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1.
Int J Mol Sci ; 20(18)2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31500317

RESUMO

d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin C oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin C reported by others.


Assuntos
Substituição de Aminoácidos , Cefalosporinas/metabolismo , D-Aminoácido Oxidase/genética , Saccharomycetales/enzimologia , Biocatálise , D-Aminoácido Oxidase/química , D-Aminoácido Oxidase/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/farmacologia , Mutação Puntual , Saccharomycetales/genética , Termodinâmica
2.
EMBO J ; 38(18): e101801, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31393028

RESUMO

From bacteria to mammalian cells, damaged DNA is sensed and targeted by DNA repair pathways. In eukaryotes, kinases play a central role in coordinating the DNA damage response. DNA damage signaling kinases were identified over two decades ago and linked to the cell cycle checkpoint concept proposed by Weinert and Hartwell in 1988. Connections between the DNA damage signaling kinases and DNA repair were scant at first, and the initial perception was that the importance of these kinases for genome integrity was largely an indirect effect of their roles in checkpoints, DNA replication, and transcription. As more substrates of DNA damage signaling kinases were identified, it became clear that they directly regulate a wide range of DNA repair factors. Here, we review our current understanding of DNA damage signaling kinases, delineating the key substrates in budding yeast and humans. We trace the progress of the field in the last 30 years and discuss our current understanding of the major substrate regulatory mechanisms involved in checkpoint responses and DNA repair.


Assuntos
Reparo do DNA , Proteínas Quinases/metabolismo , Animais , Dano ao DNA , Humanos , Saccharomycetales/enzimologia , Transdução de Sinais
3.
J Biotechnol ; 304: 10-15, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400343

RESUMO

Huimcola insolens cutinase (HiC) was heterologously expressed in Pichia pastoris. To avoid a carbon starvation step, fermentation was conducted using combinations of sorbitol with glycerol and methanol in the cell growth and induction phases, respectively. The cutinase productivity (27.71 U mL-1 h-1) was 9.93 U mL-1 h-1 greater than that achieved using traditional two-phase methods, and a cutinase activity of 2660 U mL-1, using p-nitrophenyl butyrate as substrate, was achieved after only 96 h in a 3-L bioreactor. Subsequently, the combination of HiC with Thermobifida fusca cutinase (TfC) in cotton fabric bioscouring was evaluated by monitoring the wettability and dyeability of the fabric. Treatment with 20 U mL-1 of HiC at 80 °C for 5 min followed by 30 U mL-1 of TfC at 50 °C for 1 h gave the best results. The total treatment time was shorter and performance was better than those seen with the alkali method.


Assuntos
Hidrolases de Éster Carboxílico/genética , Pichia/crescimento & desenvolvimento , Saccharomycetales/enzimologia , Biodegradação Ambiental , Reatores Biológicos/microbiologia , Carbono/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Fibra de Algodão , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Engenharia Genética , Pichia/genética , Pichia/metabolismo , Saccharomycetales/genética , Têxteis
4.
J Biotechnol ; 304: 44-51, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31419454

RESUMO

A novel type III fungal CAR was identified from the organism Thermothelomyces thermophila. High expression levels were observed in E. coli using the pETDuet-1 plasmid system in combination with an autoinduction protocol. A broad substrate scope ranging from aromatic to aliphatic carboxylic acids was tested and TtCAR showed activity for all substrates. High specific activities for aromatic substrates and short chain aliphatic substrates were observed, comparable to those of NcCAR, the first type III fungal CAR. TtCAR's pH and temperature optima were at 6.5 and 30 °C, respectively. Up to 20% (v/v) cosolvents did not show a decrease in specific activity of TtCAR using (E)-cinnamic acid as a substrate. Its half-life at 40 °C was determined to be 8.25 h and its melting temperature (Tm) was 56 °C. In vitro reactions with TtCAR reduced 95.2% of 10 mM vanillic acid, which correlated to a titer of 1.4 g L-1 of vanillin. The space time yield of 0.029 g L-1 h-1 indicates that further improvements would be necessary for an industrially relevant application. This would be especially important when competing against de novo synthesis of bio vanillin by microbial strains producing >30 g L-1. In de novo and in vivo biosynthesis systems, by-products are fairly common. By contrast, we were pleased to observe less than 0.7% of vanillyl alcohol formed, making the cell-free acid reduction in the envisaged sequential two-step bioconversion from eugenol to vanillin very attractive.


