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1.
PLoS One ; 16(12): e0260787, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34855894

RESUMO

The biosynthesis of chiral alcohols has important value and high attention. Aldo-keto reductases (AKRs) mediated reduction of prochiral carbonyl compounds is an interesting way of synthesizing single enantiomers of chiral alcohols due to the high enantio-, chemo- and regioselectivity of the enzymes. However, relatively little research has been done on characterization and apply of AKRs to asymmetric synthesis of chiral alcohols. In this study, the AKR from Candida tropicalis MYA-3404 (C. tropicalis MYA-3404), was mined and characterized. The AKR shown wider optimum temperature and pH. The AKR exhibited varying degrees of catalytic activity for different substrates, suggesting that the AKR can catalyze a variety of substrates. It is worth mentioning that the AKR could catalytic reduction of keto compounds with benzene rings, such as cetophenone and phenoxyacetone. The AKR exhibited activity on N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP), a key intermediate for biosynthesis of the antidepressant drug duloxetine. Besides, the AKR still has high activity whether in a reaction system containing 10%-30% V/V organic solvent. What's more, the AKR showed the strongest stability in six common organic solvents, DMSO, acetonitrile, ethyl acetate, isopropanol, ethanol, and methanol. And, it retains more that 70% enzyme activity after 6 hours, suggesting that the AKR has strong solvent tolerance. Furthermore, the protein sequences of the AKR and its homology were compared, and a 3D model of the AKR docking with coenzyme NADPH were constructed. And the important catalytic and binding sites were identified to explore the binding mechanism of the enzyme and its coenzyme. These properties, predominant organic solvents resistance and extensive substrate spectrum, of the AKR making it has potential applications in the pharmaceutical field.


Assuntos
Álcoois/metabolismo , Aldo-Ceto Redutases/metabolismo , Candida tropicalis/enzimologia , Códon , Proteínas Fúngicas/metabolismo , Solventes/química , Aldo-Ceto Redutases/química , Aldo-Ceto Redutases/genética , Sítios de Ligação , Catálise , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Especificidade por Substrato
2.
Chem Commun (Camb) ; 57(36): 4460-4463, 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33949502

RESUMO

We report a facile and reversible method to immobilize a broad range of His6-tagged proteins on the E. coli cell surface through Fe(iii)-metal complexes. A His6-tagged eGFP and four His6-tagged enzymes were successfully immobilized on the cell surface. Additionally, a hydrogel sheath around E. coli cells was generated by immobilized His6-tagged HRP.


Assuntos
Oxirredutases do Álcool/metabolismo , Escherichia coli/metabolismo , Compostos Férricos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Lacase/metabolismo , Lipase/metabolismo , Oxirredutases do Álcool/química , Bacillus licheniformis/enzimologia , Bacillus subtilis/enzimologia , Candida tropicalis/enzimologia , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/química , Escherichia coli/citologia , Compostos Férricos/química , Proteínas de Fluorescência Verde/química , Histidina/química , Histidina/metabolismo , Lacase/química , Lipase/química , Oligopeptídeos/química , Oligopeptídeos/metabolismo
3.
Microb Cell Fact ; 20(1): 105, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34034730

RESUMO

BACKGROUND: Commercial xylose purification produces xylose mother liquor (XML) as a major byproduct, which has become an inexpensive and abundant carbon source. A portion of this XML has been used to produce low-value-added products such as caramel but the remainder often ends up as an organic pollutant. This has become an issue of industrial concern. In this study, a uracil-deficient Candida tropicalis strain was engineered to efficiently convert XML to the commercially useful product xylitol. RESULTS: The xylitol dehydrogenase gene was deleted to block the conversion of xylitol to xylulose. Then, an NADPH regeneration system was added through heterologous expression of the Yarrowia lipolytica genes encoding 6-phosphate-gluconic acid dehydrogenase and 6-phosphate-glucose dehydrogenase. After process optimization, the engineered strain, C. tropicalis XZX-B4ZG, produced 97.10 g L- 1 xylitol in 120 h from 300 g L- 1 XML in a 5-L fermenter. The xylitol production rate was 0.82 g L- 1 h- 1 and the conversion rate was 92.40 %. CONCLUSIONS: In conclusion, this study performed a combination of metabolic engineering and process optimizing in C. tropicalis to enhance xylitol production from XML. The use of C. tropicalis XZX-B4ZG, therefore, provided a convenient method to transform the industrial by-product XML into the useful material xylitol.


