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1.
World J Microbiol Biotechnol ; 36(3): 51, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157408

RESUMO

Culture-independent molecular-based approaches can be used to identify genes of interest from environmental sources that have desirable properties such as thermo activity. For this study, a putative thermo stable endoglucanase gene was identified from a mixed culture resulting from the inoculation of Brock-CMcellulose (1%) broth with mudspring water from Mt. Makiling, Laguna, Philippines that had been incubated at 90 °C. Genomic DNA was extracted from the cellulose-enriched mixed culture and endo1949 forward and reverse primers were used to amplify the endoglucanase gene, which was cloned into pCR-script plasmid vector. Blastn alignment of the sequenced insert revealed 99.69% similarity to the glycosyl hydrolase, sso1354 (CelA1; Q97YG7) from Saccharolobus solfataricus. The endoglucanase gene (GenBank accession number MK984682) was determined to be 1,021 nucleotide bases in length, corresponding to 333 amino acids with a molecular mass of ~ 37 kDa. The endoglucanase gene was inserted into a pET21 vector and transformed in E. coli BL21 for expression. Partially purified recombinant Mt. Makiling endoglucanase (MM-Engl) showed a specific activity of 187.61 U/mg and demonstrated heat stability up to 80 °C. The thermo-acid stable endoglucanase can be used in a supplementary hydrolysis step to further hydrolyze the lignocellulosic materials that were previously treated under high temperature-dilute acid conditions, thereby enhancing the release of more glucose sugars for bioethanol production.


Assuntos
Celulase/genética , Celulase/metabolismo , Celulose/metabolismo , DNA , Genômica , Água/metabolismo , Sequência de Aminoácidos , Archaea/enzimologia , Archaea/genética , Bactérias/enzimologia , Bactérias/genética , Sequência de Bases , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Filipinas , Proteínas Recombinantes , Alinhamento de Sequência , Sulfolobales/enzimologia , Sulfolobales/genética , Temperatura , Microbiologia da Água
2.
Nucleic Acids Res ; 48(5): 2209-2219, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31943056

RESUMO

Ongoing large-scale genome sequencing projects are forecasting a data deluge that will almost certainly overwhelm current analytical capabilities of evolutionary genomics. In contrast to population genomics, there are no standardized methods in evolutionary genomics for extracting evolutionary and functional (e.g. gene-trait association) signal from genomic data. Here, we examine how current practices of multi-species comparative genomics perform in this aspect and point out that many genomic datasets are under-utilized due to the lack of powerful methodologies. As a result, many current analyses emphasize gene families for which some functional data is already available, resulting in a growing gap between functionally well-characterized genes/organisms and the universe of unknowns. This leaves unknown genes on the 'dark side' of genomes, a problem that will not be mitigated by sequencing more and more genomes, unless we develop tools to infer functional hypotheses for unknown genes in a systematic manner. We provide an inventory of recently developed methods capable of predicting gene-gene and gene-trait associations based on comparative data, then argue that realizing the full potential of whole genome datasets requires the integration of phylogenetic comparative methods into genomics, a rich but underutilized toolbox for looking into the past.


Assuntos
Biologia Computacional/métodos , Epistasia Genética , Genoma , Família Multigênica , Filogenia , Animais , Celulase/classificação , Celulase/genética , Celulase/metabolismo , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Dictyostelium/enzimologia , Dictyostelium/genética , Fungos/classificação , Fungos/enzimologia , Fungos/genética , Dosagem de Genes , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Phascolarctidae/genética , Phascolarctidae/metabolismo , Plantas/classificação , Plantas/genética , Plantas/metabolismo
4.
PLoS Genet ; 15(11): e1008510, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31765390