Assuntos
Benzaldeídos/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Saccharomycetales/enzimologia , Cinamatos/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Oxirredutases/química , Conformação Proteica , Engenharia de Proteínas , Saccharomycetales/genética , Especificidade por Substrato , Termodinâmica , Ácido Vanílico/metabolismo
5.
Food Chem ; 293: 254-262, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151609

RESUMO

This study reported a novel highly active alkaline urate oxidase (UOX) and demonstrated its application in reducing uric acid content of food under alkaline conditions. The UOX gene was cloned from Arxula adeninivorans NBRC 10858, and its N-terminally his6-tagged form (rUOX) was overexpressed in Escherichia coli. The rUOX displayed maximal activity at 40 °C and pH 10, kept more than 90% initial activity under alkaline conditions (pH 9-11) and more than 80% at temperatures below 55 °C. The apparent Km, turnover number (kcat) and catalytic efficiency (kcat/Km) values for the substrate uric acid were respective 29.15 µM, 151.16 s-1 and 5.19 s-1. µM-1, which are improvements over previously reported UOXs. The rUOX efficiently reduced uric acid and purine contents in beer, beef and yeast extract at pH 10, indicating a promising application in food with low purine and uric acid contents to prevent hyperuricemia and gout.


Assuntos
Escherichia coli/genética , Análise de Alimentos/métodos , Saccharomycetales/enzimologia , Urato Oxidase/metabolismo , Ácido Úrico/análise , Animais , Cerveja/análise , Catálise , Bovinos , Temperatura Alta , Concentração de Íons de Hidrogênio , Hiperuricemia/prevenção & controle , Carne/análise , Purinas/análise , Urato Oxidase/genética
6.
FEMS Yeast Res ; 19(2)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30753455

RESUMO

The yeast Starmerella bombicola NBRC10243 is an excellent producer of sophorolipids, which are among the most useful biosurfactants. The primary alcoholic metabolic pathway of S. bombicola has been elucidated using alcohol oxidase FAO1, but the secondary alcohol metabolic pathway remains unknown. Although the FAO1 mutant was unable to grow with secondary alcohols and seemed to be involved in the secondary alcohol metabolism pathway of S. bombicola, it had very low activity toward secondary alcohols. By analyzing the products of secondary alcohol metabolism, alkyl polyglucosides hydroxylated at the ω position in the alkyl chain of the secondary alcohol were observed in the FAO1 mutant, but not in the wild-type yeast. In the double mutant of FAO1 and UGTA1, accumulation of 1,13-tetradecandiol and 2,13-tetradecandiol was observed. The above results indicated that hydroxylation occurred first at the ω and ω-1 positions in the secondary alcohol metabolism of S. bombicola, followed by primary alcohol oxidation.


Assuntos
Oxirredutases do Álcool/metabolismo , Álcoois/metabolismo , Redes e Vias Metabólicas , Saccharomycetales/enzimologia , Saccharomycetales/metabolismo , Oxirredutases do Álcool/genética , Deleção de Genes , Hidroxilação , Saccharomycetales/genética , Saccharomycetales/crescimento & desenvolvimento
7.
Mar Biotechnol (NY) ; 21(2): 229-239, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30684102

