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1.
World J Microbiol Biotechnol ; 35(9): 135, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432264

RESUMO

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.


Assuntos
Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Thermoactinomyces/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Thermoactinomyces/genética
2.
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 Ambiente
3.
World J Microbiol Biotechnol ; 35(7): 106, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267229

RESUMO

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.


Assuntos
Antifúngicos/farmacologia , Quitinases/antagonistas & inibidores , Quitinases/genética , Quitinases/metabolismo , Xenorhabdus/metabolismo , Alternaria/efeitos dos fármacos , Animais , Ascomicetos/efeitos dos fármacos , Quitina/metabolismo , Quitinases/classificação , Clonagem Molecular , Sinergismo Farmacológico , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Micotoxinas/genética , Micotoxinas/metabolismo , Filogenia , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Verticillium/efeitos dos fármacos , Xenorhabdus/genética
4.
J Agric Food Chem ; 67(33): 9307-9313, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352784

RESUMO

Porphyra is one of the most consumed types of red algae. Porphyran is the major polysaccharide extracted from Porphyra, and it is composed of alternating 4-linked α-l-galactopyranose-6-sulfate (L6S) and 3-linked ß-d-galactopyranose (G) residues. ß-Porphyranases are promising tools for degrading porphyran; however, few enzymes have been reported, and the biochemical properties of porphyranases are still unclear. Here, a novel GH16 ß-porphyranase, designated as Por16A_Wf, was cloned from Wenyingzhuangia fucanilytica and expressed in Escherichia coli. Its biochemical properties and hydrolysis pattern were characterized. Por16A_Wf exhibited stable activity on a wide pH scale from 3.5 to 11.0. Glycomics analysis using LC-MS revealed that Por16A_Wf specifically hydrolyzed the glycosidic linkage of G-L6S, whereas it tolerated 3,6-anhydro-α-l-galactopyranose and methyl-d-galactose in -2 and +2 subsites, respectively. Por16A_Wf could be applied as a biotechnological tool for tailoring porphyran, which would serve in directional preparation of its disaccharide, producing products with various molecular weights and facilitating investigation of the structural heterogeneity of Porphyra polysaccharides.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Flavobacteriaceae/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Água do Mar/microbiologia , Sefarose/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Biocatálise , Biotecnologia , Clonagem Molecular , Estabilidade Enzimática , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Filogenia , Porphyra/química , Porphyra/metabolismo , Sefarose/química , Sefarose/metabolismo , Alinhamento de Sequência
5.
Prep Biochem Biotechnol ; 49(8): 830-836, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31274051

RESUMO

The current study evaluated the production and characterization of ß-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of ß-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5-4.5 and at 70 °C. The enzyme exhibited high thermostability, for 1 h, up to 60 °C, and good tolerance to glucose (10 mM) and ethanol (10%). The optimization of fermentative parameters on the production of ß-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0 U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH4)2SO4 solution at pH 5.5-6.0 and fungus incubated at 40 °C. A more detailed study of ß-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.


Assuntos
Fibras na Dieta/metabolismo , Microbiologia Industrial/métodos , Mucorales/metabolismo , beta-Glucosidase/metabolismo , Estabilidade Enzimática , Etanol/metabolismo , Fermentação , Glucose/metabolismo , Íons/metabolismo
6.
J Agric Food Chem ; 67(29): 8177-8185, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31290662

RESUMO

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose to trehalose, and is therefore essential for trehalose production. Consequently, dissecting the catalytic mechanism of TreS is important for enzyme optimization and industrial applications. TreS from Thermobaculum terrenum (TtTreS) is a thermostable enzyme. Here, we studied the composition of the TtTreS active site through computer calculation and enzyme analysis. The results were consistent with a two-step double-displacement mechanism, similar to that of glycoside hydrolase 13 family enzymes. However, our data suggested that glucose rotation, following breakage of the α-1,4 glycosidic bond, is a key factor determining the reaction direction and conversion rate. The N246 residue plays an important role in glucose rotation. Moreover, we established a saturated mutation model for the nonconserved amino acids around the substrate gateway domain. Finally, four TtTreS mutants (K136T, Y137D, K138N, and D139S) resulted in improved trehalose yield compared to that of the wild-type enzyme.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Glucosiltransferases/química , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Biologia Computacional , Estabilidade Enzimática , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Temperatura Alta , Especificidade por Substrato
7.
Food Chem ; 297: 125005, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253325

