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1.
J Sci Food Agric ; 100(4): 1479-1485, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31756272

RESUMO

BACKGROUND: Sweet corn cob (SCC), an agricultural by-product of the corn-processing industry, contains more than 80% insoluble bound ferulic acid (FA). Extraction of these bound phenolics can be achieved through chemical or enzymatic hydrolysis; however, the shift towards greener chemistry has raised awareness about the use of enzymatic hydrolysis. In the present study, the ability of ferulic acid esterase (FAE) and xylanase (XY) to catalyze the hydrolysis of FA from SCC was investigated. Response surface methodology (RSM), based on a five-level, four-factor central composite rotatable design (CCRD), was used to establish the optimum conditions for enzymatic hydrolysis of FA from SCC. Sweet corn cob was treated with a combination of FAE and XY at various concentrations (FAE: 0.00 to 0.04 U/g; XY: 0.00 to 18 093.5 U/g), temperatures (45 to 65 °C), and pH levels (pH 4.5 to 6.5). RESULTS: The optimum extraction conditions predicted by the model were: FAE concentration of 0.02 U/g, XY concentration of 3475.3 U/g, extraction pH of 4.5, and an extraction temperature of 45 °C. CONCLUSION: Under these conditions, the experimental yield of FA was 1.69 ± 0.02 g kg-1 of SCC, which is in agreement with the value predicted by the model. © 2019 Society of Chemical Industry.


Assuntos
Hidrolases de Éster Carboxílico/química , Ácidos Cumáricos/isolamento & purificação , Endo-1,4-beta-Xilanases/química , Química Verde/métodos , Extratos Vegetais/isolamento & purificação , Resíduos/análise , Zea mays/química , Biocatálise , Ácidos Cumáricos/química , Concentração de Íons de Hidrogênio , Hidrólise , Extratos Vegetais/química , Temperatura Ambiente
2.
Chem Biol Interact ; 316: 108914, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31837295

RESUMO

Heroin (diamorphine) is a highly addictive opioid drug synthesized from morphine. The use of heroin and incidence of heroin associated overdose death has increased sharply in the US. Heroin is primarily metabolized via deacetylation (hydrolysis) forming the active metabolites 6-monoacetylmorphine (6-MAM) and morphine. A diminution in heroin hydrolysis is likely to cause higher drug effects and toxicities. In this study, we sought to determine the contribution of the major hepatic hydrolase carboxylesterase 1 (CES1) to heroin metabolism in the liver as well as the potential influence of one of its known genetic variants, G143E (rs71647871). Furthermore, given the potential therapeutic application of cannabidiol (CBD) for heroin addiction and the frequent co-abuse of cannabis and heroin, we also assessed the effects of CBD on heroin metabolism. In vitro systems containing human liver, wild-type CES1, and G143E CES1 S9 fractions were utilized in the assessment. The contribution of CES1 to the hydrolysis of heroin to 6-MAM was determined as 3.66%, and CES1 was unable to further catalyze 6-MAM under our assay conditions. The G143E variant showed a 3.2-fold lower intrinsic clearance of heroin as compared to the WT. CBD inhibited heroin and 6-MAM hydrolysis in a reversible manner, with IC50s of 14.7 and 12.1 µM, respectively. Our study results suggested only minor involvement of CES1 in heroin hydrolysis in the liver. Therefore, the G143E variant is unlikely to cause significant impact despite a much lower hydrolytic activity. CBD exhibited potent in vitro inhibition toward both heroin and 6-MAM hydrolysis, which may be of potential clinical relevance.


Assuntos
Canabidiol/farmacologia , Hidrolases de Éster Carboxílico/metabolismo , Hepatócitos/efeitos dos fármacos , Heroína/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Cromatografia Líquida de Alta Pressão , Hepatócitos/citologia , Hepatócitos/metabolismo , Heroína/análise , Humanos , Hidrólise/efeitos dos fármacos , Cinética , Polimorfismo de Nucleotídeo Único , Espectrometria de Massas em Tandem , Ácido Valproico/farmacologia
3.
Enzyme Microb Technol ; 131: 109380, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615673