RESUMO

A screening among marine yeasts was carried out for nitrile hydrolyzing activity. Meyerozyma guilliermondii LM2 (UBOCC-A-214008) was able to efficiently grow on benzonitrile and cyclohexanecarbonitrile (CECN) as sole nitrogen sources. A two-step one-pot method for obtaining cells of M. guilliermondii LM2 (UBOCC-A-214008) endowed with high nitrilase activity was established; the resulting whole cells converted different nitriles with high molar conversions and showed interesting enantioselectivity toward racemic substrates. Nitrilase from M. guilliermondii LM2 (UBOCC-A-214008) displayed high activity on aromatic substrates, but also arylaliphatic and aliphatic substrates were accepted. Salt-resistant M. guilliermondii LM2 (UBOCC-A-214008) was used in media with different salinity, being highly active up to 1.5 M NaCl concentration. Finally, hydrolysis of nitriles was efficiently performed using a bioprocess (yeast growth and biotransformation with resting cells) entirely carried out in seawater.


Assuntos
Biocatálise , Hidrólise , Nitrilos/metabolismo , Saccharomycetales/metabolismo , Aminoidrolases , Cicloexanos/metabolismo , Saccharomycetales/enzimologia , Saccharomycetales/crescimento & desenvolvimento , Salinidade , Água do Mar
8.
J Biosci Bioeng ; 127(3): 265-272, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30243531

RESUMO

Alcohol oxidase catalyzes the oxidation of primary alcohols into the corresponding aldehydes, making it a potential biocatalyst in the chemical industry. However, the high production cost and poor operational stability of this enzyme are limitations for industrial application. Immobilization of enzyme onto solid supports is a useful strategy for improving enzyme stability. In this work, alcohol oxidase from the thermotolerant methylotrophic yeast Ogataea thermomethanolica (OthAOX) was covalently immobilized onto barium ferrite (BaFe12O19) magnetic microparticles. Among different conditions tested, the highest immobilization efficiency of 71.0 % and catalytic activity of 34.6 U/g was obtained. Immobilization of OthAOX onto magnetic support was shown by Fourier-Transformed infrared microscopy, scanning electron microscopy and X-ray diffraction. The immobilized OthAOX worked optimally at 55 °C and pH 8.0. Immobilization also improved thermostability, in which >65% of the initial immobilized enzyme activity was retained after 24 h pre-incubation at 45 °C. The immobilized enzyme showed a greater catalytic efficiency for oxidation of methanol and ethanol than free enzyme. The immobilized enzyme could be recovered by magnetization and recycled for at least three consecutive batches, after which 70% activity remained. The properties of the immobilized enzyme suggest its potential industrial application for synthesis of aldehyde.


Assuntos
Oxirredutases do Álcool/química , Compostos de Bário/química , Compostos de Bário/síntese química , Enzimas Imobilizadas/química , Compostos Férricos/química , Compostos Férricos/síntese química , Imãs/química , Microesferas , Saccharomycetales/enzimologia , Oxirredutases do Álcool/metabolismo , Biocatálise , Técnicas de Química Sintética , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Metanol/química , Temperatura Ambiente
9.
Appl Microbiol Biotechnol ; 103(1): 279-289, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30357454

RESUMO

The ß-fructofuranosidase Ffase from the yeast Schwanniomyces occidentalis produces potential prebiotic fructooligosaccharides with health-promoting properties, making it of biotechnological interest. Ffase is one of the highest and more selective known producers of 6-kestose by transfructosylation of sucrose. In this work, production of 6-kestose was simplified by directly using cultures of S. occidentalis and Saccharomyces cerevisiae expressing both the wild-type enzyme and a mutated Ffase variant including the Ser196Leu substitution (Ffase-Leu196). Best results were obtained using yeast cultures supplemented with sucrose and expressing the Ffase-Leu196, which after only 4 h produced ~ 116 g/L of 6-kestose, twice the amount obtained with the corresponding purified enzyme. 6-Kestose represented ~ 70% of the products synthesized. In addition, a small amount of 1-kestose and the neofructoligosaccharides neokestose and blastose were also produced. The Ser196Leu substitution skewed production of 6-kestose and neofructooligosaccharides resulting in an increase of ~ 2.2- and 1.5-fold, respectively, without affecting production of 1-kestose. Supplementing yeast cultures with glucose clearly showed that blastose originates from direct fructosylation of glucose, a property that has not been described for other similar proteins from yeasts. Modeling neokestose and blastose into the Ffase-active site revealed the molecular basis explaining the peculiar specificity of this enzyme.