RESUMO

Multiwalled carbon nanotubes molybdenum disulfide 3D nanocomposite (MWCNT-MoS2 NC) was successfully synthesized via eco-friendly hydrothermal method. The microstructural characterization of synthesized nanocomposite was carried out using different spectroscopic and microscopic techniques. Nanocomposite was activated using glutaraldehyde chemistry and used as a platform to immobilize Lens culinaris ß-galactosidase (Lsbgal) which resulted in 93% of immobilization efficiency. Attachment of Lsbgal onto nanocomposite was confirmed by AFM, FE-SEM, FTIR, and CLSM. The nanobiocatalyst showed broadening in operational pH and temperature working range. Remarkable increase in thermal stability was observed as compared to soluble enzyme. Nanobiocatalyst showed outstanding increase in storage stability, retained 92% of residual activity over a period of 8 months. This offers good reusability as it retained ∼50% residual activity up to 21 reuses and exhibited higher rate of lactose hydrolysis in whey. MWCNT-MoS2 NC conjugated to biomolecules can serve as a potential platform for fabrication of lactose biosensor.


Assuntos
Lactose/metabolismo , Lens (Planta)/enzimologia , Nanocompostos/química , Soro do Leite/metabolismo , beta-Galactosidase/metabolismo , Biocatálise , Dissulfetos/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Molibdênio/química , Nanotubos de Carbono/química , Temperatura Ambiente , beta-Galactosidase/química
8.
Food Chem ; 295: 311-319, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174763

RESUMO

A novel gene aga3027 from the genome of Flammeovirga sp. OC4, isolated from the deep sea, was screened and expressed in Escherichia coli BL21. This gene encoded the genetic information of a potential agarase that consists of 851 amino acids and belongs to 16 ß-agarase family of glycoside hydrolase. Purified recombinant Aga3027 demonstrated the maximum activity of agarase at 40 °C and pH 9.0, displaying excellent thermostability and pH-stability. The agarase retained more than 80% of its maximum activity after incubation at 30-40 °C for 48 h, or after incubation at pH 6.0-9.0 for 60 min, which indicated that this agarase was suitable for industrial applications. Silica gel chromatography was used to purify the hydrolysates of agar treated by agarase from the recombinant Aga3027. The hydrolysates were identified as neoagarotetraose and neoagarohexaose by thin layer chromatography and further confirmed by ion chromatography.


Assuntos
Ágar/química , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Ágar/metabolismo , Bacteroidetes/enzimologia , Bacteroidetes/genética , Cromatografia em Camada Delgada , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosídeos/metabolismo , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Hidrólise , Oligossacarídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente
9.
J Agric Food Chem ; 67(24): 6837-6846, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31180217

RESUMO

Mannooligosaccharides are released by mannan-degrading endo-ß-1,4-mannanase and are known as functional additives in human and animal diets. To satisfy demands for biocatalysis and bioprocessing in crowed environments, in this study, we employed a recently developed enzyme-engineering system, isopeptide bond-mediated molecular cyclization, to modify a mesophilic mannanase from Bacillus subtilis. The results revealed that the cyclized enzymes showed enhanced thermostability and ion stability and resilience to aggregation and freeze-thaw treatment by maintaining their conformational structures. Additionally, by using the SpyTag/SpyCatcher system, we generated a mannanase-xylanase bifunctional enzyme that exhibited a synergistic activity in substrate deconstruction without compromising substrate affinity. Interestingly, the dual-enzyme ring conformation was observed to be more robust than the linear enzyme but inferior to the single-enzyme ring conformation. Taken together, these findings provided new insights into the mechanisms of molecular cyclization on stability improvement and will be useful in the production of new functional oligosaccharides and feed additives.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , beta-Manosidase/química , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclização , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Engenharia de Proteínas , beta-Manosidase/genética , beta-Manosidase/metabolismo
10.
Food Chem ; 295: 653-661, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174809