Assuntos
Candida tropicalis/genética , Candida tropicalis/metabolismo , D-Xilulose Redutase/genética , Engenharia Metabólica , Xilitol/biossíntese , Xilose/metabolismo , Candida tropicalis/enzimologia , D-Xilulose Redutase/metabolismo , Fermentação , Glucose 1-Desidrogenase , Glucosefosfato Desidrogenase/metabolismo , Microbiologia Industrial
4.
Nucleic Acids Res ; 49(8): 4768-4781, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33856462

RESUMO

Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.


Assuntos
Motivos de Aminoácidos , Candida albicans/química , Candida tropicalis/química , Domínio Catalítico , Telomerase/química , Motivos de Aminoácidos/genética , Candida albicans/enzimologia , Candida tropicalis/enzimologia , Catálise , Domínio Catalítico/genética , Cromatografia em Gel , Cristalografia por Raios X , Difusão Dinâmica da Luz , Escherichia coli/metabolismo , Técnicas In Vitro , Modelos Moleculares , Mutação , Proteínas Recombinantes , Telomerase/genética
5.
Mol Biotechnol ; 63(6): 544-555, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33786739

RESUMO

Candida tropicalis can metabolize alkanes or fatty acids to produce long-chain dicarboxylic acids (DCAs). Fatty acid transporters located on the cell or peroxisome membrane may play an important role in this process. Using amino acid sequence homologous alignment, two putative proteins, CtFat1p and CtPxa1p, located on the cell and peroxisome membrane were found, respectively. Moreover, single- and double-knockout homologous recombination technology was used to study ctfat1p and ctpxa1p gene effects on DCA synthesis. In comparison to the wild-type strain, long-chain DCA yield decreased by 65.14%, 88.38% and 56.19% after single and double-copy knockout of ctfat1p genes and double-copy knockout of ctpxa1p genes, respectively, indicating that the knockout of ctfat1p and ctpxa1p genes had a significant effect on the conversion of oils and fats into long-chain DCAs by C. tropicalis. However, the yield of long-chain DCAs increased by 21.90% after single-knockout of the ctpxa1p gene, indicating that the single-knockout of the ctpxa1p gene may reduce fatty acid transport to peroxisome for further oxidation. Moreover, to improve the intracellular transport rate of fatty acids, ctfat1p copy number increased, increasing DCA yield by 30.10%. These results may provide useful information for enhancing the production of long-chain DCAs by C. tropicalis.


Assuntos
Alcanos/química , Candida tropicalis/química , Ácidos Graxos/química , Engenharia de Proteínas , Alcanos/metabolismo , Sequência de Aminoácidos/genética , Candida tropicalis/enzimologia , Candida tropicalis/metabolismo , Proteínas de Transporte de Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Fermentação , Redes e Vias Metabólicas/genética , Oxirredução , Peroxissomos/enzimologia , Peroxissomos/genética , Engenharia de Proteínas/métodos , Alinhamento de Sequência
6.
Prep Biochem Biotechnol ; 51(10): 1060-1070, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33724897

RESUMO

Enzymatic production of bioxylitol from lignocellulosic biomass (LCB) provides a promising alternative to both chemical and fermentative routes. This study aimed to assess the impacts of catalytic variables on bioxylitol production from wood sawdust using xylose reductase (XR) enzyme and to optimize the bioprocess. Enzyme-based xylitol production was carried out in batch cultivation under various experimental conditions to obtain maximum xylitol yield and productivity. The response surface methodology (RSM) was followed to fine-tune the most significant variables such as reaction time, temperature, and pH, which influence the synthesis of bioxylitol from sawdust hydrolysate and to optimize them. The optimum time, temperature, and pH became were 12.25 h, 35 °C, and 6.5, respectively, with initial xylose 18.8 g/L, NADPH 2.83 g/L, XR 0.027 U/mg, and agitation 100 rpm. The maximum xylitol production was attained at 16.28 g/L with a yield and productivity of 86.6% (w/w) and 1.33 g/L·h, respectively. Optimization of catalytic parameters using sequential strategies resulted in 1.55-fold improvement in overall xylitol production. This study explores a novel strategy for using sawdust hemicellulose in bioxylitol production by enzyme technology.