RESUMO

Cellulolytic fungi have evolved a complex regulatory network to maintain the precise balance of nutrients required for growth and hydrolytic enzyme production. When fungi are exposed to cellulose, the transcript levels of cellulase genes rapidly increase and then decline. However, the mechanisms underlying this bell-shaped expression pattern are unclear. We systematically screened a protein kinase deletion set in the filamentous fungus Neurospora crassa to search for mutants exhibiting aberrant expression patterns of cellulase genes. We observed that the loss of stk-12 (NCU07378) caused a dramatic increase in cellulase production and an extended period of high transcript abundance of major cellulase genes. These results suggested that stk-12 plays a critical role as a brake to turn down the transcription of cellulase genes to repress the overexpression of hydrolytic enzymes and prevent energy wastage. Transcriptional profiling analyses revealed that cellulase gene expression levels were maintained at high levels for 56 h in the Δstk-12 mutant, compared to only 8 h in the wild-type (WT) strain. After growth on cellulose for 3 days, the transcript levels of cellulase genes in the Δstk-12 mutant were 3.3-fold over WT, and clr-2 (encoding a transcriptional activator) was up-regulated in Δstk-12 while res-1 and rca-1 (encoding two cellulase repressors) were down-regulated. Consequently, total cellulase production in the Δstk-12 mutant was 7-fold higher than in the WT. These results strongly suggest that stk-12 deletion results in dysregulation of the cellulase expression machinery. Further analyses showed that STK-12 directly targets IGO-1 to regulate cellulase production. The TORC1 pathway promoted cellulase production, at least partly, by inhibiting STK-12 function, and STK-12 and CRE-1 functioned in parallel pathways to repress cellulase gene expression. Our results clarify how cellulase genes are repressed at the transcriptional level during cellulose induction, and highlight a new strategy to improve industrial fungal strains.


Assuntos
Celulase/genética , Proteínas Fúngicas/genética , Fatores de Transcrição/genética , Celulose/genética , Regulação Fúngica da Expressão Gênica/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Neurospora crassa/enzimologia , Neurospora crassa/genética
5.
Enzyme Microb Technol ; 131: 109382, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615681

RESUMO

In an effort to find a suitable genetic background for efficient cellulolytic secretion, genetically diverse strains were transformed to produce core fungal cellulases namely, ß-glucosidase (BGLI), endoglucanase (EGII) and cellobiohydrolase (CBHI) in various combinations and expression configurations. The secreted enzyme activity levels, gene copy number, substrate specificities, as well as hydrolysis and fermentation yields of the transformants were analysed. The effectiveness of the partially cellulolytic yeast transformants to convert two different pre-treated corn residues, namely corn cob and corn husk was then explored. Higher secretion titers were achieved by cellulolytic strains with the YI13 genetic background and cellulolytic transformants produced up to 1.34 fold higher glucose concentrations (g/L) than a control composed of equal amounts of each enzyme type. The transformant co-producing BGLI and EGII in a secreted ratio of 1:15 (cellulase activity unit per gram dry cell weight) converted 56.5% of the cellulose present in corn cob to glucose in hydrolysis experiments and yielded 4.05 g/L ethanol in fermentations. We demonstrate that the choice of optimal genetic background and cellulase activity secretion ratio can improve cellulosic ethanol production by consolidated bioprocessing yeast strains.


Assuntos
Celulase/metabolismo , Expressão Gênica , Leveduras/enzimologia , Leveduras/metabolismo , Zea mays/metabolismo , Biotransformação , Celulase/genética , Diploide , Fermentação , Dosagem de Genes , Hidrólise , Leveduras/genética
6.
Mol Biotechnol ; 61(11): 826-835, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31435842

RESUMO

The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.


