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1.
Acta Odontol Latinoam ; 33(2): 104-111, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32920612

RESUMO

Candida dubliniensis (Cd) and Candida albicans (Ca) are the most frequently isolated yeasts in HIV+ patients. Some of the enzymes produced by these yeasts are considered virulence factors since they contribute to pathogenicity of Candida spp. The aim of the present study was to compare production of enzymes such as phospholipase (Ph), proteinase (P), and hemolysin (H) by Cd and Ca strains isolated from periodontal HIV-positive patients receiving and not receiving highly active antiretroviral therapy (HAART). Subgingival biofilm samples were obtained using paper points, and a sample of oral mucosa was taken using a swab. Phenotypic and molecular methods were used to isolate 39 strains of Candida, including 25 strains of Cd and 14 strains of Ca, obtained from 33 periodontal pocket samples and 6 oral mucosa samples collected from 15 HIV+ patients (8 receiving and 7 not receiving HAART). Malt egg-yolk agar, albumin agar and blood agar were used to evaluate pH, P and H production respectively. The strains were inoculated in duplicate and incubated at 37 ºC. Colony and halo diameters were measured. A greater proportion of Ca was observed in patients not receiving HAART, and a higher proportion of Cd was observed in those under HAART, Chi2 p< 0.001. Phospholipase production was observed in 92.9% percent of isolated Ca strains but in none of the isolated Cd strains. Proteinase production was high in Ca and Cd strains isolated from patients not receiving HAART. Hemolysin production was observed in all the studied strains, though it was significantly higher (p=0.04) in Ca and Cd strains isolated from patients not receiving HAART. To sum up, the proportion of Candida dubliniensis strains was highest in the subgingival biofilm of patients receiving HAART, and Cd strains were found to express fewer virulence factors than Ca strains.


Assuntos
Terapia Antirretroviral de Alta Atividade/métodos , Biofilmes/crescimento & desenvolvimento , Candida albicans/enzimologia , Candida albicans/isolamento & purificação , Candida/enzimologia , Candida/isolamento & purificação , Candidíase Bucal/microbiologia , Gengiva/microbiologia , Infecções por HIV/complicações , Candida/classificação , Candida/genética , Candida albicans/genética , Candidíase Bucal/complicações , Genótipo , Infecções por HIV/microbiologia , Humanos , Mucosa Bucal/microbiologia , Fenótipo , Reação em Cadeia da Polimerase , Fatores de Virulência/genética
2.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 782-791, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32347072

RESUMO

L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.


Assuntos
Aminobutiratos , Formiato Desidrogenases , Leucina Desidrogenase , Treonina Desidratase , Treonina , Aminobutiratos/síntese química , Bacillus thuringiensis/enzimologia , Candida/enzimologia , Escherichia coli/enzimologia , Formiato Desidrogenases/metabolismo , Leucina Desidrogenase/metabolismo , Treonina/metabolismo , Treonina Desidratase/metabolismo
3.
Arch Microbiol ; 202(7): 1729-1739, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32328754

RESUMO

We isolated two Candida pseudointermedia strains from the Atlantic rain forest in Brazil, and analyzed cellobiose metabolization in their cells. After growth in cellobiose medium, both strains had high intracellular ß-glucosidase activity [~ 200 U (g cells)-1 for 200 mM cellobiose and ~ 100 U (g cells)-1 for 2 mM pNPßG] and negligible periplasmic cellobiase activity. During batch fermentation, the strain with the best performance consumed all the available cellobiose in the first 18 h of the assay, producing 2.7 g L-1 of ethanol. Kinetics of its cellobiase activity demonstrated a high-affinity hydrolytic system inside cells, with Km of 12.4 mM. Our data suggest that, unlike other fungal species that hydrolyze cellobiose extracellularly, both analyzed strains transport it to the cytoplasm, where it is then hydrolyzed by high-affinity intracellular ß-glucosidases. We believe this study increases the fund of knowledge regarding yeasts from Brazilian microbiomes.


