starch and can potentially be used with industrial amylases to convert starch into a fermentable carbohydrate. The oxidative activities of the starch-active PMOs from the fungi Neurospora crassa and Myceliophthora thermophila, NcAA13 and MtAA13, respectively, on three different starch substrates are reported here. Using high-performance anion-exchange chromatography coupled with pulsed amperometry detection, we observed that both enzymes have significantly higher oxidative activity on amylose than on amylopectin and cornstarch. Analysis of the product distribution revealed that NcAA13 and MtAA13 more frequently oxidize glycosidic linkages separated by multiples of a helical turn consisting of six glucose units on the same amylose helix. Docking studies identified important residues that are involved in amylose binding and suggest that the shallow groove that spans the active-site surface of AA13 PMOs favors the binding of helical amylose substrates over nonhelical substrates. Truncations of NcAA13 that removed its native carbohydrate-binding module resulted in diminished binding to amylose, but truncated NcAA13 still favored amylose oxidation over other starch substrates. These findings establish that AA13 PMOs preferentially bind and oxidize the helical starch substrate amylose. Moreover, the product distributions of these two enzymes suggest a unique interaction with starch substrates.


Assuntos
Proteínas Fúngicas/metabolismo , Oxigenases de Função Mista/metabolismo , Amido/metabolismo , Amilose/química , Amilose/metabolismo , Sítios de Ligação , Domínio Catalítico , Proteínas Fúngicas/química , Oxigenases de Função Mista/química , Simulação de Acoplamento Molecular , Neurospora crassa/enzimologia , Oxirredução , Conformação Proteica em alfa-Hélice , Sordariales/enzimologia , Amido/química , Especificidade por Substrato
15.
Proc Natl Acad Sci U S A ; 116(25): 12400-12409, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31147459

RESUMO

A central feature of meiosis is pairing of homologous chromosomes, which occurs in two stages: coalignment of axes followed by installation of the synaptonemal complex (SC). Concomitantly, recombination complexes reposition from on-axis association to the SC central region. We show here that, in the fungus Sordaria macrospora, this critical transition is mediated by robust interaxis bridges that contain an axis component (Spo76/Pds5), DNA, plus colocalizing Mer3/Msh4 recombination proteins and the Zip2-Zip4 mediator complex. Mer3-Msh4-Zip2-Zip4 colocalizing foci are first released from their tight axis association, dependent on the SC transverse-filament protein Sme4/Zip1, before moving to bridges and thus to a between-axis position. Ensuing shortening of bridges and accompanying juxtaposition of axes to 100 nm enables installation of SC central elements at sites of between-axis Mer3-Msh4-Zip2-Zip4 complexes. We show also that the Zip2-Zip4 complex has an intrinsic affinity for chromosome axes at early leptotene, where it localizes independently of recombination, but is dependent on Mer3. Then, later, Zip2-Zip4 has an intrinsic affinity for the SC central element, where it ultimately localizes to sites of crossover complexes at the end of pachytene. These and other findings suggest that the fundamental role of Zip2-Zip4 is to mediate the recombination/structure interface at all post-double-strand break stages. We propose that Zip2-Zip4 directly mediates a molecular handoff of Mer3-Msh4 complexes, from association with axis components to association with SC central components, at the bridge stage, and then directly mediates central region installation during SC nucleation.


Assuntos
Recombinação Genética , Sordariales/genética , Cromossomos Fúngicos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Complexo Sinaptonêmico/metabolismo
16.
Prep Biochem Biotechnol ; 49(7): 639-648, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31131710

RESUMO

There are many diseases linked to oxidative stress, including cancer. Importantly, endogenous antioxidants are insufficient to protect against this process. Peptides derived from food proteins produced by hydrolysis have been investigated as exogenous antioxidants. The present study aimed to identify novel peptides with antioxidant potential produced from egg and milk proteins hydrolysis with two new fungal proteases isolated from Eupenicillium javanicum and Myceliophthora thermophila. The degree of hydrolysis at several time points was calculated and correlated to DPPH scavenging and metal chelating assays, all hydrolysates presented antioxidant activity. Casein hydrolyzed by the M. thermophila protease showed the best antioxidant activity. The identified sequences showed that the proportions of amino acids that influence antioxidant activity support the antioxidant assay. Our data reveal the conditions necessary for the successful generation of antioxidant peptides using two novel fungal proteases. This opens a potential new avenue for the design and manufacture of antioxidant molecules.