Assuntos
Celulase/metabolismo , Ruminococcus/enzimologia , beta-Manosidase/metabolismo , Sequência de Aminoácidos/genética , Celulase/biossíntese , Celulase/química , Celulase/genética , Celulossomas/enzimologia , Quelantes , Cromatografia em Camada Delgada , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Galactanos/química , Galactanos/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Mananas/química , Mananas/metabolismo , Oligossacarídeos/química , Gomas Vegetais/química , Gomas Vegetais/metabolismo , Ruminococcus/genética , Especificidade por Substrato , Temperatura , beta-Manosidase/química , beta-Manosidase/genética
7.
J Biosci Bioeng ; 128(6): 669-676, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31257006

RESUMO

An endoglucanase was isolated from solid-state culture of the ectomycorrhizal fungus Tricholoma matsutake (TmEgl5A) grown on rolled barley and vermiculite. The enzyme was purified by ammonium sulfate fractionation, ion-exchange, hydrophobic, and gel filtration. TmEgl5A showed a molecular mass of approximately 40 kDa as determined by SDS-PAGE. The single band of the protein was analyzed by peptide-mass-finger-printing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the trypsin-digested peptide sequences were matched to a putative endoglucanase sequence (protein ID1465229) in the JGI T. matsutake 945 v3.0 genome database. Based on the sequence information, the gene encoding TmEgl was cloned and expressed in Pichia pastoris KM71H. The deduced amino acid sequence was similar to GH5 family endoglucanases from Basidiomycetes. The enzyme acts on barley ß-glucan, lichenan, and CMC-Na. The hydrolyzation products from these substrates were detected by thin-layer chromatography as oligosaccharides with minimal disaccharides. These results suggested that T. matsutake produces a typical endoglucanase in solid-state culture, and the fungus has the potential to degrade ß-linkage polysaccharides.


Assuntos
Celulase/metabolismo , Tricholoma/enzimologia , Sequência de Aminoácidos , Celulase/genética , Celulase/isolamento & purificação , Cromatografia em Camada Delgada , Eletroforese em Gel de Poliacrilamida , Glicosídeos/metabolismo , Hordeum/metabolismo , Hidrólise , Oligossacarídeos/metabolismo , Tricholoma/genética
8.
Food Chem ; 298: 124999, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31261010

RESUMO

Glycoside hydrolase family 8 (GH8) includes endoglucanases, lichenases, chitosanases and xylanases, which are essential for polysaccharides breakdown. In this work, we studied a thermally stable GH8 from the cellulose synthase complex of Enterobacter sp. R1, for deconstruction of ß-glucans. The biochemical characterization of the recombinant GH8ErCel showed high specificity towards barley ß-glucan and lichenan and lower activity on carboxymethylcellulose and swollen cellulose, yielding different length oligosaccharides. By molecular modeling, six conserved subsites for glucose binding and some possible determinants for its lack of xylanase and chitosanase activity were identified. GH8ErCel was active at a broad range of pH and temperature and presented remarkable stability at 60 °C. Additionally, it hydrolyzed ß-glucan from oat and wheat brans mainly to tri- and tetraoligosaccharides. Therefore, GH8ErCel may be a good candidate for enzymatic deconstruction of ß-glucans at high temperature in food and feed industries, including the production of prebiotics and functional foods.


Assuntos
Celulase/química , Celulase/metabolismo , Celulose/metabolismo , Enterobacter/enzimologia , beta-Glucanas/metabolismo , Argentina , Carboximetilcelulose Sódica/metabolismo , Celulase/genética , Enterobacter/genética , Enterobacter/isolamento & purificação , Estabilidade Enzimática , Glucanos/metabolismo , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Oligossacarídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Microbiologia do Solo , Especificidade por Substrato , Temperatura , beta-Glucanas/química
9.
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
10.
Chem Commun (Camb) ; 55(57): 8219-8222, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31210215

