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1.
J Med Microbiol ; 68(11): 1629-1640, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31553301

RESUMO

Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification.Aim. In the present study attempts were made to characterize ML1899 in detail.Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.Results. In silico analysis predicted ML1899 as a member of α/ß hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM-1 min-1 catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min-1 ml-1 and 27 U mg- 1, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.


Assuntos
Proteínas de Bactérias/imunologia , Esterases/imunologia , Hanseníase/microbiologia , Mycobacterium leprae/enzimologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Citocinas/genética , Citocinas/imunologia , Estabilidade Enzimática , Esterases/química , Esterases/genética , Feminino , Humanos , Concentração de Íons de Hidrogênio , Cinética , Hanseníase/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Mycobacterium leprae/química , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Óxido Nítrico/imunologia , Espécies Reativas de Oxigênio/imunologia , Alinhamento de Sequência
2.
J Agric Food Chem ; 67(31): 8548-8558, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31266305

RESUMO

Herein, we report a double enzyme system to degrade 12 phthalate esters (PAEs), particularly bulky PAEs, such as the widely used bis(2-ethylhexyl) phthalate (DEHP), in a one-pot cascade process. A PAE-degrading bacterium, Gordonia sp. strain 5F, was isolated from soil polluted with plastic waste. From this strain, a novel esterase (GoEst15) and a mono(2-ethylhexyl) phthalate hydrolase (GoEstM1) were identified by homology-based cloning. GoEst15 showed broad substrate specificity, hydrolyzing DEHP and 10 other PAEs to monoalkyl phthalates, which were further degraded by GoEstM1 to phthalic acid. GoEst15 and GoEstM1 were heterologously coexpressed in Escherichia coli BL21 (DE3), which could then completely degrade 12 PAEs (5 mM), within 1 and 24 h for small and bulky substrates, respectively. To our knowledge, GoEst15 is the first DEHP hydrolase with a known protein sequence, which will enable protein engineering to enhance its catalytic performance in the future.


Assuntos
Proteínas de Bactérias/química , Esterases/química , Ésteres/química , Gordonia (Bactéria)/enzimologia , Ácidos Ftálicos/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Biodegradação Ambiental , Dietilexilftalato/química , Dietilexilftalato/metabolismo , Esterases/genética , Esterases/metabolismo , Ésteres/metabolismo , Gordonia (Bactéria)/genética , Gordonia (Bactéria)/isolamento & purificação , Gordonia (Bactéria)/metabolismo , Hidrólise , Ácidos Ftálicos/metabolismo , Alinhamento de Sequência , Microbiologia do Solo
3.
J Sci Food Agric ; 99(14): 6644-6648, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31325326

RESUMO

BACKGROUND: Apple juice is rich in polyphenolic compounds, especially in chlorogenic acid. A sour and bitter taste has been attributed to the compound. Chlorogenic acid in coffee powder was quickly hydrolysed by a p-coumaryl esterase of Rhizoctonia solani (RspCAE) at its optimal pH of 6.0. It was unknown, however, if RspCAE would also degrade chlorogenic acid under the strongly acidic conditions (pH 3.3) present in apple juice. RESULTS: Treatment of apple juice with RspCAE led to a chlorogenic acid degradation from 53.38 ± 0.94 mg L-1 to 21.02 ± 1.47 mg L-1 . Simultaneously, the caffeic acid content increased from 6.72 ± 0.69 mg L-1 to 19.33 ± 1.86 mg/L-1 . The aroma profile of the enzymatically treated sample and a control sample differed in only one volatile. Vitispirane had a higher flavour dilution factor in the treated juice. Sensory analysis showed no significant difference in the taste profile ( p < 0.05). CONCLUSION: These results demonstrated a high stability and substrate specificity of RspCAE. An increase in caffeic acid and a concurrent decrease in chlorogenic acid concentration may exert a beneficial effect on human health. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.