Assuntos
Albuminas/química , Antioxidantes/química , Caseínas/química , Proteínas do Ovo/química , Peptídeos/química , Proteínas do Soro do Leite/química , Albuminas/farmacologia , Animais , Antioxidantes/farmacologia , Caseínas/farmacologia , Proteínas do Ovo/farmacologia , Eupenicillium/enzimologia , Peptídeo Hidrolases/química , Peptídeos/farmacologia , Proteólise , Sordariales/enzimologia , Proteínas do Soro do Leite/farmacologia
17.
Int J Biol Macromol ; 135: 768-775, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31129216

RESUMO

A codon-optimized cutinase gene (TtCutopt) from Thielavia terrestris was over-expressed in Pichia pastoris. An extracellular activity reached 10,200 U/mL using high cell density fermentation. The optimal pH and temperature of TtCutopt were 7.0 and 50 °C, respectively. It displayed high stability over a wide range of pH from 3.0 to 11.0 and up to 85 °C. Among tested p-nitrophenyl esters and triglycerides, TtCutopt showed the highest activity towards p-nitrophenyl butyrate and tributyrin, with specificity activity of 2322.4 U/mg and 1152.5 U/mg, respectively. It was extremely stable in organic solvents and surfactants. TtCutopt efficiently catalyzed the synthesis of butyl butyrate, hexyl butyrate, butyl hexanoate and hexyl hexanoate with esterification efficiency of >95%. Furthermore, it catalyzed the degradation of >90% of dimethyl phthalate, diethyl phthalate, dipropyl phthalate and dibutyl phthalate to release their corresponding monoalkyl phthalates within 24 h. Thus, high yield, high stability, and esterification efficiency of TtCutopt make it an attractive candidate for ester biosynthesis and biodegradation.


Assuntos
Biotecnologia/métodos , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Códon/genética , Ésteres/metabolismo , Sordariales/enzimologia , Alquilação , Ésteres/química , Fermentação , Expressão Gênica , Pichia/genética , Regiões Promotoras Genéticas/genética , Sordariales/genética , Especificidade por Substrato
18.
Enzyme Microb Technol ; 127: 70-74, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31088620

RESUMO

D-glucuronic acid (GlcUA) is an important intermediate with numerous applications in the food, cosmetics, and pharmaceutical industries. Its biological production routes which employ myo-inositol oxygenase (MIOX) as the key enzyme are attractive. In this study, five diverse MIOX-encoding genes, from Cryptococcus neoformans, Chaetomium thermophilum, Arabidopsis thaliana, Thermothelomyces thermophila, and Mus musculus were overexpressed in Escherichia coli, respectively. A novel MIOX from Thermothelomyces thermophila (TtMIOX) exhibited high specific activity, and efficiently converted myo-inositol to GlcUA. Meanwhile, the degradation of GlcUA was inhibited by inactivation of uxaC from the Escherichia coli genome. Finally, the BWΔuxaC whole-cell biocatalyst harboring TtMIOX resulted in the production of 106 g/L GlcUA within 12 h in a 1-L bioreactor, corresponding to a conversion of 91% and productivity of 8.83 g/L/h. This study provides a feasible method for the industrial production of GlcUA.


Assuntos
Escherichia coli/metabolismo , Expressão Gênica , Ácido Glucurônico/metabolismo , Inositol Oxigenase/metabolismo , Inositol/metabolismo , Proteínas Recombinantes/metabolismo , Sordariales/enzimologia , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Biotransformação , Chaetomium/enzimologia , Chaetomium/genética , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/genética , Escherichia coli/genética , Inositol Oxigenase/genética , Camundongos , Proteínas Recombinantes/genética , Sordariales/genética
19.
Appl Environ Microbiol ; 85(12)2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-30979837