Assuntos
Oligossacarídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/enzimologia , beta-Frutofuranosidase/metabolismo , Domínio Catalítico , Dissacaridases/metabolismo , Microrganismos Geneticamente Modificados , Modelos Moleculares , Oligossacarídeos/química , Prebióticos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomycetales/genética , Especificidade por Substrato , Sacarose/metabolismo , Trissacarídeos/metabolismo , beta-Frutofuranosidase/química , beta-Frutofuranosidase/genética
10.
Mar Biotechnol (NY) ; 21(1): 76-87, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30456695

RESUMO

Aureobasidium melanogenum strain 11-1 with a high laccase activity was isolated from a mangrove ecosystem. Under the optimal conditions, the 11-1 strain yielded the highest laccase activity up to 3120.0 ± 170 mU/ml (1.2 U/mg protein) within 5 days. A laccase gene (LAC1) of the yeast strain 11-1 contained two introns and encoded a protein with 570 amino acids and four conserved copper-binding domains typical of the fungal laccase. Expression of the LAC1 gene in the yeast strain 11-1 made a recombinant yeast strain produce the laccase activity of 6005 ± 140 mU/ml. The molecular weight of the recombinant laccase after removing the sugar was about 62.5 kDa. The optimal temperature and pH of the recombinant laccase were 40 °C and 3.2, respectively, and it was stable at a temperature less than 25 °C. The laccase was inhibited in the presence of sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), and DL-dithiothreitol (DTT). The Km and Vmax values of the laccase for 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was 6.3 × 10-2 mM and 177.4 M/min, respectively. Many synthetic dyes were greatly decolored by the laccase.


Assuntos
Organismos Aquáticos , Proteínas Fúngicas/genética , Lacase/genética , Plasmídeos/metabolismo , Saccharomycetales/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Lacase/biossíntese , Lacase/isolamento & purificação , Peso Molecular , Filogenia , Plasmídeos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomycetales/classificação , Saccharomycetales/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Temperatura Ambiente
11.
Arch Microbiol ; 201(2): 185-192, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30519708

RESUMO

Phylogenetic analysis of class III transaminases in the budding yeasts Lachancea kluyveri, Saccharomyces cerevisiae and Scheffersomyces stipitis identified a hitherto uncharacterised Sch. stipitis transaminase encoded by the PICST_54153 gene, which clustered with previously described γ-amino butyric acid (GABA) and ß-alanine transaminases. Deletion of the PICST_54153 gene in Sch. stipitis resulted in a complete loss in the utilisation of ß-alanine and ß-ureidopropionic acid as nitrogen sources, while growth on 1,3-diaminopropane displayed a significant lag phase compared to the wild-type control. It was therefore concluded that the Sch. stipitis PICST_54153 gene likely encodes a ß-alanine transaminase. However, minor growth defects when 1,4-diaminobutane or 1,5-diaminopentane was provided as the nitrogen source suggested that the Picst_54153 transaminase may also participate in the catabolism of other diamine-derived ω-amino acids. Unexpectedly, the ∆picst_54153 deletion mutant failed to grow on solid minimal medium in the presence of 5 mM ß-alanine even if a preferred nitrogen source was provided.