Assuntos
Ácido Clorogênico/química , Esterases/química , Sucos de Frutas e Vegetais/análise , Proteínas Fúngicas/química , Malus/química , Rhizoctonia/enzimologia , Aromatizantes/química , Concentração de Íons de Hidrogênio , Hidrólise , Odorantes/análise , Especificidade por Substrato
4.
Enzyme Microb Technol ; 129: 109353, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31307573

RESUMO

A three catalytic domain multi-enzyme; a CE1 ferulic acid esterase, a GH62 α-l-arabinofuranosidase and a GH10 ß-d-1,4-xylanase was identified in a metagenome obtained from wastewater treatment sludge. The capability of the CE1-GH62-GH10 multi-enzyme to degrade arabinoxylan was investigated to examine the hypothesis that CE1-GH62-GH10 would degrade arabinoxylan more efficiently than the corresponding equimolar mix of the individual enzymes. CE1-GH62-GH10 efficiently catalyzed the production of xylopyranose, xylobiose, xylotriose, arabinofuranose and ferulic acid (FA) when incubated with insoluble wheat arabinoxylan (WAX-I) (kcat = 20.8 ± 2.6 s-1). Surprisingly, in an equimolar mix of the individual enzymes a similar kcat towards WAX-I was observed (kcat = 17.3 ± 3.8 s-1). Similarly, when assayed on complex plant biomass the activity was comparable between CE1-GH62-GH10 and an equimolar mix of the individual enzymes. This suggests that from a hydrolytic point of view a CE1-GH62-GH10 multi-enzyme is not an advantage. Determination of the melting temperatures for CE1-GH62-GH10 (71.0 ± 0.05 °C) and CE1 (69.9 ± 0.02), GH62 (65.7 ± 0.06) and GH10 (71 ± 0.05 °C) indicates that CE1 and GH62 are less stable as single domain enzymes. This conclusion was corroborated by the findings that CE1 lost ˜50% activity within 2 h, while GH62 retained ˜50% activity after 24 h, whereas CE1-GH62-GH10 and GH10 retained ˜50% activity for 72 h. GH62-GH10, when appended to each other, displayed a higher specificity constant (kcat/Km = 0.3 s-1 mg-1 ml) than the individual GH10 (kcat/Km = 0.12 s-1 ± 0.02 mg-1 ml) indicating a synergistic action between the two. Surprisingly, CE1-GH62, displayed a 2-fold lower kcat towards WAX-I than GH62, which might be due to the presence of a putative carbohydrate binding module appended to CE1 at the N-terminal. Both CE1 and CE1-GH62 released insignificant amounts of FA from WAX-I, but FA was released from WAX-I when both CE1 and GH10 were present, which might be due to GH10 releasing soluble oligosaccharides that CE1 can utilize as substrate. CE1 also displayed activity towards solubilized 5-O-trans-feruloyl-α-l-Araf (kcat = 36.35 s-1). This suggests that CE1 preferably acts on soluble oligosaccharides.


Assuntos
Esterases/química , Glicosídeo Hidrolases/química , Xilanos/química , Domínio Catalítico , Endo-1,4-beta-Xilanases/química , Hidrólise , Cinética , Esgotos/análise , Especificidade por Substrato
5.
Chemphyschem ; 20(16): 2082-2092, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31233266