Assuntos
Aldeído Redutase/metabolismo , Candida tropicalis/enzimologia , Peróxidos/metabolismo , Polissacarídeos/metabolismo , Titânio/metabolismo , Óxido de Zinco/metabolismo , Biocatálise , Biomassa , Combinação de Medicamentos , Fermentação , Microbiologia Industrial/métodos
7.
Gene ; 780: 145530, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33631248

RESUMO

Candida spp. have attracted considerable attention as they cause serious human diseases in immunocompromised individuals. The genomes of the pathogenic Candida spp. have been sequenced, but systemic characterizations of their kinomes are yet to be reported. As in various eukaryotes, the protein kinases play crucial regulatory roles in pathogenicity of Candida. Increased frequency of antifungal resistance in Candida spp. requires significant attention to explore novel therapeutic molecules for their control. The present in-silico study involves novel bioinformatics strategies to identify the kinase proteins and their potential drug targets with the purpose to combat fungal infections. The study reports 103, 107 and 106 kinase proteins from 3 Candida spp., C. albicans, C. parapsilosis and C. tropicalis, respectively. Moreover, 79 common kinase proteins were identified, of which 54 proteins play essential roles in Candida spp. and 42 proteins were human non-homologues. Among the essential and human non-homologous protein kinases, 9 were found to be common essential human non-homologues, of which 6 are uniquely present in Candida. These 6 protein kinases namely, Hsl1, Npr1, Ptk2, Kin2, Ksp1 and orf19.3854 (CAALFM_CR06040WA) are involved in various molecular and cellular processes regulating virulence or pathogenicity. Further, these 6 kinases are prioritized as potential drug targets and explored for discovering new lead compounds against candidiasis. The drug repurposing approach for these 6 kinases show 13 approved drugs and investigational compounds that might play substantial inhibitory roles during combating candidiasis.


Assuntos
Antifúngicos/farmacologia , Candida albicans/enzimologia , Candida parapsilosis/enzimologia , Candida tropicalis/enzimologia , Farmacorresistência Fúngica/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Proteínas Quinases/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Testes de Sensibilidade Microbiana
8.
PLoS One ; 16(1): e0245160, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33439909

RESUMO

Increasing reports of azole resistance in Candida tropicalis, highlight the development of rapid resistance detection techniques. Nonsynonymous mutations in the lanosterol C14 alpha-demethylase (ERG11) gene is one of the predominant mechanisms of azole resistance in C. tropicalis. We evaluated the tetra primer-amplification refractory mutation system-PCR (T-ARMS-PCR), restriction site mutation (RSM), and high-resolution melt (HRM) analysis methods for rapid resistance detection based on ERG11 polymorphism in C. tropicalis. Twelve azole-resistant and 19 susceptible isolates of C. tropicalis were included. DNA sequencing of the isolates was performed to check the ERG11 polymorphism status among resistant and susceptible isolates. Three approaches T-ARMS-PCR, RSM, and HRM were evaluated and validated for the rapid detection of ERG11 mutation. The fluconazole MICs for the 12 resistant and 19 susceptible isolates were 32-256 mg/L and 0.5-1 mg/L, respectively. The resistant isolates showed A339T and C461T mutations in the ERG11 gene. The T-ARMS-PCR and RSM approaches discriminated all the resistant and susceptible isolates, whereas HRM analysis differentiated all except one susceptible isolate. The sensitivity, specificity, analytical sensitivity, time, and cost of analysis suggests that these three methods can be utilized for the rapid detection of ERG11 mutations in C. tropicalis. Additionally, an excellent concordance with DNA sequencing was noted for all three methods. The rapid, sensitive, and inexpensive T-ARMS-PCR, RSM, and HRM approaches are suitable for the detection of azole resistance based on ERG11 polymorphism in C. tropicalis and can be implemented in clinical setups for batter patient management.