RESUMO

Although ß-xylosidases have a wide range of applications, cold-active ß-xylosidases have been poorly studied. In this study, a cold active ß-xylosidase gene (xyl) from Bacillus pumilus TCCC 11,350 was cloned and overexpressed in Escherichia coli. The recombinant XYL (rXYL) was revealed to be a bifunctional enzyme with both ß-xylosidase and α-l-arabinofuranosidase activities. Purified rXYL was most active at 30 °C, demonstrating 26% and 18% of its maximum activity at 4 °C and 0 °C, respectively. Meanwhile, rXYL showed a 52% activity in 200 mM xylose, indicating a relatively strong tolerance to xylose. Moreover, rXYL exhibited a high synergistic effect (11.14-fold and 16.21-fold) with endo-xylanase to degrade beechwood xylan in both sequential and simultaneous reactions at low temperatures. As the first report on the novel cold-adapted ß-xylosidase from B. pumilus, these results suggested rXYL had attractive properties for food industrial utilizations.


Assuntos
Bacillus pumilus/enzimologia , Xilosidases/metabolismo , Sequência de Aminoácidos , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Filogenia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Temperatura Ambiente , Xilanos/metabolismo , Xilose/metabolismo , Xilosidases/classificação , Xilosidases/genética
11.
J Microbiol Biotechnol ; 29(6): 913-922, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154745

RESUMO

Magnetic Ni0.7Co0.3Fe2O4 nanoparticles that were prepared via the rapid combustion process were functionalized and modified to obtain magnetic Ni0.7Co0.3Fe2O4@SiO2-CHO nanocomposites, on which penicillin G acylase (PGA) was covalently immobilized. Selections of immobilization concentration and time of fixation were explored. Catalytic performance of immobilized PGA was characterized. The free PGA had greatest activity at pH 8.0 and 45oC while immobilized PGA's a ctivities peaked a t pH 7.5 and 45°C. Immobilized PGA had better thermal stability than free PGA at the range of 30-50°C for different time intervals. The activity of free PGA would be 0 and that of immobilized PGA still retained some activities at 60°C after 2 h. Vmax and Km of immobilized PGA were 1.55 mol/min and 0.15 mol/l, respectively. Free PGA's Vmax and Km separately were 0.74 mol/min and 0.028 mol/l. Immobilized PGA displayed more than 50% activity after 10 successive cycles. We concluded that immobilized PGA with magnetic Ni0.7Co0.3Fe2O4@SiO2-CHO nanocomposites could become a novel example for the immobilization of other amidohydrolases.


Assuntos
Cobalto/química , Enzimas Imobilizadas/química , Nanopartículas de Magnetita/química , Nanocompostos/química , Níquel/química , Penicilina Amidase/química , Penicilina Amidase/metabolismo , Catálise , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Glutaral/química , Concentração de Íons de Hidrogênio , Dióxido de Silício/química , Temperatura Ambiente
12.
Bioelectrochemistry ; 129: 116-123, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31153126

RESUMO

In this study, (1→3)(1→6)-ß-D-glucan (botryosphaeran) from Botryosphaeria rhodina MAMB-05 was used, for the first time, to immobilize laccase on a carbon black paste electrode modified with gold nanoparticles. The physicochemical characterization of the proposed laccase-biosensor was performed using transmission electron microscopy and electrochemical impedance spectroscopy. The performance of this novel bio-device was evaluated by choosing hydroquinone as a typical model of a phenolic compound. For hydroquinone determination, experimental variables such as enzyme concentration, pH and operational parameters of the electroanalytical technique were optimized. From square-wave voltammograms, a linear dependence between the cathodic current peak and the hydroquinone concentration was observed within the range 2.00-56.5µmolL-1, with a theoretical detection limit of 0.474µmolL-1. The proposed method was successfully applied to determine hydroquinone in dermatological cream, and samples from biological and environmental niches. The proposed biosensor device presented good selectivity in the presence of uric acid, various inorganic ions, as well as other phenolic compounds, demonstrating the potential application of this biosensing platform in complex matrices. Operational and analytical stability of the laccase biosensor were evaluated, and demonstrated good intra-day (SD=0.3%) and inter-day (SD=3.4%) repeatability and long storage stability (SD=4.9%).