RESUMO

The studied enzyme-based biocatalytic system mimics NXOR Boolean logic gate, which is a logical operator that corresponds to equality in Boolean algebra. It gives the functional value true (1) if both functional arguments (input signals) have the same logical value (0,0 or 1,1), and false (0) if they are different (0,1 or 1,0). The output signal producing reaction is catalyzed by pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH), which is inhibited at acidic and basic pH values. Two other reactions catalyzed by esterase and urease produce acetic acid and ammonium hydroxide, respectively, shifting solution pH from the optimum pH for PQQ-GDH to acidic and basic values (1,0 and 0,1 input combinations, respectively), thus switching the enzyme activity off (output 0). When the input signals are not applied (0,0 combination) or both applied compensating each other (1,1 combination) the optimum pH is preserved, thus keeping PQQ-GDH running at the high rate (output 1). The biocatalytic cascade mimicking the NXOR gate was characterized optically and electrochemically. In the electrochemical experiments the PQQ-GDH enzyme communicated electronically with a conducting electrode support, thus resulting in the electrocatalytic current when signal combinations 0,0 and 1,1 were applied. The logic gate operation, when it was realized electrochemically, was also extended to the biomolecular release controlled by the gate. The release system included two electrodes, one performing the NXOR gate and another one activated for the release upon electrochemically stimulated alginate hydrogel dissolution. The studied system represents a general approach to the biocatalytic realization of the NXOR logic gate, which can be included in different catalytic cascades mimicking operation of concatenated gates in sophisticated logic circuitries.


Assuntos
Computadores Moleculares , Esterases/química , Glucose Desidrogenase/química , Lógica , Urease/química , Acetatos/química , Alginatos/química , Animais , Canavalia/enzimologia , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Ferro/química , Nanotubos de Carbono/química , Suínos , Ureia/química
6.
Chem Commun (Camb) ; 55(47): 6747-6750, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31119249

RESUMO

o-Phenylazonaphthol (o-PAN) derivatives including 6-bromo-1-((4-bromophenyl)diazenyl)naphthalen-2-ol (AN-Br-OH) and 1-phenylazo-2-naphthol (AN-OH, known as Sudan I (Color Index 12055)) were synthesized to investigate their fluorogenic behaviors, in which their aggregated-induced emission (AIE) is reported. The o-PANs showed a two-photon absorption. The protection of hydroxyl groups in o-PANs was used for fluorescence imaging of esterase-expressed HepG2 cells, which is potentially suitable for sensing and two-photon cell imaging applications.


Assuntos
Esterases/metabolismo , Corantes Fluorescentes/química , Naftóis/química , Esterases/química , Corantes Fluorescentes/síntese química , Células Hep G2 , Humanos , Limite de Detecção , Microscopia de Fluorescência por Excitação Multifotônica , Naftóis/síntese química , Espectrometria de Fluorescência , Raios Ultravioleta
7.
Extremophiles ; 23(4): 407-419, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31053933

RESUMO

In the framework of the discovery of new thermophilic enzymes of potential biotechnological interest, we embarked in the characterization of a new thermophilic esterase from the thermophilic bacterium Geobacillus thermodenitrificans. The phylogenetic analysis of the GTNG_0744 esterase indicated that the sequence belongs to the enterochelin/enterobactin esterase group, which have never been recognized as a family in the lipases/esterase classification. These enzymes catalyze the last step in the acquisition of environmental Fe3+ through siderophore hydrolysis. In silico analysis revealed, for the first time, that the machinery for the uptake of siderophores is present in G. thermodenitrificans. The purified recombinant enzyme, EstGtA3, showed different substrate specificity from known enterochelin/enterobactin esterases, recognizing short chain esters with a higher specificity constant for 4-NP caprylate. The enzyme does not require cofactors for its activity, is active in the pH range 7.0-8.5, has highest activity at 60 °C and is 100% stable when incubated for 16 h at 55 °C. DTT, ß-mercaptoethanol and Triton X-100 have an activating effect on the enzymatic activity. Organic solvents have in general a negative effect on the enzyme, but n-hexane is a strong activator up to 150, making EstGtA3 a good candidate for applications in biotechnology.