Assuntos
Azóis/farmacologia , Candida tropicalis/genética , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Mutação de Sentido Incorreto , Polimorfismo Genético , Candida tropicalis/enzimologia , Farmacorresistência Fúngica/efeitos dos fármacos
9.
Int J Mol Sci ; 21(21)2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-33105833

RESUMO

Significant amounts of enolase-a cytosolic enzyme involved in the glycolysis pathway-are exposed on the cell surface of Candida yeast. It has been hypothesized that this exposed enolase form contributes to infection-related phenomena such as fungal adhesion to human tissues, and the activation of fibrinolysis and extracellular matrix degradation. The aim of the present study was to characterize, in structural terms, the protein-protein interactions underlying these moonlighting functions of enolase. The tight binding of human vitronectin, fibronectin and plasminogen by purified C. albicans and C. tropicalis enolases was quantitatively analyzed by surface plasmon resonance measurements, and the dissociation constants of the formed complexes were determined to be in the 10-7-10-8 M range. In contrast, the binding of human proteins by the S.cerevisiae enzyme was much weaker. The chemical cross-linking method was used to map the sites on enolase molecules that come into direct contact with human proteins. An internal motif 235DKAGYKGKVGIAMDVASSEFYKDGK259 in C. albicans enolase was suggested to contribute to the binding of all three human proteins tested. Models for these interactions were developed and revealed the sites on the enolase molecule that bind human proteins, extensively overlap for these ligands, and are well-separated from the catalytic activity center.


Assuntos
Fibronectinas/metabolismo , Fosfopiruvato Hidratase/metabolismo , Plasminogênio/metabolismo , Vitronectina/metabolismo , Motivos de Aminoácidos , Anticorpos/metabolismo , Ligação Competitiva , Candida albicans/enzimologia , Candida tropicalis/enzimologia , Citosol/enzimologia , Fibronectinas/química , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Proteínas Imobilizadas/metabolismo , Modelos Moleculares , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/imunologia , Plasminogênio/química , Vitronectina/química
10.
Protein Expr Purif ; 171: 105625, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32173567

RESUMO

Owing to its high-temperature tolerance, robustness, and wide use of carbon sources, Candida tropicalis is considered a good candidate microorganism for bioconversion of lignocellulose to ethanol. It also has the intrinsic ability to in situ detoxify aldehydes derived from lignocellulosic hydrolysis. However, the aldehyde reductases that catalyze this bioconversion in C. tropicalis remain unknown. Herein, we found that the uncharacterized open reading frame (ORF), CTRG_02797, from C. tropicalis encodes a novel and broad substrate-specificity aldehyde reductase that reduces at least seven aldehydes. This enzyme strictly depended on NADH rather than NADPH as the co-factor for catalyzing the reduction reaction. Its highest affinity (Km), maximum velocity (Vmax), catalytic rate constant (Kcat), and catalytic efficiency (Kcat/Km) were observed when reducing acetaldehyde (AA) and its enzyme activity was influenced by different concentrations of salts, metal ions, and chemical protective additives. Protein localization assay demonstrated that Ctrg_02797p was localized in the cytoplasm in C. tropicalis cells, which ensures an effective enzymatic reaction. Finally, Ctrg_02797p was grouped into the cinnamyl alcohol dehydrogenase (CADH) subfamily of the medium-chain dehydrogenase/reductase family. This research provides guidelines for exploring more uncharacterized genes with reduction activity for detoxifying aldehydes.