RESUMO

Here we reported a new strategy to construct synthetic metabolons using dCas9-guided assembly. Three orthogonal dCas9 proteins were exploited to guide the independent and site-specific assembly of their fusion partners onto a single DNA scaffold. This new platform was applied towards the construction of a two-component cellulosome. Because of the superior binding affinity, the resulting structures exhibited both improved assembly and reducing sugar production. Conditional enzyme assembly was made possible by utilizing toehold-gated sgRNA (thgRNA), which blocks cellulosome formation until the spacer region is unblocked by a RNA trigger. This platform is highly modular owing to the ease of target synthesis, combinations of possible Cas9-fusion arrangements, and expansion to other metabolic pathways.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , RNA Guia/metabolismo , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Celulase/química , Celulase/genética , Celulase/metabolismo , Celulossomas/química , Celulossomas/metabolismo , DNA/química , DNA/metabolismo , Ligação Proteica , Domínios Proteicos , RNA Guia/genética
11.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067833

RESUMO

In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study aims to improve on the characterization of a thermostable Aspergillus fumigatus endo-1,4-ß-glucanase GH7 (Af-EGL7). To this end, Af-EGL7 was successfully expressed in Pichia pastoris X-33. The kinetic parameters Km and Vmax were estimated and suggested a robust enzyme. The recombinant protein was highly stable within an extreme pH range (3.0-8.0) and was highly thermostable at 55 °C for 72 h. Low Cu2+ concentrations (0.1-1.0 mM) stimulated Af-EGL7 activity up to 117%. Af-EGL7 was tolerant to inhibition by products, such as glucose and cellobiose. Glucose at 50 mM did not inhibit Af-EGL7 activity, whereas 50 mM cellobiose inhibited Af-EGL7 activity by just 35%. Additionally, the Celluclast® 1.5L cocktail supplemented with Af-EGL7 provided improved hydrolysis of sugarcane bagasse "in natura", sugarcane exploded bagasse (SEB), corncob, rice straw, and bean straw. In conclusion, the novel characterization of Af-EGL7 conducted in this study highlights the extraordinary properties that make Af-EGL7 a promising candidate for industrial applications.


Assuntos
Aspergillus fumigatus/enzimologia , Celulase/metabolismo , Proteínas Fúngicas/metabolismo , Microbiologia Industrial/métodos , Lignina/metabolismo , Pichia/metabolismo , Aspergillus fumigatus/genética , Biomassa , Celulase/genética , Estabilidade Enzimática , Proteínas Fúngicas/genética , Hidrólise , Pichia/genética , Pichia/crescimento & desenvolvimento , Termotolerância
12.
Microb Cell Fact ; 18(1): 81, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31077201

RESUMO

BACKGROUND: Cellulolytic enzymes produced by the filamentous fungus Trichoderma reesei are commonly used in biomass conversion. The high cost of cellulase is still a significant challenge to commercial biofuel production. Improving cellulase production in T. reesei for application in the cellulosic biorefinery setting is an urgent priority. RESULTS: Trichoderma reesei hyper-cellulolytic mutant SS-II derived from the T. reesei NG14 strain exhibited faster growth rate and more efficient lignocellulosic biomass degradation than those of RUT-C30, another hyper-cellulolytic strain derived from NG14. To identify any genetic changes that occurred in SS-II, we sequenced its genome using Illumina MiSeq. In total, 184 single nucleotide polymorphisms and 40 insertions and deletions were identified. SS-II sequencing revealed 107 novel mutations and a full-length wild-type carbon catabolite repressor 1 gene (cre1). To combine the mutations of RUT-C30 and SS-II, the sequence of one confirmed beneficial mutation in RUT-C30, cre196, was introduced in SS-II to replace full-length cre1, forming the mutant SS-II-cre196. The total cellulase production of SS-II-cre196 was decreased owing to the limited growth of SS-II-cre196. In contrast, 57 genes mutated only in SS-II were selected and knocked out in RUT-C30. Of these, 31 were involved in T. reesei growth or cellulase production. Cellulase activity was significantly increased in five deletion strains compared with that in two starter strains, RUT-C30 and SS-II. Cellulase production of T. reesei Δ108642 and Δ56839 was significantly increased by 83.7% and 70.1%, respectively, compared with that of RUT-C30. The amount of glucose released from pretreated corn stover hydrolyzed by the crude enzyme from Δ108642 increased by 11.9%. CONCLUSIONS: The positive attribute confirmed in one cellulase hyper-producing strain does not always work efficiently in another cellulase hyper-producing strain, owing to the differences in genetic background. Genome re-sequencing revealed novel mutations that might affect cellulase production and other pathways indirectly related to cellulase formation. Our strategy of combining the mutations of two strains successfully identified a number of interesting phenotypes associated with cellulase production. These findings will contribute to the creation of a gene library that can be used to investigate the involvement of various genes in the regulation of cellulase production.