Assuntos
Proteínas de Bactérias/metabolismo , Esterases/metabolismo , Geobacillus/enzimologia , Termotolerância , Proteínas de Bactérias/química , Caprilatos/metabolismo , Estabilidade Enzimática , Esterases/química , Desnaturação Proteica , Especificidade por Substrato
8.
Eur J Pharm Sci ; 133: 145-159, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30946965

RESUMO

Non-small cell lung cancer is a major sub-type of lung cancer that is associated with a poor diagnosis resulting in poor therapy for the disorder. In order to achieve a better prognosis, innovative multi-functional systems need to be developed which will aide in diagnosis as well as therapy for the disorder. One such multi-functional delivery system fabricated is Quantum Dots (QDs). QDs are photo-luminescent inorganic nanoparticles utilized for tumor detection, preclinically. Erlotinib hydrochloride, a tyrosine kinase inhibitor, is a first-generation drug developed to treat NSCLC. Its active metabolite, Desmethyl Erlotinib (OSI-420), exhibits similar anticancer activity as erlotinib. OSI-420 was conjugated to QDs to fabricate a delivery system and was then characterized by FT-IR, H NMR, UV-VIS, particle size, zeta potential, fluorescence spectroscopy and TEM. Drug loading was estimated using UV-VIS spectroscopy (52.2 ±â€¯7.5%). A concentration-dependent release of OSI-420 was achieved using esterase enzymes, which was further confirmed using LC-MS. A cellular uptake study revealed the internalization potential of QDs and QD-OSI 420. A cellular recovery study was performed to confirm the internalization potential. Cell viability studies revealed that QD-OSI 420 conjugates had significantly better efficacy than pure drugs in all tested cell lines. QD conjugated OSI-420 demonstrated an IC60 of 2.5 µM in erlotinib-resistant A549 cell lines, where erlotinib or OSI-420 alone could not exhibit 60% inhibition when evaluated up to 20 µM. Similar cytotoxic enhancement of erlotinib was seen with QD-OSI 420 in other NSCLC cell lines as well. These results were strengthened by 3D-SCC model of A549 which revealed that QD-OSI 420 was significantly better in reducing in-vitro 3D tumor volume, as compared to pure drugs. This study, being one of its kind, explores the feasibility of conjugating OSI-420 with QDs as an alternative to traditional anti-cancer therapy, by improving intracellular drug delivery.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Pontos Quânticos/administração & dosagem , Quinazolinas/administração & dosagem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Esterases/química , Humanos , Lisossomos/metabolismo , Inibidores de Proteínas Quinases/química , Pontos Quânticos/química , Quinazolinas/química
9.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 4): 270-277, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30950828

RESUMO

The human membrane-bound α/ß-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane-bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2-AG and esters of medium-length fatty acids. PA2949 in the presence of n-octyl ß-D-glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified from Escherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space group I4122 and diffracted to a resolution of 2.54 Å. This study provides hints on the functional similarity of ABHD6-like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult-to-crystallize proteins.


Assuntos
Esterases/química , Esterases/isolamento & purificação , Monoacilglicerol Lipases/química , Pseudomonas aeruginosa/enzimologia , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Cristalização , Estabilidade Enzimática , Humanos , Cinética , Especificidade por Substrato , Temperatura Ambiente
10.
J Biol Chem ; 294(16): 6635-6644, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30814248

RESUMO

Glucuronoyl esterases (GEs) catalyze the cleavage of ester linkages found between lignin and glucuronic acid moieties on glucuronoxylan in plant biomass. As such, GEs represent promising biochemical tools in industrial processing of these recalcitrant resources. However, details on how GEs interact with their natural substrates are sparse, calling for thorough structure-function studies. Presented here is the structure and biochemical characterization of a GE, TtCE15A, from the bacterium Teredinibacter turnerae, a symbiont of wood-boring shipworms. To gain deeper insight into enzyme-substrate interactions, inhibition studies were performed with both the WT TtCE15A and variants in which we, by using site-directed mutagenesis, substituted residues suggested to have key roles in binding to or interacting with the aromatic and carbohydrate structures of its uronic acid ester substrates. Our results support the hypothesis that two aromatic residues (Phe-174 and Trp-376), conserved in bacterial GEs, interact with aromatic and carbohydrate structures of these substrates in the enzyme active site, respectively. The solved crystal structure of TtCE15A revealed features previously not observed in either fungal or bacterial GEs, with a large inserted N-terminal region neighboring the active site and a differently positioned residue of the catalytic triad. The findings highlight key interactions between GEs and complex lignin-carbohydrate ester substrates and advance our understanding of the substrate specificities of these enzymes in biomass conversion.