Assuntos
Aldeído Redutase/metabolismo , Candida tropicalis/enzimologia , Citoplasma/enzimologia , Proteínas Fúngicas/metabolismo , NADP/metabolismo , Fases de Leitura Aberta , Aldeído Redutase/genética , Candida tropicalis/genética , Citoplasma/genética , Proteínas Fúngicas/genética , NADP/genética
11.
Biotechnol Prog ; 36(3): e2972, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31990139

RESUMO

Xylitol is a five-carbon sugar alcohol that has a variety of uses in the food and pharmaceutical industries. In xylose assimilating yeasts, NAD(P)H-dependent xylose reductase (XR) catalyzes the reduction of xylose to xylitol. In the present study, XR with varying cofactor specificities was overexpressed in Saccharomyces cerevisiae to screen for efficient xylitol production. Xylose consumption and xylitol yields were higher when NADPH-dependent enzymes (Candida tropicalis XR and S. cerevisiae Gre3p aldose reductase) were expressed, indicating that heterologous enzymes can utilize the intracellular NADPH pool more efficiently than the NADH pool, where they may face competition from native enzymes. This was confirmed by overexpression of a NADH-preferring C. tropicalis XR mutant, which led to decreased xylose consumption and lower xylitol yield. To increase intracellular NADPH availability for xylitol production, the promoter of the ZWF1 gene, coding for the first enzyme of the NADPH-generating pentose phosphate pathway, was replaced with the constitutive GPD promoter in a strain expressing C. tropicalis XR. This change led to a ~12% increase in xylitol yield. Deletion of XYL2 and SOR1, whose gene products can use xylitol as substrate, did not further increase xylitol yield. Using wheat stalk hydrolysate as source of xylose, the constructed strain efficiently produced xylitol, demonstrating practical relevance of this approach.


Assuntos
Aldeído Redutase/genética , Engenharia Metabólica , Xilitol/biossíntese , Xilose/biossíntese , Candida tropicalis/enzimologia , Etanol/química , Fermentação , Regulação Fúngica da Expressão Gênica/genética , NAD/química , NADP/genética , Saccharomyces cerevisiae/enzimologia , Xilitol/genética , Xilose/genética
12.
Int J Antimicrob Agents ; 55(1): 105799, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31520783

RESUMO

This study evaluated the activity of echinocandins, azoles and amphotericin B against Candida spp. isolates and other yeasts and characterised azole resistance mechanisms in Candida parapsilosis and Candida tropicalis. Invasive Candida spp. isolates (n = 2936) collected in 60 hospitals worldwide during 2016-2017 underwent antifungal susceptibility testing by broth microdilution. Azole-resistant C. parapsilosis and C. tropicalis were submitted to qPCR for ERG11, CDR1 and MDR1, and the whole genome sequence was analysed. Results of non-susceptibility to echinocandins ranged from 0.0-2.3%, being highest in Candida glabrata. More than 99.0% of the Candida albicans isolates were susceptible to both fluconazole and voriconazole. Fluconazole resistance in C. glabrata was 6.5% overall, being highest in the USA (13.0%). Resistance to voriconazole in Candida krusei was only noted in the USA (5.0%). Azoles inhibited 89.1-91.6% of C. parapsilosis isolates, with most resistant isolates noted in Europe (15.1%), including 36 isolates from Italy (three hospitals), of which 34 harboured Erg11 Y132F mutations and overexpressed MDR1. Azole non-wild-type C. tropicalis (7/227) were found in five countries: 3 isolates from Thailand had the same Erg11 Y132F alteration. Fluconazole non-wild-type isolates were noted among 3/77 (3.9%) Candida dubliniensis, 4/17 (23.5%) Candida guilliermondii, 4/47 (8.5%) Candida lusitaniae and other less common yeast species. Echinocandin use has been recommended over fluconazole for invasive Candida infections. However, azoles are still active against the most common Candida spp. and resistance appears to be restricted to certain geographic regions and associated with Erg11 Y132 alterations in C. parapsilosis and C. tropicalis.