Assuntos
Saccharomycetales/enzimologia , Transaminases/genética , Aminoácidos/metabolismo , Nitrogênio/metabolismo , Fenótipo , Saccharomyces cerevisiae/enzimologia , Saccharomycetales/genética , Transaminases/metabolismo , beta-Alanina/metabolismo
12.
Sci Rep ; 8(1): 18051, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30575795

RESUMO

In methylotrophic yeasts, the expression of methanol-inducible genes is repressed by ethanol even in the presence of methanol, a phenomenon called ethanol repression. The mechanism of ethanol repression in Komagataella phaffii (Pichia pastoris) was studied, and acetyl-CoA synthesis from ethanol by sequential reactions of alcohol dehydrogenase, aldehyde dehydrogenase and acetyl-CoA synthetase (ACS) was involved in ethanol repression. Molecular analysis of the ACS-encoding gene product KpAcs1 revealed that its N-terminal motif, which is conserved in methylotrophic yeasts, was required for ethanol repression. ACS activity was downregulated during methanol-induced gene expression, which partially depended on autophagy. In addition, acetyl-CoA synthesis and phosphorylation of a transcription factor KpMxr1 were found to contribute to ethanol repression in a synergistic manner.


Assuntos
Acetilcoenzima A/biossíntese , Etanol/farmacologia , Metanol/farmacologia , Pichia/efeitos dos fármacos , Pichia/genética , Acetilcoenzima A/metabolismo , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Organismos Geneticamente Modificados , Pichia/enzimologia , Pichia/metabolismo , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/enzimologia , Saccharomycetales/genética
13.
World J Microbiol Biotechnol ; 35(1): 3, 2018 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-30547239

RESUMO

The occurrence of putative cyanases (EC 4.2.1.104) in the genomes of yeasts belonging to the ascomycete sub-phyla Saccharomycotina (budding yeasts) and Taphrinomycotina (fission yeasts) was investigated. Predicted gene products displaying significant sequence similarity to previously characterized cyanases were identified in the genomes of the budding yeast Lipomyces starkeyi and the fission yeasts Protomyces lactucaedebilis, Saitoella complicata and Taphrina deformans. Li. starkeyi and Sai. complicata were further tested for their ability to utilize cyanate as a nitrogen source. However, neither species displayed significant growth when cyanate was provided as the sole nitrogen source. Cyanate utilization assays of 15 yeast species whose genomes lack detectable cyanase homologs unexpectedly resulted in consistently strong growth in six species as well as variable growth in an additional three species. The present study represents the first known report of cyanase-independent utilization of cyanate as a nitrogen source in ascomycete yeasts. Implications of cyanate utilization for the ecological niches occupied by ascomycete yeasts are discussed.


Assuntos
Ascomicetos/metabolismo , Carbono-Nitrogênio Liases/metabolismo , Cianatos/metabolismo , Nitrogênio/metabolismo , Ascomicetos/enzimologia , Ascomicetos/genética , Ascomicetos/crescimento & desenvolvimento , Carbono-Nitrogênio Liases/genética , Meios de Cultura/química , DNA Fúngico/genética , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Fenótipo , Saccharomycetales/enzimologia , Saccharomycetales/genética , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Análise de Sequência de Proteína , Homologia de Sequência
14.
World J Microbiol Biotechnol ; 34(9): 131, 2018 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-30105649

RESUMO

A new yeast strain which was capable of degrading various azo dyes under high-salt conditions was identified in this study. The results showed that the yeast named S4 was identified as Cyberlindnera samutprakarnensis through 26S rDNA sequence analysis and could decolorize more than 97% of Acid Red B (ARB) within 18 h under the optimal conditions. The acute toxicity of ARB sharply decreased after degradation. NADH-DCIP reductase and lignin peroxidase were determined as the key reductase and oxidase of the yeast S4, respectively. Furthermore, it was proposed that ARB was degraded by strain S4 successively through reduction of azo bonds, hydroxylation, deamination, desulfonation and finally to the TCA cycle.