Assuntos
Candida/enzimologia , Celobiose/metabolismo , Madeira/metabolismo , Madeira/microbiologia , beta-Glucosidase/metabolismo , Brasil , Candida/isolamento & purificação , Candida/metabolismo , Metabolismo dos Carboidratos , Etanol/metabolismo , Fermentação , Hidrólise , Cinética
4.
World J Microbiol Biotechnol ; 36(3): 45, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32130535

RESUMO

Entrapment of halloysite nanotubes (HNTs) loaded with enzyme, into a polymer matrix (PVA/Alg), is a way to produce an environment surrounding the adsorbed enzyme molecules which improves the enzyme properties such as storage and operational stability. Hence, in this study, we optimised the factors affecting lipase adsorption onto halloysite nanotubes including halloysite amounts (5, 42.5 and 80 mg), lipase concentrations (30, 90 and 150 µg/ml), temperatures (5, 20 and 35 °C) and adsorption times (30, 165 and 300 min). The optimal conditions were determined as an halloysite amount of 50 to 80 mg, a lipase concentration of 30 to 57 µg/ml, an adsorption temperature of 20 °C and an adsorption time of 165 min, which resulted in a specific activity and adsorption efficiency of 15,000 (U/g protein) and 70%, respectively. Then, lipase adsorbed under optimal conditions was entrapped in a PVA/Alg hydrogel. The formation mechanism of immobilized lipase was investigated by FESEM and FTIR. Subsequent entrapment of adsorbed lipase improved the lipase storage and operational stability. Km, Vmax, Kcat and Kcat/Km values showed an increase in the entrapped HNT-lipase performance in comparison with the free and adsorbed lipase.


Assuntos
Argila/química , Lipase/química , Álcool de Polivinil/química , Adsorção , Candida/enzimologia , Estabilidade Enzimática , Enzimas Imobilizadas/química , Nanotubos
5.
Ecotoxicol Environ Saf ; 195: 110480, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32203774

RESUMO

Arsenic (As) is a serious threat for environment and human health. Rice, the main staple crop is more prone to As uptake. Bioremediation strategies with heavy metal tolerant rhizobacteria are well known. The main objective of the study was to characterize arsenic-resistant yeast strains, capable of mitigating arsenic stress in rice. Three yeast strains identified as Debaryomyces hansenii (NBRI-Sh2.11), Candida tropicalis (NBRI-B3.4) and Candida dubliniensis (NBRI-3.5) were found to have As reductase activity. D. hansenii with higher As tolerance has As expulsion ability as compared to other two strains. Inoculation of D. hansenii showed improved detoxification through scavenging of reactive oxygen species (ROS) by the modulation of SOD and APX activity under As stress condition in rice. Modulation of defense responsive gene (NADPH, GST, GR) along with arsR and metal cation transporter are the probable mechanism of As detoxification as evident with improved membrane (electrolyte leakage) stability. Reduced grain As (~40% reduction) due to interaction with D. hansenii (NBRI-Sh2.11) further validated it's As mitigation property in rice. To the best of our knowledge D. hansenii has been reported for the first time for arsenic stress mitigation in rice with improved growth and nutrient status of the plant.


Assuntos
Arsênico/toxicidade , Debaromyces/enzimologia , Oryza/efeitos dos fármacos , Inoculantes Agrícolas , Arseniato Redutases/metabolismo , Arsênico/metabolismo , Biodegradação Ambiental , Candida/enzimologia , Debaromyces/efeitos dos fármacos , Debaromyces/genética , Debaromyces/metabolismo , Oryza/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo
6.
J Enzyme Inhib Med Chem ; 35(1): 398-403, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31899979

RESUMO

(2-(2,4-Dichlorophenyl)-3-(1H-indol-1-yl)-1-(1,2,4-1H-triazol-1-yl)propan-2-ol (8 g), a new 1,2,4-triazole-indole hybrid molecule, showed a broad-spectrum activity against Candida, particularly against low fluconazole-susceptible species. Its activity was higher than fluconazole and similar to voriconazole on C. glabrata (MIC90 = 0.25, 64 and 1 µg/mL, respectively), C. krusei (MIC90 = 0.125, 64 and 0.125 µg/mL, respectively) and C. albicans (MIC90 = 0.5, 8 and 0.25 µg/mL, respectively). The action mechanisms of 8 g were also identified as inhibition of ergosterol biosynthesis and phospholipase A2-like activity. At concentration as low as 4 ng/mL, 8g inhibited ergosterol production by 82% and induced production of 14a-methyl sterols, that is comparable to the results obtained with fluconazole at higher concentration. 8 g demonstrated moderate inhibitory effect on phospholipase A2-like activity being a putative virulence factor. Due to a low MRC5 cytotoxicity, this compound presents a high therapeutic index. These results pointed out that 8 g is a new lead antifungal candidate with potent ergosterol biosynthesis inhibition.


Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Indóis/farmacologia , Triazóis/farmacologia , Animais , Antifúngicos/química , Candida/enzimologia , Candida/metabolismo , Linhagem Celular , Ergosterol/antagonistas & inibidores , Ergosterol/biossíntese , Feminino , Humanos , Indóis/química , Camundongos , Testes de Sensibilidade Microbiana , Especificidade da Espécie , Triazóis/química
7.
J Oleo Sci ; 69(1): 43-53, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31902894

RESUMO

Ferulic acid (FA), 4-hydroxyl-3-methoxy-2-benzylacrylic acid, has antioxidant, anticancer and ultraviolet absorption activities. However, the low hydrophilicity of FA has limited its application. Glyceryl ferulate (FG), which is an all-natural hydrophilic derivative of FA, can be used as an antioxidant and UV filter in food and cosmetic formulations. However, the applications of FG in these fields are limited due to its low content in nature. In this work, free liquid lipase was firstly used as a catalyst for FG preparation. Several different free liquid lipases (Candida antartica lipase-B, Candida antartica lipase-A, Thermomyces lanuginosus (Lipozyme TL 100L)) were screened and compared. The effects of the transesterification parameters (time, temperature, enzyme load and substrate ratio) were optimized and evaluated by response surface methodology. A reaction thermodynamic investigation was also performed. The results showed that, among the tested free lipases, the maximum FG yield (84.8±1.5%) was achieved using free Candida antartica lipase-B. Under the optimized conditions (an atmospheric system, an enzyme load of 11.1% and a 20:1 molar ratio of glycerol to EF at 70°C for 39.5 h), the FG yield and EF conversion were 84.8±1.5% and 95.7±1.2%, respectively. The activation energies of FG formation and EF conversion were 56.4 and 58.0kJ/mol, respectively.


Assuntos
Candida/enzimologia , Proteínas Fúngicas/química , Lipase/química , Monoglicerídeos/química , Catálise , Interações Hidrofóbicas e Hidrofílicas
8.
Appl Biochem Biotechnol ; 190(1): 218-231, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31332676

RESUMO

Yeast surface display has emerged as a viable approach for self-immobilization enzyme as whole-cell catalysts. Herein, we displayed Candida rugosa lipase 1 (CRL LIP1) on the cell wall of Pichia pastoris for docosahexaenoic acid (DHA) enrichment in algae oil. After a 96-h culture, the displayed CRL LIP1 achieved the highest activity (380 ± 2.8 U/g) for hydrolyzing olive oil under optimal pH (7.5) and temperature (45 °C) conditions. Additionally, we improved the thermal stability of displayed LIP1, enabling retention of 50% of its initial bioactivity following 6 h of incubation at 45 °C. Furthermore, the content of DHA enhanced from 40.61% in original algae oil to 50.44% in glyceride, resulting in a 1.24-fold increase in yield. The displayed CRL LIP1 exhibited an improved thermal stability and a high degree of bioactivity toward its native macromolecule substrates algae oil and olive oil, thereby expanding its potential for industrial applications in fields of food and pharmaceutical. These results suggested that surface display provides an effective strategy for simultaneous convenient expression and target protein immobilization.


Assuntos
Proteínas de Bactérias/metabolismo , Candida/enzimologia , Ácidos Docosa-Hexaenoicos/metabolismo , Temperatura Alta , Lipase/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo
9.
Appl Biochem Biotechnol ; 190(2): 686-702, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31456111

RESUMO

Hexyl laurate is employed in several cosmetics having great demand. It could be synthesized catalytically like a "natural" perfume using a lipase. The use of mesocellular foam silica (MCF) for immobilization of lipases could be made using supercritical CO2 as a medium to enhance its activity in comparison with the normal techniques. Three different catalysts were supported on MCF such as Candida antractica B (CALB), Amano AYS, and Porcine pancreas (PPL), and their activity was evaluated in the preparation of hexyl laurate from lauric acid and hexyl alcohol. CALB@ MCF was the best among all. A systematic study was conducted to assess the effects of different operating parameters. It was ternary complex mechanism with inhibition by hexyl alcohol. The enzyme was reusable and the process is green.