Assuntos
Antifúngicos/farmacologia , Candida parapsilosis/enzimologia , Candida tropicalis/enzimologia , Candidíase Invasiva/microbiologia , Candidíase/microbiologia , Farmacorresistência Fúngica , Esterol 14-Desmetilase/genética , Substituição de Aminoácidos , Anfotericina B/farmacologia , Azóis/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/genética , Candida parapsilosis/efeitos dos fármacos , Candida parapsilosis/genética , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/genética , Candidíase/tratamento farmacológico , Candidíase Invasiva/tratamento farmacológico , Equinocandinas/farmacologia , Fluconazol/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Voriconazol/farmacologia
13.
J Biol Chem ; 295(2): 570-583, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31806705

RESUMO

Copper (Cu)-only superoxide dismutases (SOD) represent a newly characterized class of extracellular SODs important for virulence of several fungal pathogens. Previous studies of the Cu-only enzyme SOD5 from the opportunistic fungal pathogen Candida albicans have revealed that the active-site structure and Cu binding of SOD5 strongly deviate from those of Cu/Zn-SODs in its animal hosts, making Cu-only SODs a possible target for future antifungal drug design. C. albicans also expresses a Cu-only SOD4 that is highly similar in sequence to SOD5, but is poorly characterized. Here, we compared the biochemical, biophysical, and cell biological properties of C. albicans SOD4 and SOD5. Analyzing the recombinant proteins, we found that, similar to SOD5, Cu-only SOD4 can react with superoxide at rates approaching diffusion limits. Both SODs were monomeric and they exhibited similar binding affinities for their Cu cofactor. In C. albicans cultures, SOD4 and SOD5 were predominantly cell wall proteins. Despite these similarities, the SOD4 and SOD5 genes strongly differed in transcriptional regulation. SOD5 was predominantly induced during hyphal morphogenesis, together with a fungal burst in reactive oxygen species. Conversely, SOD4 expression was specifically up-regulated by iron (Fe) starvation and controlled by the Fe-responsive transcription factor SEF1. Interestingly, Candida tropicalis and the emerging fungal pathogen Candida auris contain a single SOD5-like SOD rather than a pair, and in both fungi, this SOD was induced by Fe starvation. This unexpected link between Fe homeostasis and extracellular Cu-SODs may help many fungi adapt to Fe-limited conditions of their hosts.


Assuntos
Candida/enzimologia , Candidíase/microbiologia , Ferro/metabolismo , Superóxido Dismutase/metabolismo , Candida/metabolismo , Candida albicans/enzimologia , Candida albicans/metabolismo , Candida tropicalis/enzimologia , Candida tropicalis/metabolismo , Cobre/metabolismo , Humanos , Modelos Moleculares , Espécies Reativas de Oxigênio/metabolismo
14.
J Agric Food Chem ; 68(1): 235-241, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31822063

RESUMO

Enzymatic production of xylitol is a promising alternative to the chemical hydrogenation process. However, it encounters problems that are largely due to protein susceptibility to environmental factors. In this study, to develop a robust, practical enzymatic process for xylitol production, a coupled enzyme system consisting of formate dehydrogenase (FDH), glucose dehydrogenase (GDH), and xylose reductase (XR) was constructed, wherein the alkaline product produced by FDH and the acidic product produced by GDH could neutralize each other during cofactor regeneration. After optimization of conditions, a pH-neutralization, redox-balanced process was developed that could be carried out in pure water requiring no pH regulation. As a result, a xylitol production of 273.6 g/L that is much higher than those yet reported was obtained from 2 M xylose in 24 h, with a relatively high productivity of 11.4 g/(L h). The strategy demonstrated here can be adapted for the production of other NADH-consuming products.


Assuntos
Formiato Desidrogenases/química , Glucose 1-Desidrogenase/química , Água/química , Xilitol/química , Aldeído Redutase/química , Bacillus/enzimologia , Proteínas de Bactérias/química , Biocatálise , Candida tropicalis/enzimologia , Proteínas Fúngicas/química , Concentração de Íons de Hidrogênio , Oxirredução
15.
Sci Rep ; 9(1): 20088, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31882753