Assuntos
Celulase , Genômica/métodos , Trichoderma , Biomassa , Celulase/genética , Celulase/metabolismo , Glucose/metabolismo , Mutação , Trichoderma/genética , Trichoderma/crescimento & desenvolvimento , Trichoderma/metabolismo
13.
Extremophiles ; 23(4): 479-486, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31119430

RESUMO

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.


Assuntos
Proteínas de Bactérias/metabolismo , Biocombustíveis/microbiologia , Celulase/metabolismo , Metagenoma , Termotolerância , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Celulase/química , Celulase/genética , Estabilidade Enzimática , Microbiota , Centrais Elétricas , Especificidade por Substrato
14.
Food Chem ; 292: 81-89, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31054696

RESUMO

How to effectively increase or decrease the ability of A. oryzae to produce enzymes was the key to improve the quality of soy sauce. However, multi-core property of A. oryzae resulted in genetic instability of the new strain. Here, A. oryzae 3.042-3 which can stably produce mononuclear spores was constructed based on A. oryzae 3.042. A. oryzae 3.042-3-c obtained by transformation of the fragment of cis-CreA into A. oryzae 3.042-3 exhibited genetic stability. The fragment containing the cis-acting and the promoter CreA from A. oryzae was connected to chromosome VII in A. oryzae 3.042-3-c. Compared with A. oryzae 3.042-3, the cellulase activity of A. oryzae 3.042-3-c was reduced by 50.5% and the pectinase activity was decreased by 10.0%. At the end of the soy sauce fermentation, the salt-free solid content of A. oryzae 3.042-3-c was higher 58.9% than that of A. oryzae 3.042-3. The kinds and contents of the flavor components of the soy sauce from the fermentation by A. oryzae 3.042-3-c were higher than those of the A. oryzae 3.042 and A. oryzae 3.042-3, especially in alcohols and esters. HEMF was only found in the soy sauce from A. oryzae 3.042-3-c. The results indicated that the new strain A. oryzae 3.042-3-c could improve the quality of soy sauce from the low-salt solid fermentation by decreasing enzyme activity of cellulase and pectinase.


Assuntos
Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Alimentos de Soja/análise , Aspergillus oryzae/genética , Celulase/genética , Celulase/metabolismo , Cromossomos Fúngicos , Qualidade dos Alimentos , Proteínas Fúngicas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alimentos de Soja/microbiologia
15.
J Biosci Bioeng ; 128(3): 264-273, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30975564

RESUMO

The ascomycete Trichoderma reesei is known to produce a variety of cellulases and hemicellulases and the hyper-cellulolytic mutants of this fungus are useful as industrial cellulase producers. In Japan, PC-3-7, derived from the early mutant QM9414, is well-known as a cellulase hyperproducing mutant. In addition to the productivity of enzymes, the composition of secreted enzymes greatly influences biomass saccharification. Therefore, we evaluated the cellulase productivity of T. reesei mutants in Japan at different pH as a factor influencing enzyme production. At higher pH values, QM9414 exhibited reduced cellulase productivity whereas PC-3-7 maintained high cellulase productivity and gene expression at the transcriptional level. The gene encoding the pH-responsive transcription factor PACI did not mutate in PC-3-7, and its expression pattern against different pH conditions was similar between QM9414 and PC-3-7. Furthermore, the deletion of pac1 encoding PACI caused different expression patterns of cellulase genes between QM9414 and PC-3-7. Therefore, we suggest that T. reesei possesses a pH-responsive cellulase production mechanism that is different from the PACI-related mechanism. Finally, we identified that N-25, a strain developed at an early stage of mutant development acquired cellulase productivity at a higher pH. In this investigation, we also found and tested candidate genes possibly affecting pH response using comparative genome analysis.