Assuntos
Dióxido de Carbono/química , Enzimas Imobilizadas/metabolismo , Ácidos Láuricos/química , Lipase/metabolismo , Adsorção , Animais , Candida/enzimologia , Esterificação , Cromatografia Gasosa-Espectrometria de Massas , Cinética , Microscopia Eletrônica de Varredura , Espectroscopia de Infravermelho com Transformada de Fourier , Suínos , Temperatura
10.
Appl Biochem Biotechnol ; 190(3): 982-996, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31650356

RESUMO

This work presents a novel lipase immobilization method via polylactic acid (PLA) modified by polyethylene glycol (PEG). The immobilization performance of lipase was characterized by SEM and FTIR. The results indicated that lipase from Candida rugosa type VII was successfully immobilized on the biocompatible PLA/PEG film in the presence of 1, 6-hexamethylene diamine and glutaraldehyde. In addition, the presence of 1, 6-hexylenediamime (8%, w/w) could maintain the maximum enzymatic activity. Moreover, the optimum temperature of lipase shifted from 45 to 50 °C after immobilization. The thermal inactivation experiment illustrated that the immobilized lipase retained up to 63% of the original activity after treated at 50 °C in buffer for 120 min, significantly higher than that of the control (33%) (p < 0.05). The optimum pH value of the immobilized lipase shifted from 6.5 to 7.5. Additionally, compared with the free lipase maintaining 23% of its original activity, the immobilized lipase successfully retained up to 70% after 30 days of storage. Furthermore, the immobilized lipase displayed the excellent reusability of 82% after six cycles. In conclusion, the proposed immobilization approach developed can be potentially used as a sustainable alternative for the immobilization of lipases and the utilization of biocompatible polymer. Graphical Abstract.


Assuntos
Enzimas Imobilizadas/metabolismo , Lipase/metabolismo , Poliésteres/química , Polietilenoglicóis/química , Candida/enzimologia , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio
11.
Appl Biochem Biotechnol ; 190(3): 839-850, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31502107

RESUMO

This work aims to study the immobilization of Candida rugosa lipase (CRL) onto corn straw residue. For this purpose, chemical, morphological, and textural characteristics of the corn straw; immobilization process by adsorption; and immobilized enzyme activity and storage stability were evaluated. The corn straw presented isoelectric point of 7.0, surface with hydroxyl bands being favorable to the immobilization process. An irregular surface was also observed with fibers and pores, which are mesoporous and macroporous, characteristics that demonstrate efficiency in mass transfer mechanisms. Upon immobilization, it was observed that adsorption velocity is proportional to the square of the available adsorption sites (pseudo-second-order), and that the immobilization occurs in monolayers (Langmuir isotherm). The adsorption process was favorable and considered as a chemical adsorption mechanism. After immobilization, the optimum temperature increased, the optimum pH reduced, and the affinity of the biocatalyst for the substrate decreased. Corn straw derivative demonstrated good thermal stability. Regarding storage stability, there was approximately 12% loss of activity after 60 days of storage at 4 °C. Considering that no treatment was applied to the corn straw, this result is satisfactory and shows good affinity between this support and CRL.


Assuntos
Enzimas Imobilizadas/metabolismo , Lipase/metabolismo , Zea mays/química , Adsorção , Candida/enzimologia , Temperatura Baixa , Estabilidade Enzimática , Ponto Isoelétrico
12.
Enzyme Microb Technol ; 132: 109439, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731963