Assuntos
Ascomicetos/enzimologia , Técnicas Biossensoriais/métodos , Enzimas Imobilizadas/química , Glucanos/química , Hidroquinonas/análise , Lacase/química , Fuligem/química , Técnicas Biossensoriais/instrumentação , Estabilidade Enzimática , Desenho de Equipamento , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química
13.
J Enzyme Inhib Med Chem ; 34(1): 946-954, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31039618

RESUMO

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes present in all living organisms on the planet. They are classified into seven genetically distinct families and catalyse the hydration reaction of carbon dioxide to bicarbonate and protons, as well as the opposite reaction. CAs were proposed to be used for biotechnological applications, such as the post-combustion carbon capture processes. In this context, there is a great interest in searching CAs with robust chemical and physical properties. Here, we describe the enhancement of thermostability of the α-CA from Sulfurihydrogenibium yellowstonense (SspCA) by using the anchoring-and-self-labelling-protein-tag system (ASLtag). The anchored chimeric H5-SspCA was active for the CO2 hydration reaction and its thermostability increased when the cells were heated for a prolonged period at high temperatures (e.g. 70 °C). The ASLtag can be considered as a useful method for enhancing the thermostability of a protein useful for biotechnological applications, which often need harsh operating conditions.


Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Bactérias Gram-Negativas Quimiolitotróficas/enzimologia , Coloração e Rotulagem/métodos , Temperatura Ambiente , Estabilidade Enzimática , Modelos Moleculares , Relação Estrutura-Atividade
14.
J Microbiol ; 57(6): 521-531, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31073894

RESUMO

Characteristics of naringinase nano-encapsulated forms on different carrier materials (chitosan and alginate polymers) were investigated in this study. Screening of twelve fungal isolates for naringinase production indicated that Trichoderma longibrachiatum was the most promising. Grapefruit rind was used as a substrate containing naringin for naringinase production. TEM micrographs showed that chitosan nano-capsules were applied for the production of morphologically homogeneous enzymatic nano-particles with high enzyme encapsulation efficiency, small asymmetric sizes (from 15.09 to 27.07 nm with the mean of 21.8 nm) and rough surfaces compared to nano-encapsulated naringinase in alginate which showed nano-particle size (from 33.37 to 51.01 nm with the mean of 43.03 nm). It was revealed that the highest naringinase activity was found in case of chitosan nano-capsule naringinase compared to alginate nano-capsule one. Thermogram analysis (TGA) showed that the free enzyme loses about 92% of its weight at approximately 110°C, while the nano-encapsulated ones show more stability at higher temperatures. Conclusively, the nano-capsulation process improves the kinetics and operational stability so could be useful as a debittering agent for various thermal processing applications in citrus juices industries which makes the fruit juice more acceptable and cost-effective to the consumer.


Assuntos
Biopolímeros/química , Enzimas Imobilizadas/química , Sucos de Frutas e Vegetais , Complexos Multienzimáticos/metabolismo , Trichoderma/metabolismo , beta-Glucosidase/metabolismo , Quitosana/química , Citrus , Citrus paradisi , Estabilidade Enzimática , Flavanonas/metabolismo , Indústria Alimentícia , Concentração de Íons de Hidrogênio , Cinética , Complexos Multienzimáticos/isolamento & purificação , Tamanho da Partícula , Temperatura Ambiente , beta-Glucosidase/isolamento & purificação
15.
Chem Commun (Camb) ; 55(44): 6293-6296, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31086931

RESUMO

A novel strategy for enhancing the activity and stability of metal-activated enzymes by allosteric control and confinement of metal-nulceobase hybrid coordination was reported in this study. Despite the lack of active Mg2+ ions, methionine adenosyltransferase (MAT) immobilized by Zn2(adenine) still exhibited robust catalytic activity and stability at pH 3 and 80 °C.