RESUMO

Cytochrome P450 reductases (CPRs) are diflavin oxidoreductases that supply electrons to type II cytochrome P450 monooxygenases (CYPs). In addition, it can also reduce other proteins and molecules, including cytochrome c, ferricyanide, and different drugs. Although various CPRs have been functionally and structurally characterized, the overall mechanism and its interaction with different redox acceptors remain elusive. One of the main problems regarding electron transfer between CPRs and CYPs is the so-called "uncoupling", whereby NAD(P)H derived electrons are lost due to the reduced intermediates' (FAD and FMN of CPR) interaction with molecular oxygen. Additionally, the decay of the iron-oxygen complex of the CYP can also contribute to loss of reducing equivalents during an unproductive reaction cycle. This phenomenon generates reactive oxygen species (ROS), leading to an inefficient reaction. Here, we present the study of the CPR from Candida tropicalis (CtCPR) lacking the hydrophobic N-terminal part (Δ2-22). The enzyme supports the reduction of cytochrome c and ferricyanide, with an estimated 30% uncoupling during the reactions with cytochrome c. The ROS produced was not influenced by different physicochemical conditions (ionic strength, pH, temperature). The X-ray structures of the enzyme were solved with and without its cofactor, NADPH. Both CtCPR structures exhibited the closed conformation. Comparison with the different solved structures revealed an intricate ionic network responsible for the regulation of the open/closed movement of CtCPR.


Assuntos
Candida tropicalis/enzimologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Cristalografia por Raios X , Transporte de Elétrons , NADPH-Ferri-Hemoproteína Redutase/química , Oxirredução , Conformação Proteica
16.
J Biotechnol ; 304: 31-37, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31421146

RESUMO

2,3-Butanediol (2,3-BD) can be produced at high titers by engineered Saccharomyces cerevisiae by abolishing the ethanol biosynthetic pathway and introducing the bacterial butanediol-producing pathway. However, production of 2,3-BD instead of ethanol by engineered S. cerevisiae has resulted in glycerol production because of surplus NADH accumulation caused by a lower degree of reduction (γ = 5.5) of 2,3-BD than that (γ = 6) of ethanol. In order to eliminate glycerol production and resolve redox imbalance during 2,3-BD production, both GPD1 and GPD2 coding for glycerol-3-phosphate dehydrogenases were disrupted after overexpressing NADH oxidase from Lactococcus lactis. As disruption of the GPD genes caused growth defects due to limited supply of C2 compounds, Candida tropicalis PDC1 was additionally introduced to provide a necessary amount of C2 compounds while minimizing ethanol production. The resulting strain (BD5_T2 nox_dGPD1,2_CtPDC1) produced 99.4 g/L of 2,3-BD with 0.5 g/L glycerol accumulation in a batch culture. The fed-batch fermentation led to production of 108.6 g/L 2,3-BD with a negligible amount of glycerol production, resulting in a high BD yield (0.462 g2,3-BD/gglucose) corresponding to 92.4 % of the theoretical yield. These results demonstrate that glycerol-free production of 2,3-BD by engineered yeast is feasible.


Assuntos
Butileno Glicóis/metabolismo , Deleção de Genes , Glicerolfosfato Desidrogenase/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Técnicas de Cultura Celular por Lotes , Candida tropicalis/enzimologia , Fermentação , Proteínas Fúngicas/genética , Engenharia Genética , Glicerol/metabolismo , Glicerol-3-Fosfato Desidrogenase (NAD+)/genética , Lactococcus lactis/enzimologia , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Piruvato Descarboxilase/deficiência , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
17.
Phys Chem Chem Phys ; 21(19): 10105-10113, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31062768

RESUMO

Enoyl thioester reductase from Candida tropicalis (Etr1p) catalyzes the NADPH-dependent conversion of enoyl thioesters into acyl thioesters, which are essential in fatty acid and second metabolite biosynthesis. In this paper, we explored the detailed catalytic mechanism of Etr1p by performing QM/MM calculations. Here, we focused on the formation of the covalent ene adduct intermediate and the proton transfer from Tyr79 to the substrate. Our calculation results reveal that the formation of the stable covalent ene adduct follows the Michael addition mechanism rather than the electrocyclic ene reaction. In addition, the ene adduct intermediate can reversibly decompose into the carbanion, and the proton of Tyr79 undertakes a direct electrophilic attack on the substrate to yield the product. In addition, three crystal water molecules do not participate in the catalytic reaction, but they play a crucial role in the hydride transfer and the proton transfer processes by forming a hydrogen bond network. These findings presented here would benefit our understanding of the catalytic mechanism of the NADPH-dependent enzyme.