Assuntos
Adaptação Fisiológica , Compostos Azo/metabolismo , Biodegradação Ambiental , Saccharomycetales/metabolismo , Aliivibrio fischeri/efeitos dos fármacos , Compostos Azo/química , Compostos Azo/toxicidade , Corantes/química , Corantes/metabolismo , Corantes/toxicidade , DNA Ribossômico/genética , Inativação Metabólica , Naftalenossulfonatos/química , Naftalenossulfonatos/metabolismo , Peroxidases/metabolismo , Filogenia , Quinona Redutases/metabolismo , Saccharomycetales/enzimologia , Saccharomycetales/genética , Saccharomycetales/isolamento & purificação , Análise de Sequência , Cloreto de Sódio/metabolismo , Águas Residuárias , Purificação da Água
15.
FEMS Microbiol Lett ; 365(17)2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29982432

RESUMO

Cadmium (Cd) is a heavy metal that is the cause of irreversible hazards to living organisms. Cadmium ions can induce the phosphorylation of MAPKs pathway molecules such as Hog1 and Slt2, but downstream effectors and potential activation pathways are still unclear. In this study, the RNA-seq data analysis in Cd-stressed yeast was performed to predict and screen the signal transduction pathway and the potential effect molecules regulated by MAPKs. Based on differentially expressed genes and Venn diagrams, 31 genes regulated by Hog1p and two genes induced by Slt2p, which related to carbohydrate metabolism, oxidative damage, DNA replication stress and detoxification, were characterized under Cd exposure to yeast. A cysteine-specific transporter (Yct1) modulated by Hog1 was confirmed via RNA-seq results. Meanwhile, we tested the Cd-sensitivity, intracellular Cd concentrations and ß-galactosidase assay, and results indicated that the hypersensitivity of the hog1 mutant to Cd was partly abrogated in YCT1 gene deletion, induction of YCT1 was dependent on Hog1 and its transcription factors, and Yct1p would be epistatic to the Hog1p in Cd-tolerance. The investigation of the transcriptome of MAPKs under Cd stress provided valuable information for future molecular studies of Cd-tolerance.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Cádmio/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/enzimologia , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Saccharomycetales/genética , Saccharomycetales/metabolismo , Transdução de Sinais
16.
Prep Biochem Biotechnol ; 48(6): 549-555, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29889602

RESUMO

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22 ± 2 °C) under static conditions which resulted in 1.41 U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 98% of the maximal activity after 100 min of incubation. The amylase activity was marginally enhanced in presence of Cu2+ and Na+ and slightly reduced by K+, Ca2+, Fe2+, Mg2+, Co2+, Zn2+, and Mn2+ ions at 1 mM concentration.


Assuntos
Amilases/biossíntese , Espaço Extracelular/enzimologia , Fermentação , Micélio/enzimologia , Saccharomycetales/enzimologia , Amilases/metabolismo , Biocatálise , Biomassa , Cátions , Meios de Cultura , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio
17.
Macromol Biosci ; 18(7): e1800095, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29870597

RESUMO

Herein, the synthesis of enzyme-polymer conjugates is reported. Four different activated polymers (mPEG-aldehyde, mPEG-NHS, maltodextrin-aldehyde, carboxymethyl cellulose aldehyde) are conjugated to the surface of protease, α-amylase, and lipase using two different strategies (reductive amination and alkylation with NHS-activated acid). Although the chemical modification of the enzymes is accompanied by losses in enzyme activity (maximum loss 40%), the covalent attachment of polymers increases the thermal stability and the stability in a standard detergent formulation compared to the unmodified enzymes. The enzyme-polymer conjugates are characterized by asymmetrical-flow field-flow fractionation and differential scanning microcalorimetry. Furthermore, it is demonstrated that conjugated enzymes still show performance in a real washing process. Enzyme-polymer conjugates show a potential as a stabilizing system for enzymes in detergents.