Assuntos
Adenina/química , Ativadores de Enzimas/química , Nanopartículas Metálicas/química , Metionina Adenosiltransferase/metabolismo , Zinco/química , Regulação Alostérica , Catálise , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Espectrofotometria Infravermelho , Thermus thermophilus/enzimologia , Difração de Raios X
16.
Int J Nanomedicine ; 14: 3235-3244, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31118633

RESUMO

Purpose: Here, we present the successful preparation of a highly efficient gallic acid resin grafted with magnetic nanoparticles (MNPs) and containing a branched brush polymeric shell. Methods: Using a convenient co-precipitation method, we prepared Fe3O4 nanoparticles stabilized by citric acid. These nanoparticles underwent further silica modification and amino functionalization followed by gallic acid functionalization on their surface. Under alkaline conditions, we used a condensation reaction that combined formaldehyde and gallic, to graft the gallic acid-formaldehyde resin on the surface. We then evaluated the polymer-grafted MNPs to assay the Candida Antarctica B lipase(Cal-B) immobilization via physical adsorption. Conclusion: Furthermore, during optimization of parameters that defined conditions of immobilization, we found that the optimum immobilization was achieved in 15 mins. Also, optimal immobilization temperature and pH were 38ºC and 7.5, respectively. In addition, the reusability study of immobilized lipase polymer-grafted MNPs was done by isolating the MNPs from the reaction medium using magnetic separation, which showed that grafted MNPs reached 5 cycles with 91% activity retention.


Assuntos
Enzimas Imobilizadas/metabolismo , Compostos Férricos/química , Proteínas Fúngicas/metabolismo , Ácido Gálico/química , Lipase/metabolismo , Nanopartículas de Magnetita/química , Resinas Sintéticas/química , Adsorção , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Nanopartículas de Magnetita/ultraestrutura , Polímeros , Dióxido de Silício/química , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura Ambiente , Termogravimetria , Fatores de Tempo , Difração de Raios X
17.
J Basic Microbiol ; 59(7): 667-679, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31087565

RESUMO

A psychrotolerant yeast strain Mrakia robertii A2-3 isolated from cryoconites of Hamtah glacier, Himalaya, India was investigated for the production of cold-tolerant endoglucanase. Optimum endoglucanase production was found at 15°C with an initial pH of 5.5, and potent inducers were 1% wt/vol of xylose and KNO3 and 0.1% wt/vol of NaCl. Under optimum conditions, the enzyme production was 1.81-fold higher than the unoptimized conditions. Crude enzyme was partially purified by ammonium sulfate precipitation followed by dialysis. The enzyme was purified to 2.53-fold and the yield was 6.03% with specific activity of 17.38 U/mg and molecular weight ~57 kDa. The Km and Vmax values of the partially purified enzyme were found to be 1.57 mg/ml and 142.85 U/mg, respectively. The characterization study revealed that the best temperature was 15°C for activity and stability. Furthermore, the enzyme showed the highest activity at pH 11.0 and was stable at pH 6.0. Fe2+ , Mn2+ , Na2+ , Cu2+ , Co2+ , Ca2+ proved to be activators of endoglucanase. Ethylenediamine tetraacetic acid showed very low effect on the enzyme activity whereas it was active with Tween-80 and sodium deoxycholate. The present study successfully produced a cold-active endoglucanase with novel properties making it promising as a biocatalyst for industrial processes.


Assuntos
Basidiomycota/enzimologia , Celulase/fisiologia , Temperatura Baixa , Camada de Gelo/microbiologia , Basidiomycota/classificação , Basidiomycota/fisiologia , Carboximetilcelulose Sódica/metabolismo , Celulase/química , Celulase/isolamento & purificação , Celulase/metabolismo , DNA Fúngico/genética , DNA Ribossômico/genética , Detergentes , Ativadores de Enzimas , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Índia , Cinética , Peso Molecular , Filogenia , Análise de Sequência de DNA
18.
Biochimie ; 163: 33-49, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31078582

RESUMO

Snake venom L-amino acid oxidases (svLAAOs) are an interesting class of enzymes with important biological activities. Their participation in key metabolic processes, including pathological disorders, suggest that svLAAOs are potential lead compounds in drug discovery. However, their short-term stability defies their applications. This paper describes the stability studies together with functional and structural characterization of the LAAO bordonein-L. It has 498 amino acid residues, one N-glycosylation site and two disulfide bonds, revealed by high-resolution MS/MS. Molecular modeling approach showed its monomer folds into three conserved domains: FAD, substrate and helical domains. Differential scanning fluorimetry showed the enzyme tends to destabilize from neutral to basic pHs and in presence of mono/bivalent ions and it is highly stabilized by acid pHs and its substrates. However, high concentrations of L-amino acids decrease bordonein-L enzyme activity. Dynamic light scattering revealed bordonein-L remains in the dimeric and monodisperse form, so aggregation does not cause the rapidly decrease of enzyme activity. In vitro, the enzyme exhibited cytotoxicity against fibroblast cell line and killed Leishmania amazonensis promastigotes, intensified by substrate addition. Concluding, our results provide biochemistry and biophysical insights to improve LAAOs stability and better approaches to long-term storage. Moreover, our study emphasizes the importance of proper buffers choice mainly in cell-based assays.