Assuntos
Celulase/genética , Celulase/metabolismo , Engenharia de Proteínas/métodos , Trichoderma , Biomassa , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Japão , Organismos Geneticamente Modificados , Fatores de Transcrição/genética , Trichoderma/genética , Trichoderma/metabolismo
16.
Int J Mol Sci ; 20(7)2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30935060

RESUMO

Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15⁻21% increase in specific activity against carboxymethylcellulose and ß-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52⁻58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15⁻22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.


Assuntos
Celulase/química , Dissulfetos/química , Proteínas Fúngicas/química , Penicillium/enzimologia , Termotolerância , Celulase/genética , Celulase/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Simulação de Dinâmica Molecular , Penicillium/genética , Penicillium/metabolismo , Engenharia de Proteínas/métodos , Especificidade por Substrato
17.
Appl Microbiol Biotechnol ; 103(11): 4511-4523, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30982107

RESUMO

XYR1 is the key transcription activator for cellulase gene expression in the model filamentous fungus Trichoderma reesei, which is widely applied in the industry due to its excellent capability of secreting a large quantity of cellulases. Despite the essential role of XYR1, the regulation of its expression in T. reesei cellulolytic response is poorly understood. In this study, we identified a transcription factor RXE1 exhibiting strong binding activity to the xyr1 promoter using yeast one-hybrid screen. RXE1 homologs exist in quite a few filamentous fungi but none of them have been assessed regarding their functional involvement in plant cell wall degradation. Knockdown of rxe1 in T. reesei using a copper-mediated RNAi system not only abrogated conidiation, but also remarkably compromised xyr1 and cellulase gene expression. The defective cellulase but not conidia production in the rxe1-knockdown strain was fully rescued by the constitutive expression of XYR1. Our study thus identified a novel transcriptional regulator controlling xyr1 and cellulase gene expression, which will contribute to elaborating the intricate network of cellulase gene regulation in T. reesei.


Assuntos
Celulase/biossíntese , Regulação Fúngica da Expressão Gênica , Genes Reguladores , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Trichoderma/genética , Trichoderma/metabolismo , Celulase/genética , DNA Fúngico/metabolismo , Técnicas de Silenciamento de Genes , Testes Genéticos , Regiões Promotoras Genéticas , Ligação Proteica
18.
Bioresour Technol ; 282: 494-501, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30897487

RESUMO

Site-directed mutagenesis of ß-1,4-endoglucanase from family 5 glycoside hydrolase (CtGH5) from Clostridium thermocellum was performed to develop a mutant CtGH5-F194A that gave 40 U/mg specific activity against carboxymethyl cellulose, resulting 2-fold higher activity than wild-type CtGH5. CtGH5-F194A was fused with a ß-1,4-glucosidase, CtGH1 from Clostridium thermocellum to develop a chimeric enzyme. The chimera (CtGH1-L1-CtGH5-F194A) expressed as a soluble protein using E. coli BL-21cells displaying 3- to 5-fold higher catalytic efficiency for endoglucanase and ß-glucosidase activities. TLC analysis of hydrolysed product of CMC by chimera 1 revealed glucose as final product confirming both ß-1,4-endoglucanase and ß-1,4-glucosidase activities, while the products of CtGH5-F194A were cellobiose and cello-oligosaccharides. Protein melting studies of CtGH5-F194A showed melting temperature (Tm), 68 °C and of CtGH1, 79 °C, whereas, chimera showed 78 °C. The improved structural integrity, thermostability and enhanced bi-functional enzyme activities of chimera makes it potentially useful for industrial application in converting biomass to glucose and thus bioethanol.