RESUMO

We previously described the fungus Penicillium chrysogenum 31B, which has high performance to produce the ferulic acid esterase (FAE) for de-esterifying ferulic acids (FAs) from sugar beet pulp. However, the characteristics of this fungus have not yet been determined. Therefore, in this study, we evaluated the molecular characteristics and natural substrate specificity of the Pcfae1 gene from Penicillium chrysogenum and examined its synergistic effects on sugar beet pectin. The Pcfae1 gene was cloned and overexpressed in Pichia pastoris KM71H, and the recombinant enzyme, named PcFAE1, was characterized. The 505 amino acids of PcFAE1 possessed a GCSTG motif (Gly164 to Gly168), characteristic of the serine esterase family. By comparing the amino acid sequence of PcFAE1 with that of the FAE (AoFaeB) of Aspergillus oryzae, Ser166, Asp379, and His419 were identified as the catalytic triad. PcFAE1 was purified through two steps using anion-exchange column chromatography. Its molecular mass without the signal peptide was 75 kDa. Maximum PcFAE1 activity was achieved at pH 6.0-7.0 and 50 °C. The enzyme was stable up to 37 °C and at a pH range of 3-8. PcFAE1 activity was only inhibited by Hg2+, and the enzyme had activity toward methyl FA, methyl caffeic acid, and methyl p-coumaric acid, with specific activities of 6.97, 4.65, and 9.32 U/mg, respectively, but not on methyl sinapinic acid. These results indicated that PcFAE1 acted similar to FaeB type according the Crepin classification. PcFAE1 de-esterified O-[6-O-feruloyl-ß-d-galactopyranosyl-(1→4)]-d-galactopyranose, O-[2-O-feruloyl-α-l-arabinofuranosyl-(1→5)]-l-arabinofuranose, and O-[5-O-feruloyl-α-l-arabinofuranosyl-(1→3)]-O-ß-d-xylopyranosyl-(1→4)-d-xylopyranose, indicating that the enzyme could de-esterify FAs decorated with both ß-d-galactopyranosidic and α-l-arabinofuranosidic residues in pectin and xylan. PcFAE1 acted in synergy with endo-α-1,5-arabinanase and α-l-arabinofuranosidase, which releases FA linked to arabinan, to digest the sugar beet pectin. Moreover, when PcFAE1 was allowed to act on sugar beet pectin together with Driselase, approximately 90% of total FA in the substrate was released. Therefore, PcFAE1 may be an interesting candidate for hydrolysis of lignocellulosic materials and could have applications as a tool for production of FA from natural substrates.


Assuntos
Arabinose/análogos & derivados , Hidrolases de Éster Carboxílico/metabolismo , Ácidos Cumáricos/metabolismo , Galactose/metabolismo , Pectinas/metabolismo , Penicillium chrysogenum/enzimologia , Arabinose/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/isolamento & purificação , Clonagem Molecular , Estabilidade Enzimática , Expressão Gênica , Concentração de Íons de Hidrogênio , Pichia/genética , Pichia/metabolismo , Especificidade por Substrato , Temperatura Ambiente
4.
Anal Chim Acta ; 1089: 108-114, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31627807

RESUMO

Droplet microfluidics has the ability to greatly increase the throughput of screening and sorting of enzymes by carrying reagents in picoliter droplets flowing in inert oils. It was found with the use of a specific surfactant, the interfacial tension of droplets can be very sensitive to droplet pH. This enables the sorting of droplets of different pH when confined droplets encounter a microfabricated trench. The device can be extended to sort enzymes, as a large number of enzymatic reactions lead to the production of an acidic or basic product and a concurrent change in solution pH. The progress of an enzymatic reaction is tracked from the position of a flowing train of droplets. We demonstrate the sorting of esterase isoenzymes based on their enzymatic activity. This label-free technology, that we dub droplet sorting by interfacial tension (SIFT), requires no active components and would have applications for enzyme sorting in high-throughput applications that include enzyme screening and directed evolution of enzymes.