Assuntos
Aldeídos/química , Enzimas Imobilizadas/química , Lipase/química , Polietilenoglicóis/química , Serina Endopeptidases/química , alfa-Amilases/química , Alquilação , Aminação , Bacillus licheniformis/química , Bacillus licheniformis/enzimologia , Bacillus subtilis/química , Bacillus subtilis/enzimologia , Carboximetilcelulose Sódica/química , Detergentes/química , Ensaios Enzimáticos , Estabilidade Enzimática , Enzimas Imobilizadas/isolamento & purificação , Cinética , Lipase/isolamento & purificação , Polissacarídeos/química , Saccharomycetales/química , Saccharomycetales/enzimologia , Serina Endopeptidases/isolamento & purificação , Succinimidas/química , Termodinâmica , alfa-Amilases/isolamento & purificação
18.
Chembiochem ; 19(17): 1845-1848, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-29944204

RESUMO

An improved sol-gel process involving the use of hollow silica microspheres as a supporting additive was applied for the co-immobilization of whole cells of Escherichia coli with Chromobacterium violaceum ω-transaminase activity and Lodderomyces elongisporus with ketoreductase activity. The co-immobilized cells with two different biocatalytic activities could perform a cascade of reactions to convert racemic 4-phenylbutan-2-amine or heptan-2-amine into a nearly equimolar mixture of the corresponding enantiomerically pure R amine and S alcohol even in continuous-flow mode. The novel co-immobilized whole-cell system proved to be an easy-to-store and durable biocatalyst.


Assuntos
Aldo-Ceto Redutases/metabolismo , Células Imobilizadas/metabolismo , Transaminases/metabolismo , Aminas/química , Aminas/metabolismo , Biocatálise , Reatores Biológicos , Células Imobilizadas/enzimologia , Chromobacterium/enzimologia , Chromobacterium/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Microesferas , Saccharomycetales/enzimologia , Saccharomycetales/metabolismo , Dióxido de Silício/química , Estereoisomerismo
19.
Protein Expr Purif ; 150: 26-32, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29738827

RESUMO

Alcohol oxidase (AOX) functions in oxidation of primary alcohols into the corresponding aldehydes with potential on catalyzing synthesis reactions in chemical industry. In this study, AOX from a thermotolerant methylotrophic yeast, Ogataea thermomethanolica (OthAOX) was purified to high homogeneity using a single step chromatographic separation on a DEAE-Sepharose column. The purified OthAOX had a specific activity of 15.34 U/mg with 77.5% recovery yield. The enzyme worked optimally at 50 °C in an alkaline range (pH 9.0). According to kinetic analysis, OthAOX showed a higher affinity toward short-chain aliphatic primary alcohol with the Vmax, Km, and kcat of 0.24 nmol/min, 0.27 mM, and 3628.8 min-1, respectively against methanol. Addition of alginic acid (0.35%) showed a protective effect on enhancing thermal stability of the enzyme, resulting in 72% increase in its half-life at 40 °C under the operational conditions. This enzyme represents a promising candidate for conversion of bioethanol to acetaldehyde as secondary chemical in biorefinery.


Assuntos
Oxirredutases do Álcool , Proteínas Fúngicas , Saccharomycetales/enzimologia , Oxirredutases do Álcool/química , Oxirredutases do Álcool/isolamento & purificação , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação
20.
Appl Microbiol Biotechnol ; 102(12): 5235-5244, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29680898

RESUMO

Inorganic polyphosphate (polyP) is a significant regulatory and metabolic compound in yeast cells. We compared polyP content and localization, polyphosphatase activities, and transcriptional profile of polyP-related genes in industrially important methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The increased need for phosphate, the decrease of long-chain polyP level, the accumulation of short-chain polyP, and enhanced endopolyphosphatase activity in the crude membrane fraction were observed in methanol-grown cells compared with glucose-grown cells of both species. Transcriptome analysis revealed notable differences in the expression patterns of key genes encoding proteins related to polyP metabolism. In methanol-grown cells, the genes encoding endopolyphosphatases and phosphate transporters were upregulated. The changes in polyP metabolism are probably related to the peculiarities of bioenergetics of methanol-grown cells.


Assuntos
Pichia , Polifosfatos/metabolismo , Saccharomycetales , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Metanol/metabolismo , Pichia/química , Pichia/enzimologia , Pichia/genética , Polifosfatos/análise , Saccharomycetales/química , Saccharomycetales/enzimologia , Saccharomycetales/genética
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