Assuntos
Proteínas de Bactérias/química , Carboidratos/química , Esterases/química , Gammaproteobacteria/enzimologia , Hidrocarbonetos Aromáticos/química , Ácidos Urônicos/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Esterases/genética , Gammaproteobacteria/genética , Mutagênese Sítio-Dirigida , Domínios Proteicos , Relação Estrutura-Atividade
11.
Prep Biochem Biotechnol ; 49(3): 270-278, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794034

RESUMO

The present study focusses on the enhancement of the catalytic activity and stability of an acetylesterase enzyme isolated from Staphylococcus spp. as Cross-Linked Enzyme Aggregates (CLEAs). The various parameters governing the activity of CLEAs were optimized. The magnetite and graphene oxide nanoparticles were successfully prepared via the chemical co-precipitation and Hummer's method, respectively. These nanoparticles supported the preparation as magnetite nanoparticle-supported cross-Linked Enzyme Aggregates (MGNP-CLEAs) and graphene oxide-supported Cross-Linked Enzyme Aggregates (GO-CLEAs). The activity and stability of these immobilized CLEAs were compared with the free enzyme at various temperature, pH, and organic solvents along with its storage stability and reusability. The immobilized preparations were analyzed by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared spectroscopy (FT-IR) techniques. Acetylesterase precipitated with 60% saturated ammonium sulfate salt (SAS) solution and cross-linked with 100 mM glutaraldehyde for 4 h at 30 °C was found to be optimal to produce CLEAs with highest activity recovery of 99.8%. The optimal pH at 8.0 and temperature at 30 °C remained the same for both the free and immobilized enzyme, respectively. Storage stability significantly improved for the immobilized enzyme as compared to free enzyme. SEM showed type-I aggregate and FT-IR revealed the successful immobilization of the enzyme. MGNP-CLEAs were found to have better activity and stability in comparison to other immobilized preparations.


Assuntos
Enzimas Imobilizadas/química , Esterases/química , Nanopartículas de Magnetita/química , Precipitação Química , Reagentes para Ligações Cruzadas/química , Estabilidade Enzimática , Glutaral/química , Grafite/química , Concentração de Íons de Hidrogênio , Óxidos/química , Staphylococcus/enzimologia , Temperatura Ambiente
12.
Appl Microbiol Biotechnol ; 103(8): 3421-3437, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30809711

RESUMO

Owing to the functional versatility and potential applications in industry, interest in lipolytic enzymes tolerant to organic solvents is increasing. In this study, functional screening of a compost soil metagenome resulted in identification of two lipolytic genes, est1 and est2, encoding 270 and 389 amino acids, respectively. The two genes were heterologously expressed and characterized. Est1 and Est2 are thermostable enzymes with optimal enzyme activities at 80 and 70 °C, respectively. A second-order rotatable design, which allows establishing the relationship between multiple variables with the obtained responses, was used to explore the combined effects of temperature and pH on esterase stability. The response curve indicated that Est1, and particularly Est2, retained high stability within a broad range of temperature and pH values. Furthermore, the effects of organic solvents on Est1 and Est2 activities and stabilities were assessed. Notably, Est2 activity was significantly enhanced (two- to tenfold) in the presence of ethanol, methanol, isopropanol, and 1-propanol over a concentration range between 6 and 30% (v/v). For the short-term stability (2 h of incubation), Est2 exhibited high tolerance against 60% (v/v) of ethanol, methanol, isopropanol, DMSO, and acetone, while Est1 activity resisted these solvents only at lower concentrations (below 30%, v/v). Est2 also displayed high stability towards some water-immiscible organic solvents, such as ethyl acetate, diethyl ether, and toluene. With respect to long-term stability, Est2 retained most of its activity after 26 days of incubation in the presence of 30% (v/v) ethanol, methanol, isopropanol, DMSO, or acetone. All of these features indicate that Est1 and Est2 possess application potential.