Assuntos
Crotalus/metabolismo , L-Aminoácido Oxidase/metabolismo , Venenos de Serpentes/enzimologia , Sequência de Aminoácidos , Animais , Estabilidade Enzimática , L-Aminoácido Oxidase/química , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato , Espectrometria de Massas em Tandem
19.
Enzyme Microb Technol ; 127: 22-31, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31088613

RESUMO

The recombinant rAgaZC-1 was a family GH50 ß-agarase from Vibrio sp. ZC-1 (CICC 24670). In this paper, the mutant D622G (i.e., mutate the aspartic acid at position 622 to glycine) had better thermo-stability than rAgaZC-1, showing 1.5℃ higher T5010 (the temperature at which the half-time is 10 min) and 4-folds of half-time at 41℃, while they had almost same optimum temperature (38.5℃), optimum pH (pH6.0) and catalytic efficiency. Thermal deactivation kinetical analysis showed that D622G had higher activation energy for deactivation, enthalpy and Gibbs free energy than rAgaZC-1, indicating that more energy is required by D622G for deactivation. Substrate can protect agarase against thermal inactivation, especially D622G. Hence the yield of agarose hydrolysis catalyzed by D622G was higher than that by rAgaZC-1. The models of D622G and rAgaZC-1 predicted by homology modeling were compared to find that it is the improved distribution of surface electrostatic potential, great symmetric positive potential and more hydrophobic interactions of D622G that enhance the thermo-stability.


Assuntos
Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Mutagênese , Vibrio/enzimologia , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Conformação Proteica , Estabilidade Proteica , Sefarose/metabolismo
20.
Enzyme Microb Technol ; 127: 32-42, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31088614

RESUMO

Currently, hyperstable endo-1,4-ß-xylanase has been the focus of attention as potent biocatalyst as well as utilization in bioconversion process. Therefore, the gene (1035 bp) of a monomeric glycoside hydrolase family 10 (GH10) endo-1,4-ß-xylanase (TnXynB) from a hyperthermophilic eubacterium Thermotoga naphthophila RKU-10T was cloned and overexpressed in a mesophilic host system. The extracellular TnXynB was purified to homogeneity with a molecular mass of 40 kDa, and showed peak activity at pH 6.0 and 95 °C temperature. Purified TnXynB has prodigious stability over a broad range of pH (5.5-8.0) and temperature (50-85 °C) even after 8 h incubation, and also revealed great tolerance toward different modulators (metal cations, surfactants and organic solvents). TnXynB exhibited great affinity towards various heteroglycans and para-nitrophenyl glycosides substrates. The values of K m, Vmax, kcat, and kcat K m-1 were 2.75 mg mL-1, 3146.7 µmol mg-1 min-1, 40,342.3 s-1, 14,669.93 mL mg-1 s-1, respectively using birchwood xylan as substrate. Thermodynamic parameters for birchwood xylan hydrolysis at 95 °C as ΔS*, ΔH*, and ΔG* were -22.88 J mol-1 K-1, 62.44 kJ mol-1, and 70.86 kJ mol-1 respectively. TnXynB displayed a half-life (t1/2) of 54.15 min at 96 °C with ΔS*D, ΔH*D, and ΔG*D values of 1.074 kJ mol-1 K-1, 513.23 kJ mol-1 and 116.92 kJ mol-1, respectively. All noteworthy features of TnXynB make this new recombinant enzyme an appropriate candidate for the biodegradation of lignocellulosic substrates as well as various other industrial bioprocesses.


Assuntos
Endo-1,4-beta-Xilanases/isolamento & purificação , Endo-1,4-beta-Xilanases/metabolismo , Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/enzimologia , Clonagem Molecular , Endo-1,4-beta-Xilanases/química , Estabilidade Enzimática , Expressão Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
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