Assuntos
Celulase/metabolismo , Clostridium thermocellum/enzimologia , beta-Glucosidase/metabolismo , Biomassa , Celobiose/metabolismo , Celulase/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrólise , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Temperatura , beta-Glucosidase/genética
19.
J Biotechnol ; 296: 42-52, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-30885654

RESUMO

The biological conversion of lignocellulose into fermentable sugars is a key process for the sustainable production of biofuels from plant biomass. Polysaccharides in plant feedstock can be valorized using thermostable mixtures of enzymes that degrade the cell walls, thus avoiding harmful and expensive pre-treatments. (Hyper)thermophilic bacteria of the phylum Thermotogae provide a rich source of enzymes for such industrial applications. Here we selected T. neapolitana as a source of hyperthermophilic hemicellulases for the degradation of lignocellulosic biomass. Two genes encoding putative hemicellulases were cloned from T. neapolitana genomic DNA and expressed in Escherichia coli. Further characterization revealed that the genes encoded an endo-1,4-ß-galactanase and an α-l-arabinofuranosidase with optimal temperatures of ˜90 °C and high turnover numbers during catalysis (kcat values of ˜177 and ˜133 s-1, respectively, on soluble substrates). These enzymes were combined with three additional T. neapolitana hyperthermophilic hemicellulases - endo-1,4-ß-xylanase (XynA), endo-1,4-ß-mannanase (ManB/Man5A) and ß-glucosidase (GghA) - to form a highly thermostable hemicellulolytic blend. The treatment of barley straw and corn bran with this enzymatic cocktail resulted in the solubilization of multiple hemicelluloses and boosted the yield of fermentable sugars by up to 65% when the complex substrates were further degraded by cellulases.


Assuntos
Celulase/química , Glicosídeo Hidrolases/química , Lignina/química , Polissacarídeos/química , Biocombustíveis , Biomassa , Celulase/genética , Estabilidade Enzimática/genética , Escherichia coli/genética , Fermentação , Glicosídeo Hidrolases/genética , Hidrólise/efeitos dos fármacos , Polissacarídeos/genética , Temperatura , Thermotoga neapolitana/enzimologia , Thermotoga neapolitana/genética
20.
J Biotechnol ; 295: 55-62, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30853632

RESUMO

Endoglucanase, an important component of cellulases, is used as additives in ensiling of forage crops. However, its detailed role is unclear in ensilages. In the present study, two endoglucanases Cel5 and Cel9 produced by strain Paenibacillus panacisoli SDMCC050309, previously isolated from ensiled corn stover, were identified in the cultures by microcrystalline cellulose absorption coupled with zymogram analysis. After heterologously expressed in Escherichia coli DE3 and purified, these two proteins were biochemically characterized. Cel5 was 61 kDa and showed maximal activity at pH 7.0 and 45 °C, while the maximum activity was at pH 8.0 and 65 °C for Cel9 with 97 kDa in size. Both of them could degrade carboxymethyl cellulose into cellooligosaccharides, in which cellobiose and cellotriose could be used as substrates for the growth of homofermentative strains Lactobacillus plantarum CGMCC6888 and L. farciminis CCTCC AB2016237, but not for the heterofermentative strains L. brevis SDMCC050297 and L. parafarraginis SDMCC050300. Therefore, we concluded that the added endoglucanase contributed to enhance the growth of homofermentative lactic acid bacteria for high level of lactic acid production in ensilages.


Assuntos
Celulase/química , Celulase/metabolismo , Lactobacillus/metabolismo , Silagem/microbiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulase/genética , Celulose/metabolismo , Estabilidade Enzimática , Fermentação , Concentração de Íons de Hidrogênio , Oligossacarídeos/metabolismo , Paenibacillus/enzimologia , Paenibacillus/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura
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