Assuntos
Compostagem , Esterases/química , Esterases/metabolismo , Metagenoma/genética , Solventes/química , Sequência de Bases , Clonagem Molecular , Ativação Enzimática , Estabilidade Enzimática , Esterases/genética , Esterases/isolamento & purificação , Biblioteca Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , Lipólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
13.
Int J Biol Macromol ; 126: 1192-1200, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30625356

RESUMO

A new bacterial lipolytic enzyme Est903 was obtained from paper mill sludge via metagenomic approach. Est903 displayed moderate similarities to two lipolytic enzymes from Rhodopirellula islandica and contained a distinctive pentapeptide motif (GFSAG) that differed from those of all known fourteen families of bacterial lipolytic enzymes. Est903 was regarded as from a new bacterial lipolytic enzyme family through multiple sequence alignment and phylogenetic analysis. The recombinant Est903 showed the highest activity for ρ-nitrophenol butyrate. The pH optimum and temperature optimum of the recombinant enzyme was 9.0 and 51 °C, respectively. Also, this enzyme displayed moderate thermostability, high activity under alkaline conditions, and good tolerance against several organic solvents. In addition, the compatibility test and washing performance analysis revealed that Est903 had good compatibility with commercial laundry detergent and high cleaning ability of oil stains. These good properties make Est903 a potential candidate in organic synthesis or detergent industry.


Assuntos
Esterases/isolamento & purificação , Esterases/metabolismo , Biblioteca Gênica , Resíduos Industriais , Metagenômica , Papel , Esgotos , Sequência de Aminoácidos , Detergentes/farmacologia , Esterases/química , Esterases/genética , Indicadores e Reagentes , Íons , Metais/farmacologia , Filogenia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Soluções , Solventes/química , Têxteis
14.
Org Biomol Chem ; 17(5): 1058-1061, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30637418

RESUMO

SurE is a new, stand-alone thioesterase (TE) offloading the non-ribosomal peptide (NRP) assembly line found in surugamide biosynthesis. It is homologous to penicillin binding protein (PBP) and capable of cyclizing two structurally unrelated substrates derived from two different NRP assembly lines, highlighting the broad substrate tolerance of the SurE offloading cyclase.


Assuntos
Esterases/química , Oligopeptídeos/química , Peptídeos Cíclicos/química , Compostos de Sulfidrila/química , Vias Biossintéticas , Cromatografia Líquida/métodos , Ciclização , Genes Bacterianos , Espectrometria de Massas/métodos , Estrutura Molecular , Espectrofotometria Ultravioleta , Streptomyces/química , Streptomyces/genética , Especificidade por Substrato
15.
Curr Top Med Chem ; 19(1): 74-97, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30686257

RESUMO

Enzymatic dysregulation in tumor and intracellular microenvironments has made this property a tremendously promising responsive element for efficient diagnostics, carrier targeting, and drug release. When combined with nanotechnology, enzyme-responsive drug delivery systems (DDSs) have achieved substantial advancements. In the first part of this tutorial review, changes in tumor and intracellular microenvironmental factors, particularly the enzymatic index, are described. Subsequently, the peptide sequences of various enzyme-triggered nanomaterials are summarized for their uses in various drug delivery applications. Then, some other enzyme responsive nanostructures are discussed. Finally, the future opportunities and challenges are discussed. In brief, this review can provide inspiration and impetus for exploiting more promising internal enzyme stimuli-responsive nanoDDSs for targeted tumor diagnosis and treatment.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos , Nanoestruturas/química , Neoplasias/tratamento farmacológico , Peptídeos/química , Proliferação de Células/efeitos dos fármacos , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Esterases/química , Esterases/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Humanos , Lipase/química , Lipase/metabolismo , Neoplasias/patologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Fosfolipases/química , Fosfolipases/metabolismo
16.
Int J Biol Macromol ; 125: 87-91, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30529348