Assuntos
Candida tropicalis/enzimologia , Ésteres/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Compostos de Enxofre/metabolismo , Biocatálise , Ésteres/química , Simulação de Dinâmica Molecular , Estrutura Molecular , Teoria Quântica , Compostos de Enxofre/química
18.
Ultrason Sonochem ; 48: 321-328, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30080557

RESUMO

This study has investigated ultrasound-assisted xylitol production through fermentation of dilute acid (pentose-rich) hydrolysate of sugarcane bagasse using free cells of Candida tropicalis. Sonication of fermentation mixture at optimum conditions was carried out in ultrasound bath (37 kHz and 10% duty cycle). Time profiles of substrate and product in control (mechanical shaking) and test (mechanical shaking + sonication) fermentations were fitted to kinetic model using Genetic Algorithm (GA) optimization. Max. xylitol yield of 0.56 g/g and 0.61 g/g of xylose was achieved in control and test fermentations, respectively. The biomass yield also increased marginally (∼17%) with sonication. However, kinetics of fermentation increased drastically (2.5×) with sonication with 2× rise in xylose uptake and utilization by the cells. With comparative analysis of kinetic parameters in control and test experiments, this result was attributed to enhanced permeability of cell membrane that allowed faster diffusion of nutrients, substrates and products across cell membrane, higher enzyme-substrate affinity, dilution of toxic components and reduced inhibition of intracellular enzymes by substrate.


Assuntos
Candida tropicalis/metabolismo , Fermentação , Sonicação/métodos , Xilitol/metabolismo , Aldeído Redutase/metabolismo , Algoritmos , Candida tropicalis/citologia , Candida tropicalis/enzimologia , Permeabilidade da Membrana Celular , Citometria de Fluxo , Cinética , Saccharum/metabolismo , Especificidade por Substrato
19.
Artigo em Inglês | MEDLINE | ID: mdl-29774204

RESUMO

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.


Assuntos
Candida albicans/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Fatores Matadores de Levedura/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Candida albicans/enzimologia , Candida albicans/genética , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/enzimologia , Candida tropicalis/genética , Catalase/metabolismo , Debaryomyces/genética , Debaryomyces/metabolismo , Proteínas Fúngicas/genética , Glicerol/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Pressão Osmótica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
20.
Rev Iberoam Micol ; 35(1): 17-21, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29287631

RESUMO

BACKGROUND: Candida tropicalis is an increasingly important human pathogen which usually affects neutropenic oncology patients with common hematogenous seeding to peripheral organs and high mortality rates. Candida pathogenicity is facilitated by several virulence attributes, including secretion of hydrolytic enzymes; however, little is known regarding the C. tropicalis ability to secrete them and their role in the disease. AIMS: To confirm by molecular means the identification of 187 clinical isolates (127 from blood, 52 from urine, and 8 from diverse clinical origins) phenotypically identified as C. tropicalis, and to investigate their in vitro aspartyl proteinase, phospholipase, esterase, hemolysin, DNase and coagulase activities. METHODS: The molecular confirmation was performed by ITS sequencing, and the enzymatic determinations were conducted using plate assays with specific substrates, with the exception of coagulase, which was determined by the classical tube test. RESULTS: The majority of the strains exhibited a very strong or strong activity of aspartyl proteinase, phospholipase and esterase. A 4.7% of the bloodstream isolates were hemolysin producers, and all were negative for the coagulase and DNase assays. CONCLUSIONS: Very strong activities of aspartyl proteinase, phospholipase and esterase profiles were detected, and a statistical association between phospholipase production and blood and urine isolates was found.


Assuntos
Candida tropicalis/isolamento & purificação , Candidíase/microbiologia , Líquidos Corporais/microbiologia , Candida tropicalis/enzimologia , Candida tropicalis/genética , Candidemia/microbiologia , DNA Fúngico/análise , Proteínas Fúngicas/análise , Humanos , Fenótipo , Análise de Sequência de DNA , Infecções Urinárias/microbiologia
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