Assuntos
Hidrolases de Éster Carboxílico/isolamento & purificação , Ensaios Enzimáticos/métodos , Acetatos/química , Animais , Hidrolases de Éster Carboxílico/química , Ensaios Enzimáticos/instrumentação , Fluorcarbonetos/química , Isoenzimas/química , Isoenzimas/isolamento & purificação , Dispositivos Lab-On-A-Chip , Fígado/enzimologia , Microfluídica/instrumentação , Microfluídica/métodos , Óleos/química , Fenóis/química , Reprodutibilidade dos Testes , Tensão Superficial , Suínos , Água/química
5.
J Oleo Sci ; 68(8): 781-792, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31366855

RESUMO

The possibility of improving brain function coupled with its preferential uptake in the brain has garnered attention for docosahexaenoic acid-bound lysophosphatidylcholine (DHA-LPC). However, studies focusing on the health benefits of dietary DHA-LPC are lacking. We prepared a dietary oil rich in DHA-LPC (DHA-LPC rich oil) via enzymatic modification of phospholipids (PL) extracted from squid (Todarodes pacificus) meal and purification of active carbon, ion exchange resin, and silica gel. We then examined the effects of dietary DHA-LPC rich oil on male Wistar rats by evaluating serum and liver lipid profiles, fatty acid (FA) metabolizing enzyme activity, and the FA composition of serum and brain. The rats were fed a basal diet containing either soybean oil alone (7%) or soybean oil (4.5%) with DHA-LPC rich oil (2.5%) for 28 days, and then evaluated. The rats fed the diet containing DHA-LPC rich oil showed reduced triacylglycerol concentration due, in part, to the enhancement of carnitine palmitoyltransferase 2 and acyl-CoA oxidase activities and suppression of acetyl-CoA carboxylase and glucose-6-phosphate dehydrogenase activities in the liver. Moreover, the dietary DHA-LPC rich oil moderately increased DHA in the FA composition of the rat hippocampus, which may be due to elevated DHA composition in serum LPC. These results suggest that DHA-LPC rich oil has hypolipidemic effect and moderate increase in hippocampal DHA amount in normal rats.


Assuntos
Encéfalo/metabolismo , Gorduras Insaturadas na Dieta/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Hipolipemiantes/farmacologia , Fígado/metabolismo , Lisofosfatidilcolinas/farmacologia , Administração Oral , Animais , Química Encefálica , Hidrolases de Éster Carboxílico/química , Decapodiformes/química , Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Docosa-Hexaenoicos/administração & dosagem , Hipocampo/química , Hipocampo/metabolismo , Hipolipemiantes/administração & dosagem , Fígado/química , Lisofosfatidilcolinas/administração & dosagem , Masculino , Fosfolipídeos/química , Fosfolipídeos/isolamento & purificação , Ratos Wistar , Rhizopus/enzimologia
6.
Molecules ; 24(15)2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31362390

RESUMO

The human carboxylesterase 1 (CES1), responsible for the biotransformation of many diverse therapeutic agents, may contribute to the occurrence of adverse drug reactions and therapeutic failure through drug interactions. The present study is designed to address the issue of potential drug interactions resulting from the inhibition of CES1. Based on an ensemble of 10 crystal structures complexed with different ligands and a set of 294 known CES1 ligands, we used docking (Autodock Vina) and machine learning methodologies (LDA, QDA and multilayer perceptron), considering the different energy terms from the scoring function to assess the best combination to enable the identification of CES1 inhibitors. The protocol was then applied on a library of 1114 FDA-approved drugs and eight drugs were selected for in vitro CES1 inhibition. An inhibition effect was observed for diltiazem (IC50 = 13.9 µM). Three others drugs (benztropine, iloprost and treprostinil), exhibited a weak CES1 inhibitory effects with IC50 values of 298.2 µM, 366.8 µM and 391.6 µM respectively. In conclusion, the binding site of CES1 is relatively flexible and can adapt its conformation to different types of ligands. Combining ensemble docking and machine learning approaches improves the prediction of CES1 inhibitors compared to a docking study using only one crystal structure.