RESUMO

In this paper, the catalytic performance of non-purified esterase from wheat bran immobilized on glass fibre membrane carrier is established, the immobilization conditions observed were enzyme 1 mL, phosphate buffer 3 mL (pH 7.0), immobilization time 1 h, immobilization temperature 29 °C. After carrier functionalization some characteristics of immobilized enzyme were studied, the results showed that immobilized enzyme presenting improved characteristic than that of free enzyme. The optimum pH for free and immobilized enzymes were found to be 8 and 7, respectively. As for optimum temperature for free and immobilized enzymes were observed to be 30 °C and 40 °C, respectively. When the enzyme was immobilized on glass fibre membranes, its Km increased about 7 times. In addition, storage and thermal stability of the free wheat esterase were increased by as a result of membrane immobilization, after 12 days of storage, the immobilized enzyme still retained about 91.10% of its original activity at 4 °C, indicating a great potential in industrial application.


Assuntos
Enzimas Imobilizadas , Esterases/química , Vidro , Triticum/química , Ativação Enzimática , Estabilidade Enzimática , Enzimas Imobilizadas/química , Esterases/isolamento & purificação , Esterases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Extração Líquido-Líquido , Temperatura Ambiente
17.
J Agric Food Chem ; 67(3): 836-843, 2019 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-30585487

RESUMO

Esterase SulE detoxicates a variety of sulfonylurea herbicides through de-esterification. SulE exhibits high activity against thifensulfuron-methyl but low activity against other sulfonylureas. In this study, two variants, m2311 (P80R) and m0569 (P80R and G176A), with improved activity were screened from a mutation library constructed by error-prone PCR. Variant m2311 showed a higher activity against sulfonylureas in comparison variant m0569 and was further investigated. The kcat/ Km value of variant m2311 for metsulfuron-methyl, sulfometuron-methyl, chlorimuron-ethyl, tribenuron-methyl, and ethametsulfuron-methyl increased by 3.20-, 1.72-, 2.94-, 2.26- and 2.96-fold, respectively, in comparison with the wild type. Molecular modeling suggested that the activity improvement of variant m2311 is due to the substitution of Pro80 by arginine, leading to the formation of new hydrogen bonds between the enzyme and substrate. This study facilitates further elucidation of the structure and function of SulE and provides an improved gene resource for the detoxification of sulfonylurea residues and the genetic engineering of sulfonylurea-resistant crops.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Esterases/genética , Esterases/metabolismo , Methylocystaceae/enzimologia , Compostos de Sulfonilureia/metabolismo , Proteínas de Bactérias/química , Evolução Molecular Direcionada , Esterases/química , Variação Genética , Herbicidas/química , Herbicidas/metabolismo , Cinética , Methylocystaceae/química , Methylocystaceae/genética , Pirimidinas/química , Pirimidinas/metabolismo , Compostos de Sulfonilureia/química , Tiofenos/química , Tiofenos/metabolismo
18.
Int J Biol Macromol ; 127: 66-75, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30578903

RESUMO

DHH superfamily proteins play pivotal roles in various cellular processes like replication, recombination, repair and nucleic acids metabolism. These proteins are important for homeostasis maintenance and stress tolerance in prokaryotes and eukaryotes. The prominent members of DHH superfamily include single-strand specific exonuclease RecJ, nanoRNases, polyphosphatase PPX1, pyrophosphatase, prune phosphodiesterase and cell cycle protein Cdc45. The mutations of genes coding for DHH superfamily proteins lead to severe growth defects and in some cases, may be lethal. The members of superfamily have a wide substrate spectrum. The spectrum of substrate for DHH superfamily members ranges from smaller molecules like pyrophosphate and cyclic nucleotides to longer single-stranded DNA molecule. Several genetic, structural and biochemical studies have provided interesting insights about roles of DHH superfamily members. However, there are still various unexplored members in both prokaryotes and eukaryotes. Many aspects of this superfamily associated with homeostasis maintenance and stress tolerance are still not clearly understood. A comprehensive understanding is pre-requisite to decipher the physiological significance of members of DHH superfamily. This article provides the current understanding of DHH superfamily members and their significance in nucleic acids metabolism and stress tolerance across diverse forms of life.