RESUMO

Lipase-immobilized nanomaterials with high activity and stable reusability would have a great impact in different fields. However, developing such materials has proven to be challenging. Herein, polymer (pAcDED)-coated magnetic nanoparticles (MNPs) displaying long alkyl chains, either octyl (C8) or hexadecyl (C16), have been prepared and used for immobilization of Candida rugosa lipase. The aim of the study was to develop magnetic supports able to bind enzyme via interfacial activation thus to stabilized the lipase open conformation. Among the developed nanosupports, the one endowed with the longest alkyl chains (MNPs-pAcDED-C16) provided the best efficiencies of the immobilized enzyme (70% vs. tributyrin and 130% vs. ethyl butyrate). Such results suggest both enzyme adsorption in open conformation and a change of enzyme specificity during immobilization. The MNPs-pAcDED-C16 system also showed better resistance to temperature inactivation in the 25-70 °C temperature range compared to free lipase and good reusability (4 consecutive cycles). The overall performances together with the convenience in the recovery offered by magnetic separation indicate our surface-modified MNPs as efficient and environmentally compatible materials for lipase immobilization.


Assuntos
Candida/enzimologia , Enzimas Imobilizadas/metabolismo , Lipase/metabolismo , Nanocompostos/química , Adsorção , Estabilidade Enzimática , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Nanopartículas de Magnetita , Temperatura
13.
Int J Biol Macromol ; 144: 183-189, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31843602

RESUMO

Poly(ε-caprolactone) (PCL) particles were melt-pressed into films using a hot press and then subjected to degradation by lipase from Candida antarctica and cutinase from Fusarium solani, respectively. The differences in weight loss, degradation modes, thermal stability, and crystallization were investigated after degradation by two kinds of enzymes. The result showed that mass loss of PCL films degraded by lipase was higher than that degraded by cutinase at the same enzyme concentrations. The degradation mode of PCL films is layered for cutinase degradation and penetrated for lipase degradation. Crystallinity of PCL had no obvious decrease after degradation by cutinase, but it markedly decreased after lipase-degradation. PCL films occurred one-step decomposition during heating and the cutinase-degraded products had similar thermal stability. Whereas the thermal stability of lipase-degraded PCL decreased significantly and the weight loss of the PCL occurred in several steps with increasing lipase hydrolysis time.


Assuntos
Candida/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Fusarium/enzimologia , Lipase/metabolismo , Poliésteres/metabolismo , Varredura Diferencial de Calorimetria , Hidrólise , Peso Molecular , Espectroscopia de Infravermelho com Transformada de Fourier , Termogravimetria , Difração de Raios X
14.
Molecules ; 24(23)2019 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-31771200

RESUMO

Lipases are among the most frequently used biocatalysts in organic synthesis, allowing numerous environmentally friendly and inexpensive chemical transformations. Here, we present a biomimetic strategy based on iron(III)-catalyzed oxidative coupling and selective ester monohydrolysis using lipases for the synthesis of unsymmetric biphenyl-based esters under mild conditions. The diverse class of biphenyl esters is of pharmaceutical and technical relevance. We explored the potency of a series of nine different lipases of bacterial, fungal, and mammalian origin on their catalytic activities to cleave biphenyl esters, and optimized the reaction conditions, in terms of reaction time, temperature, pH, organic solvent, and water-organic solvent ratios, to improve the chemoselectivity, and hence control the ratio of unsymmetric versus symmetric products. Elevated temperature and increased DMSO content led to an almost exclusive monohydrolysis by the four lipases Candida rugosa lipase (CRL), Mucor miehei lipase (MML), Rhizopus niveus lipase (RNL), and Pseudomonas fluorescens lipase (PFL). The study was complemented by in silico binding predictions to rationalize the observed differences in efficacies of the lipases to convert biphenyl esters. The optimized reaction conditions were transferred to the preparative scale with high yields, underlining the potential of the presented biomimetic approach as an alternative strategy to the commonly used transition metal-based strategies for the synthesis of diverse biphenyl esters.


Assuntos
Candida/enzimologia , Ésteres/química , Lipase/metabolismo , Mucor/enzimologia , Pseudomonas fluorescens/enzimologia , Rhizopus/enzimologia , Proteínas de Bactérias/metabolismo , Mimetismo Biológico , Catálise , Simulação por Computador , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Temperatura
15.
J Agric Food Chem ; 67(51): 14121-14128, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31775508

RESUMO

Heliotropin, a compound with important roles in the spice and fragrance industries and broad application prospects, is mainly produced through chemical methods. Here, we established a novel process for the synthesis of heliotropin by Escherichia coli whole cells through biotransformation of isosafrole. Directed evolution and high-throughput screening based on 2,4-dinitrophenylhydrazine were used to improve the activity of trans-anethole oxygenase toward isosafrole, and a mutant (TAO3G2) was obtained that had a high ability to oxidize isosafrole. Formate dehydrogenase (FDH) and TAO3G2 were coexpressed in E. coli, significantly increasing the catalytic efficiency by regenerating more NADH to promote isosafrole oxidation. Furthermore, after optimizing the molar ratio of isosafrole to the auxiliary substrate, the final concentration of heliotropin was increased from 9.15 to 19.45 g/L, and the maximum yield and space-time yield reached 96.02% and 3.89 g/L/h, respectively. These results suggest that the biosynthesis of heliotropin should have excellent industrial application value.


Assuntos
Benzaldeídos/metabolismo , Benzodioxóis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Formiato Desidrogenases/genética , Proteínas Fúngicas/genética , Oxigenases/genética , Candida/enzimologia , Formiato Desidrogenases/metabolismo , Proteínas Fúngicas/metabolismo , Engenharia Metabólica , NAD/metabolismo , Oxigenases/metabolismo , Safrol/metabolismo
16.
Chemistry ; 25(69): 15863-15870, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31596001

RESUMO

Sustainability in chemistry heavily relies on heterogeneous catalysis. Enzymes, the main catalyst for biochemical reactions in nature, are an elegant choice to catalyze reactions due to their high activity and selectivity, although they usually suffer from lack of robustness. To overcome this drawback, enzyme-decorated nanoporous heterogeneous catalysts were developed. Three different approaches for Candida antarctica lipase B (CAL-B) immobilization on a covalent organic framework (PPF-2) were employed: physical adsorption on the surface, covalent attachment of the enzyme in functional groups on the surface and covalent attachment into a linker added post-synthesis. The influence of the immobilization strategy on the enzyme uptake, specific activity, thermal stability, and the possibility of its use through multiple cycles was explored. High specific activities were observed for PPF-2-supported CAL-B in the esterification of oleic acid with ethanol, ranging from 58 to 283 U mg-1 , which was 2.6 to 12.7 times greater than the observed for the commercial Novozyme 435.


Assuntos
Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Lipase/química , Estruturas Metalorgânicas/química , Adsorção , Biocatálise , Candida/enzimologia , Esterificação , Modelos Moleculares , Nanoporos/ultraestrutura , Ácido Oleico/química
17.
An Acad Bras Cienc ; 91(3): e20180735, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31553366

RESUMO

Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) consists of emergent multidrug-resistant pathogens that cause bloodstream and deep-seated infections. However, little is known about their virulence factors. Herein, we evaluated the presence of extracellular serine peptidases in this fungal complex. Serine peptidase activity was measured by spectrophotometry using chromogenic peptide substrates to the S1 family. Chymotrypsin-, trypsin- and elastase-like activities were detected in all fungal isolates. Since higher chymotrypsin- and trypsin-like activities were observed from the cleavage of N-succinyl-Ala-Ala-Pro-Phe-pNa and N-benzoyl-Phe-Val-Arg-pNa, respectively, these substrates were selected for further experiments. Overall, pHs 7.0 and 9.0 were those in which higher chymotrypsin- and trypsin-like activities were observed, respectively, displaying higher hydrolytic activities at 37-45°C. Additionally, the serine peptidases produced by C. haemulonii complex were inhibited by PMSF and AEBSF in a typically concentration-dependent manner. Although the Michaelis constant (Km) values obtained for chymotrypsin-like peptidases were similar, greater differences were observed for trypsin-like enzymes secreted by the different fungal isolates. This is the first time that peptidases belonging to the S1 family are described in the C. haemulonii species complex. Thus, these data open the doors for more detailed studies into potential roles of these peptidases in fungal virulence.


Assuntos
Candida/enzimologia , Quimotripsina/metabolismo , Farmacorresistência Fúngica Múltipla , Tripsina/metabolismo , Candida/classificação , Meios de Cultura , Espectrofotometria , Temperatura
18.
J Enzyme Inhib Med Chem ; 34(1): 1474-1480, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31414611

RESUMO

The discovery of allosteric modulators is a multi-disciplinary approach, which is time- and cost-intensive. High-throughput screening combined with novel computational tools can reduce these factors. Thus, we developed an enzyme activity assay, which can be included in the drug discovery work-flow subsequent to the in-silico library screening. While the in-silico screening yields in the identification of potential allosteric modulators, the developed in-vitro assay allows for the characterisation of them. Candida rugosa lipase (CRL), a glyceride hydrolysing enzyme, has been selected for the pilot development. The assay conditions were adjusted to CRL's properties including pH, temperature and substrate specificity for two different substrates. The optimised assay conditions were validated and were used to characterise Tropolone, which was identified as an allosteric modulator. In conclusion, the assay is a reliable, reproducible, and robust tool, which can be streamlined with in-silico screening and incorporated in an automated high-throughput screening workflow.


Assuntos
Lipase/metabolismo , Miniaturização , Regulação Alostérica , Candida/enzimologia , Cristalografia por Raios X , Estabilidade Enzimática , Ensaios de Triagem em Larga Escala , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Limite de Detecção , Lipase/química , Reprodutibilidade dos Testes , Especificidade por Substrato , Temperatura
19.
Enzyme Microb Technol ; 130: 109367, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421729

RESUMO

Currently, the chemically-assisted esterification to manufacture butyl butyrate employs corrosive homogeneous acid catalyst and liberates enormous quantities of hazardous by-products which complicate downstream treatment processes. This study aimed to identify the optimized esterification conditions, and the kinetic aspects of the enzyme-assisted synthesis of butyl butyrate using immobilized Candida rugosa lipase activated by chitosan-reinforced nanocellulose derived from raw oil palm leaves (CRL/CS-NC). The best process variables that gave the maximum conversion degree of butyl butyrate by CRL/CS-NC (90.2%) in just 3 h, as compared to free CRL (62.9%) are as follows: 50 °C, 1:2 M ratio of acid/alcohol, stirring rate of 200 rpm and a 3 mg/mL enzyme load. The enzymatic esterification followed the ping pong bi-bi mechanism with substrate inhibition, revealing a ˜1.1-fold higher Ki for CRL/CS-NC (55.55 mM) over free CRL (50.68 mM). This indicated that CRL/CS-NC was less inhibited by the substrates. Butanol was preferred over butyric acid as reflected by the higher apparent Michaelis-Menten constant of CRL/CS-NC for butanol (137 mM) than butyric acid (142.7 mM). Thus, the kinetics data conclusively showed that CRL/CS-NC (Vmax 0.48 mM min-1, Keff 0.07 min-1 mM-1) was catalytically more efficient than free CRL (Vmax 0.35 mM min-1, Keff 0.06 min-1 mM-1).


Assuntos
Butiratos/metabolismo , Candida/enzimologia , Celulose/química , Quitosana/química , Lipase/metabolismo , Biocatálise , Estabilidade Enzimática , Enzimas Imobilizadas , Esterificação , Cinética , Óleo de Palmeira/química , Folhas de Planta/química
20.
Int J Mol Sci ; 20(16)2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31426510

RESUMO

In this communication, lipase A from Candida antarctica (CALA) was immobilized by covalent bonding on magnetic nanoparticles coated with chitosan and activated with glutaraldehyde, labelled CALA-MNP, (immobilization parameters: 84.1% ± 1.0 for immobilization yield and 208.0 ± 3.0 U/g ± 1.1 for derivative activity). CALA-MNP biocatalyst was characterized by X-ray Powder Diffraction (XRPD), Fourier Transform Infrared (FTIR) spectroscopy, Thermogravimetry (TG) and Scanning Electron Microscope (SEM), proving the incorporation of magnetite and the immobilization of CALA in the chitosan matrix. Besides, the immobilized biocatalyst showed a half-life 8-11 times higher than that of the soluble enzyme at pH 5-9. CALA showed the highest activity at pH 7, while CALA-MNP presented the highest activity at pH 10. The immobilized enzyme was more active than the free enzyme at all studied pH values, except pH 7.


Assuntos
Candida/enzimologia , Quitosana/química , Lipase/metabolismo , Nanopartículas de Magnetita/química , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Proteínas Fúngicas/metabolismo
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