Assuntos
Hidrolases de Éster Carboxílico/química , Aprendizado de Máquina , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Descoberta de Drogas , Ativação Enzimática/efeitos dos fármacos , Humanos , Inibidores de Proteases/farmacologia , Relação Quantitativa Estrutura-Atividade , Curva ROC , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas
7.
Mar Drugs ; 17(7)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261836

RESUMO

N-Acylhomoserine lactonase degrades the lactone ring of N-acylhomoserine lactones (AHLs) and has been widely suggested as a promising candidate for use in bacterial disease control. While a number of AHL lactonases have been characterized, none of them has been developed as a commercially available enzymatic product for in vitro AHL quenching due to their low stability. In this study, a highly stable AHL lactonase (AhlX) was identified and isolated from the marine bacterium Salinicola salaria MCCC1A01339. AhlX is encoded by a 768-bp gene and has a predicted molecular mass of 29 kDa. The enzyme retained approximately 97% activity after incubating at 25 °C for 12 days and ~100% activity after incubating at 60 °C for 2 h. Furthermore, AhlX exhibited a high salt tolerance, retaining approximately 60% of its activity observed in the presence of 25% NaCl. In addition, an AhlX powder made by an industrial spray-drying process attenuated Erwinia carotovora infection. These results suggest that AhlX has great potential for use as an in vitro preventive and therapeutic agent for bacterial diseases.


Assuntos
Antibacterianos/farmacologia , Organismos Aquáticos/enzimologia , Proteínas de Bactérias/farmacologia , Hidrolases de Éster Carboxílico/farmacologia , Halomonadaceae/enzimologia , Acil-Butirolactonas/química , Antibacterianos/química , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Biotecnologia , Brassica rapa/microbiologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/isolamento & purificação , Ensaios Enzimáticos , Estabilidade Enzimática , Pectobacterium carotovorum/efeitos dos fármacos , Pectobacterium carotovorum/fisiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Percepção de Quorum/efeitos dos fármacos , Solanum tuberosum/microbiologia , Temperatura Ambiente
8.
Food Chem ; 300: 125194, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325749

RESUMO

The effects of near freezing temperature (NFT) storage at -1.9 °C on cell wall degradation of 'Shushanggan' apricot was studied comparing to 0 °C and 5 °C storage. Our results indicated that NFT storage strongly inhibited the solubilization of Na2CO3-soluble pectin and cellulose, by the suppression of cell wall modifying enzymes (polygalacturonase, ß-Galactosidase, pectin methyl esterase and cellulase) and related genes expressions. The loss of side chains was the main modification in CDTA (Cyclohexane-diamine-tetraacetic Acid)-soluble pectin during storage and made the main contribution to the softening of apricot, while the loss of side chain was suppressed by NFT storage. Microscopic observation showed that NFT storage delayed the degradation of pectin fraction and protected cell wall structure from loosing. This study proves that NFT storage is an effective technology to suppress the cell wall polysaccharides degradation and ultrastructure modification of apricot.


Assuntos
Parede Celular/ultraestrutura , Armazenamento de Alimentos/métodos , Polissacarídeos/química , Prunus armeniaca/química , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Celulose/química , Temperatura Baixa , Congelamento , Frutas/química , Frutas/citologia , Frutas/ultraestrutura , Pectinas/química , Células Vegetais/química , Células Vegetais/ultraestrutura , Poligalacturonase/química , Poligalacturonase/metabolismo , Polissacarídeos/metabolismo , Prunus armeniaca/citologia , Solubilidade , beta-Galactosidase/química , beta-Galactosidase/metabolismo
9.
J Environ Sci Health B ; 54(11): 883-891, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31311415

RESUMO

The characterization of soluble cholinesterases (ChEs) together with carboxylesterases (CEs) in Ficopomatus enigmaticus as suitable biomarkers of neurotoxicity was the main aim of this study. ChEs of F. enigmaticus were characterized considering enzymatic activity, substrate affinity (acetyl-, butyryl-, propionylthiocholine), kinetic parameters (Km and Vmax) and in vitro response to model inhibitors (eserine hemisulfate, iso-OMPA, BW284C51), and carbamates (carbofuran, methomyl, aldicarb, and carbaryl). CEs were characterized based on enzymatic activity, kinetic parameters and in vitro response to carbamates (carbofuran, methomyl, aldicarb, and carbaryl). Results showed that cholinesterases from F. enigmaticus showed a substrate preference for acetylthiocholine followed by propionylthiocholine; butyrylthioline was not hydrolyzed differently from other Annelida species. CE activity was in the same range of cholinesterase activity with acetylthiocholine as substrate; the enzyme activity showed high affinity for the substrate p-nytrophenyl butyrate. Carbamates inhibited ChE activity with propionylthiocholine as substrate to a higher extent than with acetylthiocoline. Also CE activity was inhibited by all tested carbamates except carbaryl. In vitro data highlighted the presence of active forms of ChEs and CEs in F. enigmaticus that could potentially be inhibited by pesticides at environmentally relevant concentration.


Assuntos
Anelídeos/enzimologia , Inibidores da Colinesterase/toxicidade , Colinesterases/química , Neurotoxinas/toxicidade , Animais , Anelídeos/efeitos dos fármacos , Biomarcadores/química , Carbamatos/química , Carbaril/química , Carbaril/toxicidade , Carbofurano/química , Carbofurano/toxicidade , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Inibidores da Colinesterase/química , Colinesterases/metabolismo , Cinética , Metomil/química , Metomil/toxicidade , Neurotoxinas/química
10.
Carbohydr Polym ; 218: 126-135, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31221313

RESUMO

In this study, maltoheptaose (G7)-based sugar esters were synthesized from maltoheptaose and fatty acids (C10-C16) using a commercial lipase. With the exception of dimethyl sulfoxide (DMSO; 76.4%, w/v), G7 showed only limited solubility in organic solvents. Among the fatty acids, palmitic acid (PA) was the best substrate for G7-based ester formation. G7-PA ester was successfully synthesized as the monoester structure exclusively in 10% DMSO of t-butanol with a 22% conversion yield. NMR and enzymatic analyses of the purified monoester product revealed that the ester bond in the G7 was located at C-6 of the glucose at the reducing end. The G7-PA monoester showed the melting temperature at 56.3 °C that was 6.5 °C lower than that of the free PA and exhibited a different endothermic pattern from the free G7. The G7-PA monoester exhibited excellent emulsifier potential with more even droplet size distribution compared with the commercial sucrose esters for an oil-in-water emulsion system.


Assuntos
Hidrolases de Éster Carboxílico/química , Ésteres/química , Proteínas Fúngicas/química , Glucanos/química , Candida/enzimologia , Emulsificantes/síntese química , Emulsificantes/química , Emulsões , Esterificação , Ésteres/síntese química , Ácidos Graxos/química , Glucanos/síntese química , Solubilidade , Temperatura de Transição
11.
Carbohydr Polym ; 218: 324-332, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31221337

RESUMO

Dialdehyde starch (DAS) is a kind of modified starch which contains many active aldehyde groups and has good biocompatibility. In this study, magnetic dialdehyde starch nanoparticles were successfully used to immobilize lipase. The lipase was immobilized onto magnetic nanoparticles by using DAS instead of glutaraldehyde as a crosslinker. The parameters like DAS dosage, enzyme concentration and immobilization time were optimized. Enzymatic properties studies exhibited that after DAS cross-linking, the storage stability of the immobilized enzyme reached 82.5%, and the recycling rate reached 53.6%, whereas in case of glutaraldehyde cross linker, it was 79.4% and 46.8%, the former also exhibited better stability and durability. Compared with the free enzyme, the immobilized enzyme indicated higher acid-base tolerance and thermal stability, and had good enzymatic properties. Magnetic dialdehyde starch nanoparticles may have application prospects as an excellent enzyme carrier, which provides a reference for the preparation of other immobilized enzymes with excellent performance.


Assuntos
Hidrolases de Éster Carboxílico/química , Reagentes para Ligações Cruzadas/química , Enzimas Imobilizadas/química , Nanopartículas de Magnetita/química , Amido/análogos & derivados , Ensaios Enzimáticos , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Oxirredução , Ácido Periódico/química , Rhizopus/enzimologia , Amido/química , Temperatura Ambiente
12.
Appl Biochem Biotechnol ; 189(4): 1304-1317, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31254227

RESUMO

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


Assuntos
Hidrolases de Éster Carboxílico , Clonagem Molecular , Proteínas Fúngicas , Sordariales , Biomassa , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/isolamento & purificação , Estabilidade Enzimática , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sordariales/enzimologia , Sordariales/genética
13.
Mol Biol Rep ; 46(4): 4385-4395, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31201678

RESUMO

The ferulic acid esterase (FAE) gene from Geobacillus thermoglucosidasius DSM 2542T was cloned into pET28a(+) expression vector and characterized and is being reported in this study for the first time in Geobacillus. The enzyme, designated as GthFAE, was purified by heat shock and ion-exchange column chromatography. In addition, a second clone containing a Histidine tag was expressed and purified by affinity column chromatography demonstrating future potential for scale-up. FAE gene contains an open reading frame (ORF) of 759-bp encoding a hypothetical 252 amino acid protein, a molecular mass of 28.11 kDa and an isoelectric point of 5.53. From this study it was found that GthFAE had optimal activity at 50 °C and pH of 8.5. Furthermore, the enzyme has been found to retain 64% of its activity after two days incubation at 50 °C and exhibited a high level of functionality with p-nitrophenyl caprylate (C8). Km, Vmax, kcat and kcat/Km values for p-nitrophenyl caprylate were determined as 0.035 mM, 11,735 µmol/min/mg protein, 5491 (1/s) and 156,885 s-1 mM-1 respectively. The combination of higher activity and stability compared to previously reported FAEs makes GthFAE a potential candidate for use in the paper manufacturing industry.


Assuntos
Bacillaceae/enzimologia , Bacillaceae/genética , Hidrolases de Éster Carboxílico/química , Sequência de Aminoácidos , Clonagem Molecular , Estabilidade Enzimática , Geobacillus/genética , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Especificidade por Substrato
14.
Environ Sci Pollut Res Int ; 26(24): 24946-24957, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31243662

RESUMO

Paraoxonase 1 (PON1) is an A-esterase calcium-dependent enzyme that is associated with high-density lipoprotein (HDL) and capable of hydrolyzing a wide variety of substrates, including organophosphate (OP) pesticides. The PON1 phenotype can be modulated by multiple internal and external factors, thereby affecting the catalytic capacity of the enzyme. The aim of this study was to evaluate factors that could modulate PON1 activity in a sample occupationally exposed to pesticides. A cross-sectional, descriptive, and analytical study was carried out with 240 workers. The participants were stratified according to their level of pesticide exposure as reference, moderate-exposure, and high-exposure groups. PON1 activities (arylesterase/AREase, CMPAase, and ssPONase (salt-stimulated)) were determined by spectrophotometry, and the Q192R and L55MPON1 genotypes by real-time PCR. The most frequent genotypes were heterozygous (QR) and homozygous (LL) for PON1Q192R and PON1L55M polymorphisms, respectively. The internal factors associated with the activity of PON1 were the PON1 genotypes (55 and 192) and biochemical parameters related to the lipid profile, in contrast, various external factors related to diet and harmful habits as well as with exposure to pesticides were associated with the activity of PON1. However, using a multivariate mixed ordinal regression model, we found a significant reduction of ssPONase activity in the high-exposure group compared with the reference group only in haplotypes QQLL and RRLL.


Assuntos
Arildialquilfosfatase/genética , Hidrolases de Éster Carboxílico/química , Compostos Organofosforados/química , Praguicidas/química , Arildialquilfosfatase/química , Arildialquilfosfatase/metabolismo , Estudos Transversais , Genótipo , Humanos , Exposição Ocupacional , Fenótipo , Polimorfismo Genético
15.
Biotechnol Lett ; 41(8-9): 995-1006, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31102076

RESUMO

OBJECTIVES: To discover novel feruloyl esterases (FAEs) by the function-driven screening procedure from soil metagenome. RESULTS: A novel FAE gene bds4 was isolated from a soil metagenomic library and over-expressed in Escherichia coli. The recombinant enzyme BDS4 was purified to homogeneity with a predicted molecular weight of 38.8 kDa. BDS4 exhibited strong activity (57.05 U/mg) toward methyl ferulate under the optimum pH and temperature of 8.0 and 37°C. Based on its amino acid sequence and model substrates specificity, BDS4 was classified as a type-C FAE. The quantity of the releasing ferulic acid can be enhanced significantly in the presence of xylanase compared with BDS4 alone from de-starched wheat bran. In addition, BDS4 can also hydrolyze several phthalates such as diethyl phthalate, dimethyl phthalate and dibutyl phthalate. CONCLUSION: The current investigation discovered a novel FAE with phthalate-degrading activity and highlighted the usefulness of metagenomic approaches as a powerful tool for discovery of novel FAEs.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , DNA/isolamento & purificação , Metagenômica , Ácidos Ftálicos/metabolismo , Microbiologia do Solo , Biotransformação , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/isolamento & purificação , Clonagem Molecular , DNA/genética , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Temperatura Ambiente
16.
Mar Drugs ; 17(5)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117226

RESUMO

MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various N-acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomLI144V and MomLV149A. These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of Pectobacterium carotovorum subsp. carotovorum (Pcc). Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on Pcc-infected Chinese cabbage. The results indicated that MomL and its mutants (MomLL254R, MomLI144V, MomLV149A) significantly decreased the pathogenicity of Pcc. This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase.


Assuntos
Aminoácidos/análise , Hidrolases de Éster Carboxílico , Pectobacterium carotovorum/enzimologia , Pectobacterium carotovorum/genética , Engenharia de Proteínas , Substituição de Aminoácidos , Brassica rapa/microbiologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Ativação Enzimática/genética , Mutação , Pectobacterium carotovorum/patogenicidade , Virulência/genética
17.
Appl Microbiol Biotechnol ; 103(14): 5533-5547, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31144014

RESUMO

Potato proteins are well known for their nutritional, emulsifying, foaming, gel forming or antioxidant properties that all make from them valuable protein source for food industry. Antifungal, antimicrobial and also antiviral properties, described for potato proteins in the review, enrich the possibilities of potato protein usage. Potato proteins were divided into patatin, protease inhibitors and fraction of other proteins that also included, besides others, proteins involved in potato defence physiology. All these proteins groups provide proteins and peptides with antifungal and/or antimicrobial actions. Patatins, obtained from cultivars with resistance to Phytophthora infestans, were able to inhibit spore germination of this pathogen. Protease inhibitors represent the structurally heterogeneous group with broad range of antifungal and antimicrobial activities. Potato protease inhibitors I and II reduced the growth of Phytophthora infestans, Rhizoctonia solani and Botrytis cinerea or of the fungi of Fusarium genus. Members of Kunitz family (proteins Potide-G, AFP-J, Potamin-1 or PG-2) were able to inhibit serious pathogens such as Staphylococcus aureus, Listeria monocytogenes, Escherichia coli or Candida albicans. Potato snakins, defensins and pseudothionins are discussed for their ability to inhibit serious potato fungi as well as bacterial pathogens. Potato proteins with the ability to inhibit growth of pathogens were used for developing of pathogen-resistant transgenic plants for crop improvement. Incorporation of potato antifungal and antimicrobial proteins in feed and food products or food packages for elimination of hygienically risk pathogens brings new possibility of potato protein usage.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Fungos/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Proteínas de Plantas/farmacologia , Solanum tuberosum/química , Antibacterianos/química , Antifúngicos/química , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/farmacologia , Fungicidas Industriais/química , Listeria monocytogenes/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Phytophthora/efeitos dos fármacos , Proteínas de Plantas/química , Staphylococcus aureus/efeitos dos fármacos
18.
PLoS One ; 14(5): e0217059, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31095643

RESUMO

Microbial colonization can be detrimental to the integrity of metal surfaces and lead to microbiologically influenced corrosion (MIC). Biocorrosion is a serious problem for aquatic and marine industries in the world. In Minnesota (USA), where this study was conducted, biocorrosion severely affects the maritime transportation industry. The anticorrosion activity of a variety of compounds, including chemical (magnesium peroxide) and biological (surfactin, capsaicin, and gramicidin) molecules were investigated as coating additives. We also evaluated a previously engineered, extremely stable, non-biocidal enzyme known to interfere in bacterial signaling, SsoPox (a quorum quenching lactonase). Experimental steel coupons were submerged in water from the Duluth Superior Harbor (DSH) for 8 weeks in the laboratory. Biocorrosion was evaluated by counting the number and the coverage of corrosion tubercles on coupons and also by ESEM imaging of the coupon surface. Three experimental coating additives significantly reduced the formation of corrosion tubercles: surfactin, magnesium peroxide and the quorum quenching lactonase by 31%, 36% and 50%, respectively. DNA sequence analysis of the V4 region of the bacterial 16S rRNA gene revealed that these decreases in corrosion were associated with significant changes in the composition of bacterial communities on the steel surfaces. These results demonstrate the potential of highly stable quorum quenching lactonases to provide a reliable, cost-effective method to treat steel structures and prevent biocorrosion.


Assuntos
Bactérias/efeitos dos fármacos , Percepção de Quorum , Aço/química , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Capsaicina/química , Hidrolases de Éster Carboxílico/química , Corrosão , Gramicidina/química , Lipopeptídeos/química , Compostos de Magnésio/química , Minnesota , Peptídeos Cíclicos/química , Peróxidos/química , RNA Ribossômico 16S/metabolismo , Propriedades de Superfície , Microbiologia da Água
19.
Int J Mol Sci ; 20(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018531

RESUMO

The evolution of release factors catalyzing the hydrolysis of the final peptidyl-tRNA bond and the release of the polypeptide from the ribosome has been a longstanding paradox. While the components of the translation apparatus are generally well-conserved across extant life, structurally unrelated release factor peptidyl hydrolases (RF-PHs) emerged in the stems of the bacterial and archaeo-eukaryotic lineages. We analyze the diversification of RF-PH domains within the broader evolutionary framework of the translation apparatus. Thus, we reconstruct the possible state of translation termination in the Last Universal Common Ancestor with possible tRNA-like terminators. Further, evolutionary trajectories of the several auxiliary release factors in ribosome quality control (RQC) and rescue pathways point to multiple independent solutions to this problem and frequent transfers between superkingdoms including the recently characterized ArfT, which is more widely distributed across life than previously appreciated. The eukaryotic RQC system was pieced together from components with disparate provenance, which include the long-sought-after Vms1/ANKZF1 RF-PH of bacterial origin. We also uncover an under-appreciated evolutionary driver of innovation in rescue pathways: effectors deployed in biological conflicts that target the ribosome. At least three rescue pathways (centered on the prfH/RFH, baeRF-1, and C12orf65 RF-PH domains), were likely innovated in response to such conflicts.


Assuntos
Hidrolases de Éster Carboxílico/genética , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/genética , Ribossomos/genética , Sequência de Aminoácidos , Animais , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Evolução Molecular , Humanos , Modelos Moleculares , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Filogenia , Biossíntese de Proteínas , Domínios Proteicos , Ribossomos/metabolismo
20.
Molecules ; 24(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018540

RESUMO

Jelly fig (Ficus awkeotsang Makino) is used to prepare drinks and desserts in Asia, owing to the gelling capability of its pectin via endogenous pectin methylesterase (PE) catalyzation. Meanwhile, substances with PE inhibitory activity (SPEI) in jelly fig achenes (JFA) residue were noticed to be able to impede the gelation. In this study, we characterized and isolated SPEI from JFA by a series of PE inhibition-guided isolations. Crude aqueous extract of JFA residue was mixed with acetone, and 90% acetone-soluble matter was further fractionated by Diaion HP-20 chromatography. The retained fraction with dominant PE inhibitory activity was collected from 100% methanol eluate. Results from high-performance liquid chromatography mass spectrometry (HPLC/MS) and hydrolysis-induced chromogenic transition revealed the SPEI as complex tannins. Total tannins content was determined in each isolated fraction, and was closely related to PE inhibitory activity. In addition, SPEI in this study could inhibit activities of digestive enzymes in vitro and may, therefore, be assumed to act as non-specific protein binding agent.


Assuntos
Hidrolases de Éster Carboxílico/antagonistas & inibidores , Inibidores Enzimáticos/isolamento & purificação , Ficus/química , Frutas/química , Proteínas de Plantas/antagonistas & inibidores , Taninos/isolamento & purificação , Acetona/química , Bebidas/análise , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/isolamento & purificação , Cromatografia por Troca Iônica , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Ficus/enzimologia , Frutas/enzimologia , Géis , Humanos , Metanol/química , Pectinas/química , Transição de Fase , Extratos Vegetais/química , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Solventes/química , Taiwan , Taninos/química , Água/química
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