Assuntos
Proteínas Arqueais , Proteínas de Bactérias , Esterases , Células Eucarióticas/enzimologia , Ácidos Nucleicos/metabolismo , Células Procarióticas/enzimologia , Estresse Fisiológico , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Esterases/química , Esterases/metabolismo , Ácidos Nucleicos/química , Ácidos Nucleicos/genética
19.
Biotechnol Lett ; 40(9-10): 1395-1406, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30062528

RESUMO

OBJECTIVE: To isolate putative lipase enzymes by screening a Cerrado soil metagenomic library with novel features. RESULTS: Of 6720 clones evaluated, Clone W (10,000 bp) presented lipolytic activity and four predicted coding sequences, one of them LipW. Characterization of a predicted esterase/lipase, LipW, showed 28% sequence identity with an arylesterase from Pseudomonas fluorescens (pdb|3HEA) from protein database (PDB). Phylogenetic analysis showed LipW clustered with family V lipases; however, LipW was clustered in different subclade belonged to family V, suggesting a different subgroup of family V. In addition, LipW presented a difference in family V GH motif, a glycine replaced by a serine in GH motif. Estimated molecular weight and stokes radius values of LipW were 29,338.67-29,411.98 Da and 2.58-2.83 nm, respectively. Optimal enzyme activity was observed at pH 9.0-9.5 and at 40 °C. Circular dichroism analysis estimated secondary structures percentages as approximately 45% α-helix and 15% ß-sheet, consistent with the 3D structure predicted by homology. CONCLUSION: Our results demonstrate the isolation of novel family V lipolytic enzyme with biotechnological applications from a metagenomic library.


Assuntos
Esterases/genética , Esterases/metabolismo , Microbiologia do Solo , Motivos de Aminoácidos , Brasil , Dicroísmo Circular , Clonagem Molecular , Esterases/química , Metagenoma , Modelos Moleculares , Peso Molecular , Filogenia , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência
20.
Extremophiles ; 22(5): 781-793, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30014242

RESUMO

The biotechnological and industrial uses of thermostable and organic solvent-tolerant enzymes are extensive and the investigation of such enzymes from microbiota present in oil reservoirs is a promising approach. Searching sequence databases for esterases from such microbiota, we have identified in silico a potentially secreted esterase from Acetomicrobium hydrogeniformans, named AhEst. The recombinant enzyme was produced in E. coli to be used in biochemical and biophysical characterization studies. AhEst presented hydrolytic activity on short-acyl-chain p-nitrophenyl ester substrates. AhEst activity was high and stable in temperatures up to 75 °C. Interestingly, high salt concentration induced a significant increase of catalytic activity. AhEst still retained ~ 50% of its activity in 30% concentration of several organic solvents. Synchrotron radiation circular dichroism and fluorescence spectroscopies confirmed that AhEst displays high structural stability in extreme conditions of temperature, salinity, and organic solvents. The enzyme is a good emulsifier agent and is able to partially reverse the wettability of an oil-wet carbonate substrate, making it of potential interest for use in enhanced oil recovery. All the traits observed in AhEst make it an interesting candidate for many industrial applications, such as those in which a significant hydrolytic activity at high temperatures is required.


Assuntos
Proteínas de Bactérias/metabolismo , Esterases/metabolismo , Ambientes Extremos , Desnaturação Proteica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Esterases/química , Esterases/genética , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salinidade , Solventes/química , Especificidade por Substrato
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