RESUMO

Sordaria fimicola, a coprophilous ascomycete, is a homothallic fungus that can undergo sexual differentiation with cellular and morphological changes followed by multicellular tissue development to complete its sexual cycle. In this study, we identified and characterized the blue-light photoreceptor gene in S. fimicola The S. fimicola white collar-1 photoreceptor (SfWC-1) contains light-oxygen-voltage-sensing (LOV), Per-Arnt-Sim (PAS), and other conserved domains and is homologous to the WC-1 blue-light photoreceptor of Neurospora crassa The LOV domain of Sfwc-1 was deleted by homologous recombination using Agrobacterium-mediated protoplast transformation. The Sfwc-1 ( Δlov ) mutant showed normal vegetative growth but produced less carotenoid pigment under illumination. The mutant showed delayed and less-pronounced fruiting-body formation, was defective in phototropism of the perithecial beaks, and lacked the fruiting-body zonation pattern compared with the wild type under the illumination condition. Gene expression analyses supported the light-induced functions of the Sfwc-1 gene in the physiology and developmental process of perithecial formation in S. fimicola Moreover, green fluorescent protein (GFP)-tagged SfWC-1 fluorescence signals were transiently strong upon light induction and prominently located inside the nuclei of living hyphae. Our studies focused on the putative blue-light photoreceptor in a model ascomycete and contribute to a better understanding of the photoregulatory functions and networks mediated by the evolutionarily conserved blue-light photoreceptors across diverse fungal phyla.IMPORTANCE Sordaria sp. has been a model for study of fruiting-body differentiation in fungi. Several environmental factors, including light, affect cellular and morphological changes during multicellular tissue development. Here, we created a light-oxygen-voltage-sensing (LOV) domain-deleted Sfwc-1 mutant to study blue-light photoresponses in Sordaria fimicola Phototropism and rhythmic zonation of perithecia were defective in the Sfwc-1 ( Δlov ) mutant. Moreover, fruiting-body development in the mutant was reduced and also significantly delayed. Gene expression analysis and subcellular localization study further revealed the light-induced differential gene expression and cellular responses upon light stimulation in S. fimicola.


Assuntos
Carpóforos/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Fotorreceptores Microbianos/genética , Processos Fototróficos/genética , Sordariales/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Carpóforos/genética , Proteínas Fúngicas/metabolismo , Fotorreceptores Microbianos/metabolismo , Sordariales/crescimento & desenvolvimento , Sordariales/efeitos da radiação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Appl Microbiol Biotechnol ; 103(10): 4065-4075, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30949809

RESUMO

Glucuronoyl esterases (CE15 family) enable targeted cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), particularly those linking lignin and glucuronoyl residues in xylan. A substantial challenge in characterization and kinetic analysis of CE15 enzymes has been the lack of proper substrates. Here, we present an assay using an insoluble LCC-rich lignin fraction from birch; lignin-rich pellet (LRP). The assay employs quantification of enzyme reaction products by LC-MS. The kinetics of four fungal CE15 enzymes, PsGE, CuGE, TtGE, and AfuGE originating from lignocellulose-degrading fungi Punctularia strigosozonata, Cerrena unicolor, Thielavia terrestris, and Armillaria fuscipes respectively were characterized and compared using this new assay. All four enzymes had activity on LRP and showed a clear preference for the insoluble substrate compared with smaller soluble LCC mimicking esters. End-product profiles were near identical for the four enzymes but differences in kinetic parameters were observed. TtGE possesses an alternative active site compared with the three other enzymes as it has the position of the catalytic glutamic acid occupied by a serine. TtGE performed poorly compared with the other enzymes. We speculate that glucuronoyl LCCs are not the preferred substrate of TtGE. Removal of an N-terminal CBM on CuGE affected the catalytic efficiently of the enzyme by reducing Kcat by more than 30%. Reaction products were detected from all four CE15s on a similar substrate from spruce indicating a more generic GE activity not limited to the hardwood. The assay with natural substrate represents a novel tool to study the natural function and kinetics of CE15s.


Assuntos
Basidiomycota/enzimologia , Metabolismo dos Carboidratos , Esterases/metabolismo , Lignina/metabolismo , Sordariales/enzimologia , Betula/química , Cromatografia Líquida , Esterases/isolamento & purificação , Cinética , Lignina/isolamento & purificação , Espectrometria de Massas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA