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1.
Enzyme Microb Technol ; 134: 109476, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32044023

RESUMO

Soybean is a most promising sustainable protein source for feed and food to help meet the protein demand of the rapidly rising global population. To enrich soy protein, the environment-friendly enzymatic processing requires multiple carbohydrases including cellulase, xylanase, pectinase, α-galactosidase and sucrase. Besides enriched protein, the processing adds value by generating monosaccharides that are ready feedstock for biofuel/bioproducts. Aspergillus could produce the required carbohydrases, but with deficient pectinase and α-galactosidase. Here we address this critical technological gap by focused evaluation of the suboptimal productivity of pectinase and α-galactosidase. A carbohydrases-productive strain A. niger (NRRL 322) was used with soybean hull as inducing substrate. Temperatures at 20 °C, 25 °C and 30 °C were found to affect cell growth on sucrose with an Arrhenius-law activation energy of 28.7 kcal/mol. The 30 °C promoted the fastest cell growth (doubling time = 2.1 h) and earliest enzyme production, but it gave lower final enzyme yield due to earlier carbon-source exhaustion. The 25 °C gave the highest enzyme yield. pH conditions also strongly affected enzyme production. Fermentations made with initial pH of 6 or 7 were most productive, e.g., giving 1.9- to 2.3-fold higher pectinase and 2.2- to 2.3-fold higher α-galactosidase after 72 h, compared to the fermentation with a constant pH 4. Further, pH must be kept above 2.6 to avoid limitation in pectinase production and, in the later substrate-limiting stage, kept below 5.5 to avoid pectinase degradation. α-Galactosidase production always followed the pectinase production with a 16-24 h lag; presumably, the former relied on pectin hydrolysis for inducers generation. Optimal enzyme production requires controlling the transient availability of inducers.


Assuntos
Aspergillus niger/enzimologia , Poligalacturonase/biossíntese , Proteínas de Soja/metabolismo , alfa-Galactosidase/biossíntese , Biocombustíveis , Fermentação , Hidrólise , Soja , Temperatura
2.
Arch Microbiol ; 202(1): 197-203, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31372664

RESUMO

Galactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans and Aspergillus fumigatus. BLASTp searches identified three putative Galf-transferases in Aspergillus niger. Phylogenetic analysis showed that they group in three distinct groups. Characterization of the three Galf-transferases in A. niger by constructing single, double, and triple mutants revealed that gfsA is most important for Galf biosynthesis. The growth phenotypes of the ΔgfsA mutant are less severe than that of the ΔgfsAC mutant, indicating that GfsA and GfsC have redundant functions. Deletion of gfsB did not result in any growth defect and combining ΔgfsB with other deletion mutants did not exacerbate the growth phenotype. RT-qPCR experiments showed that induction of the agsA gene was higher in the ΔgfsAC and ΔgfsABC compared to the single mutants, indicating a severe cell wall stress response after multiple gfs gene deletions.


Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Transferases/genética , Transferases/metabolismo , Aspergillus fumigatus/classificação , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Aspergillus nidulans/classificação , Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Aspergillus niger/classificação , Parede Celular , Deleção de Genes , Mutação , Filogenia
3.
Biochimie ; 168: 231-240, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31756400

RESUMO

A novel bgl1 gene, encoding GH3 family ß-glucosidase from Penicillium verruculosum (PvBGL), was cloned and heterologously expressed in P. canescens RN3-11-7 (niaD-) strain under the control of the strong xylA gene promoter. The recombinant rPvBGL was purified and their properties were studied in comparison with those of rAnBGL from Aspergillus niger expressed previously in the same fungal host. The rPvBGL had an observed molecular mass of 90 kDa (SDS-PAGE data) and displayed the enzyme maximum activity at pH 4.6 and 65 °C. The enzyme half-life time at 60 °C was found to be 87 min. Unlike the rAnBGL, the rPvBGL was not adsorbed on microcrystalline cellulose, which gives the latter enzyme an advantage in cellulose conversion with a longer time of hydrolysis.


Assuntos
Aspergillus niger/enzimologia , Proteínas Fúngicas , Penicillium/enzimologia , Proteínas Recombinantes , beta-Glucosidase , Celulose/química , Clonagem Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , beta-Glucosidase/química , beta-Glucosidase/isolamento & purificação
4.
Food Chem ; 310: 125970, 2020 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31838375

RESUMO

Glucose oxidases are widely used in various industrial processes, including bread baking. In this study, a novel glucose oxidase gene, CngoxA, from Cladosporium neopsychrotolerans SL16, was cloned and expressed in Pichia pastoris. Recombinant CnGOXA exhibited maximal activity at 20 °C and pH 7.0, and was stable at 30 °C and pH 6.0-9.0 for 1 h, with a half-life of 15 min at 40 °C. Compared with CnGOXA, the half-life of its mutant CnGOXA-M1 (Y169C-A211C), at 40 °C increased approximately 48-fold, and was stable at 30 °C and pH 3.0-12.0 for 1 h. The kcat and catalytic efficiency of CnGOXA-M1 were enhanced 0.7- and 1.6-fold, respectively. Both enzymes were cold-adapted and highly resistant to SDS. Furthermore, CnGOXA-M1 had a more significant effect on bread volume than that of GOX from Aspergillus niger. These favorable enzymatic properties of CnGOXA-M1 make it a potentially useful enzyme for many industrial applications.


Assuntos
Pão , Cladosporium/enzimologia , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Aspergillus niger/enzimologia , Catálise , Cladosporium/genética , Estabilidade Enzimática , Microbiologia de Alimentos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucose Oxidase/genética , Concentração de Íons de Hidrogênio , Cinética , Mutação , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Microbiologia do Solo , Temperatura
5.
Carbohydr Polym ; 229: 115471, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826427

RESUMO

Micro/nano celluloses (MC)/NC) were magnetized by nanoγ-Fe2O3 into the nanoγ-Fe2O3@MC (NMMC) and nanoγ-Fe2O3@NC (NMMC) which oxidized to NMMCD and NMNCD dialdehydes for Schiff-base immobilization of urease as NMMCD/urease and NMMCD/urease. The relative enzyme-activity of the immobilized ureases were comparable with the free-urease, although 75%-80% of the enzyme activity preserved for NMMCD/urease and NMNCD/urease after six cycles. The compared catalytic activities of the NMMCD/urease and NMMCD/urease in Biginelli/Hantzsch reactions in water at 60 °C surprised us by 100% selectivity for the Biginelli product 3,4-dihydropyrimidin-2(1H)-one (DHPM1). With the superiority of NMNCD/urease, this high selectivity using immobilized ureases is owing to the admirable urease inhibitory of the formed Biginelli product DHPM1 by urea condensation instead of urea hydrolysis. The robust enzyme inhibitory of the DHPM1 for free urease was also confirmed by phenol red test to show the deactivation of enzyme for enzymatic hydrolysis of urea and ammonia production in water.


Assuntos
Celulose/química , Enzimas Imobilizadas/química , Ureia/química , Urease/química , Aspergillus niger/enzimologia , Catálise , Celulose/isolamento & purificação , Inibidores Enzimáticos/química , Enzimas Imobilizadas/antagonistas & inibidores , Compostos Férricos/química , Gossypium/química , Concentração de Íons de Hidrogênio , Fenômenos Magnéticos , Estabilidade Proteica , Temperatura , Urease/antagonistas & inibidores
6.
Food Chem ; 305: 125382, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31525590

RESUMO

During the mashing process for brewing beer, incomplete degradation of arabinoxylan in barley malt may cause an intense filterability problem. The present study cloned a putative arabinofuranosidase (AnAbf), one of the debranching enzymes, from Aspergillus niger, to explore its application for improving filterability. Recombinant AnAbf (rAnAbf) showed activity towards both synthetic and natural substrates, such as 4-nitrophenyl α-l-arabinofuranoside (pNPαAraf) and malt water extractable arabinoxylan (WEAX), which was maximized at a temperature of 50 °C and pH of 5.5. Metal ions did not increase the activity of rAnAbf, indicating a difference in its C-terminal domain from that of type II GH43 family members. rAnAbf also exhibited a synergistic effect with ß-xylanase against WEAX. The filtration rate of the wort increased by 12.8% after supplementing with rAnAbf during the initial stage of mashing. A slight decrease in viscosity and an unexpected increase in turbidity were observed.


Assuntos
Aspergillus niger/enzimologia , Cerveja/análise , Glicosídeo Hidrolases/metabolismo , Xilanos/metabolismo , Arabinose/análogos & derivados , Arabinose/metabolismo , Endo-1,4-beta-Xilanases , Proteínas Fúngicas/metabolismo , Temperatura Alta , Proteínas Recombinantes/metabolismo
7.
Biosens Bioelectron ; 148: 111764, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31707325

RESUMO

We are reporting an original supramolecular architecture based on a rationally designed new nanohybrid with enhanced peroxidase-like activity and site-specific biorecognition properties using avidin-functionalized multi-walled carbon nanotubes (MWCNTs-Av) and Ru nanoparticles (RuNPs). The nanohybrid-electrochemical interface was obtained by drop-coating of MWCNTs-Av dispersion at glassy carbon electrodes (GCE) followed by solvent evaporation and further electrodeposition of RuNPs (50 ppm RuCl2 for 15 s at -0.600 V). The simultaneous presence of MWCNTs and RuNPs produces a synergic effect on the non-enzymatic catatalytic reduction of H2O2 and allows the quantification of H2O2 in a wide linear range (from 5.0 × 10-7 M to 1.75 × 10-3 M) with a low limit of detection (65 nM). The avidin residues present in MWCNTs-Av/RuNPs hybrid nanomaterial allowed the anchoring by bioaffinity of biotinylated glucose oxidase (biot-GOx) as proof-of-concept of the analytical application of MWCNTs-Av platform for biosensors development. The resulting nanoarchitecture behaves as a bienzymatic-like glucose biosensor with a competitive analytical performance: linear range between 2.0 × 10-5 M and 1.23 × 10-3 M, sensitivity of (0.343 ±â€¯0.002) µA mM-1 or (2.60 ±â€¯0.02) µA mM-1 cm-2, detection limit of 3.3 µM, and reproducibility of 5.2% obtained with five different GCE/MWCNTs-Av/RuNPs/biot-GOx bioplatforms prepared the same day using the same MWCNTs-Av dispersion, and 9.1% obtained with nine biosensors prepared in different days with nine different MWCNTs-Av dispersions. The average concentrations of glucose in Gatorade®, Red bull® and Pepsi® with the biosensor demonstrated excellent agreement with those reported in the commercial beverages.


Assuntos
Avidina/química , Técnicas Biossensoriais/métodos , Nanopartículas/química , Nanotubos de Carbono/química , Rutênio/química , Aspergillus niger/enzimologia , Bebidas/análise , Materiais Biomiméticos/química , Biotinilação , Catálise , Técnicas Eletroquímicas/métodos , Glucose/análise , Glucose Oxidase/química , Peróxido de Hidrogênio/análise , Limite de Detecção , Nanopartículas/ultraestrutura , Nanotubos de Carbono/ultraestrutura , Peroxidase/química
8.
Mater Sci Eng C Mater Biol Appl ; 105: 110069, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31546439

RESUMO

The development of biosensing interfaces based on copolymerization of benzenamine-2,5-di(thienyl)pyrrole (SNS-An) with 3,4-ethylenedioxythiophene (EDOT) is reported. Both homopolymer P(SNS-An) and copolymer P(SNS-An-co-EDOT) films were prepared and evaluated, in terms of biosensing efficiency, upon incorporation of carbon nanoelements (carbon nanotubes and fullerene) and cross-linking of glucose oxidase. The copolymer revealed superior performance as a biosensing interface as compared to the homopolymer structure or previously reported P(SNS) biosensors. The analytical characteristics and stability studies were performed both at cathodic potential, monitoring O2 consumption, as a result of catalytic reaction of glucose oxidase towards glucose and at anodic potential, following the oxidation of the H2O2 produced during the catalytic reaction. Whilst the measurements on the positive side offered an extended linear range (0.01-5.0 mM), the negative side provided sensitivity up to 104.96 µA/mMcm-1 within a shorter range. Detection limits were as low as 1.9 µM with Km value of 0.49 mM. Lastly, the most performant biosensing platforms, including copolymeric structure and CNTs were employed for analysis in real samples.


Assuntos
Aspergillus niger/enzimologia , Técnicas Biossensoriais , Enzimas Imobilizadas/química , Fulerenos/química , Proteínas Fúngicas/química , Glucose Oxidase/química , Glucose/análise , Nanotubos de Carbono/química , Polímeros , Pirróis
9.
J Dairy Sci ; 102(12): 10925-10933, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31563320

RESUMO

The objective of this experiment was to evaluate the effects of treating whole-plant corn at harvest with various doses of an exogenous acidic protease on fermentation and changes in nutritive value after a short period (45 d) of ensiling. Whole-plant corn (37% dry matter) was chopped and treated with 0, 20, 200, 1,000, or 2,000 mg of protease/kg of wet forage. Forages (~500 g) were packed in bag silos and ensiled at 22 to 23°C for 45 d. Data were analyzed as a 5 × 2 factorial arrangement of treatments with the main effects of the dose of protease, day of ensiling, and their interaction. Treatment with protease did not alter the concentrations of dry matter, neutral detergent fiber, acid detergent fiber, starch, lactic acid, or acetic acid compared with untreated silage, with the exception that the concentration of starch was lower in silage treated with 20 mg of protease/kg compared with untreated silage. However, the 2 highest doses of protease resulted in silages with higher concentrations of ethanol and more yeasts compared with untreated silage. Protease treatment did not affect the ruminal in vitro digestibility of neutral detergent fiber. Concentrations of soluble protein (percentage of crude protein) increased after ensiling for all treatments but was not different between silage treated with the lowest dose of protease and untreated silage. Soluble protein increased in a dose-dependent manner above the low dose of protease in silages. Concentrations of NH3-N were higher only in silages treated with the 2 highest doses of protease compared with untreated silage. Silages treated with the 3 highest doses of protease were higher in ruminal in vitro digestibility of starch compared with untreated silage but were similar to each other. The concentrations of total AA were determined in fresh forage and silages for the untreated and 200 and 2,000 mg/kg doses of protease. Neither amount of added protease affected the total concentrations of essential, nonessential, or total AA in silage. However, of the essential AA, treatment with protease resulted in silages with lower concentrations of lysine and arginine but higher concentrations of leucine compared with untreated silage. The 200 mg/kg dose of protease substantially improved ruminal in vitro starch digestion in corn silage after a short period of ensiling without affecting concentrations or numbers of ethanol and yeasts, respectively.


Assuntos
Ração Animal , Bovinos/metabolismo , Aditivos Alimentares/farmacologia , Peptídeo Hidrolases/farmacologia , Silagem , Zea mays , Animais , Aspergillus niger/enzimologia , Fermentação , Valor Nutritivo , Rúmen/metabolismo , Silagem/análise , Zea mays/metabolismo
10.
Bioprocess Biosyst Eng ; 42(12): 1993-2005, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31414183

RESUMO

Inulinases are used for the production of high-fructose syrup and fructooligosaccharides, and are widely utilized in food and pharmaceutical industries. In this study, different carbon sources were screened for inulinase production by Aspergillus niger in shake flask fermentation. Optimum working conditions of the enzyme were determined. Additionally, some properties of produced enzyme were determined [activation (Ea)/inactivation (Eia) energies, Q10 value, inactivation rate constant (kd), half-life (t1/2), D value, Z value, enthalpy (ΔH), free energy (ΔG), and entropy (ΔS)]. Results showed that sugar beet molasses (SBM) was the best in the production of inulinase, which gave 383.73 U/mL activity at 30 °C, 200 rpm and initial pH 5.0 for 10 days with 2% (v/v) of the prepared spore solution. Optimum working conditions were 4.8 pH, 60 °C, and 10 min, which yielded 604.23 U/mL, 1.09 inulinase/sucrase ratio, and 2924.39 U/mg. Additionally, Ea and Eia of inulinase reaction were 37.30 and 112.86 kJ/mol, respectively. Beyond 60 °C, Q10 values of inulinase dropped below one. At 70 and 80 °C, t1/2 of inulinase was 33.6 and 7.2 min; therefore, inulinase is unstable at high temperatures, respectively. Additionally, t1/2, D, ΔH, ΔG values of inulinase decreased with the increase in temperature. Z values of inulinase were 7.21 °C. Negative values of ΔS showed that enzymes underwent a significant process of aggregation during denaturation. Consequently, SBM is a promising carbon source for inulinase production by A. niger. Also, this is the first report on the determination of some properties of A. niger A42 (ATCC 204,447) inulinase.


Assuntos
Aspergillus niger/enzimologia , Carbono/química , Meios de Cultura/química , Glicosídeo Hidrolases/biossíntese , Sacarase/biossíntese , Tampões (Química) , Fermentação , Glucose/química , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Cinética , Açúcares , Temperatura , Termodinâmica
11.
J Agric Food Chem ; 67(37): 10392-10400, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31461615

RESUMO

The specificity of fructooligosaccharides as prebiotics depends on their size and structure, which in turn depend on their origin or the synthesis procedure. In this work we describe the application of an inulosucrase (IslA) from Leuconostoc citreum CW28 to produce high molecular weight inulin from sucrose alongside a commercial endoinulinase (Novozym 960) produced by Aspergillus niger for a simultaneous or sequential reaction to synthesize fructooligosaccharides (FOS). The simultaneous reaction resulted in a higher substrate conversion and a wide diversity of FOS when compared to the sequential reaction. A shotgun MS analysis of the commercial endoinulinase preparation surprisingly revealed an additional enzymatic activity: a fructosyltransferase, responsible for the synthesis of FOS from sucrose. Consequentially, the range of FOS obtained in reactions combining inulosucrase from Ln. citreum with the fructosyltransferase and endoinulinase from A. niger with sucrose as substrate may be extended and regulated.


Assuntos
Proteínas de Bactérias/química , Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Hexosiltransferases/química , Inulina/química , Leuconostoc/enzimologia , Oligossacarídeos/química , Aspergillus niger/enzimologia , Biocatálise , Sacarose/química
12.
Anal Chim Acta ; 1079: 164-170, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31387707

RESUMO

Ultrathin two-dimensional (2D) metal-organic framework (MOF) nanosheets have attracted numerous attention due to their unparalleled advantages such as unique structures, large surface areas, good stability and high catalytic efficiency. In this paper, 2D Cu(bpy)2(OTf)2 nanosheets (Cu-MOF, bpy = 4,4-bipyridine, OTf = trifluoromethanesulfonate) are first utilized to achieve the fluorescent detection for H2O2 and glucose with their intrinsic peroxidase-like activity. The fluorescent biosensor Cu-MOF nanosheets were achieved by the efficient catalysis of non-fluorescent thiamine (TH) to strong fluorescent thiochrome in the presence of H2O2. With the combination of glucose oxidase, highly selective and sensitive fluorescent detection for glucose could be established with a detection limit of 0.41 µM and two separate linear ranges of 10-100 µM and 100-1000 µM, respectively. Furthermore, the developed method has been applied to human serum for the quantitative determination of glucose as a clinical diagnosis indicator of diabetes mellitus.


Assuntos
Materiais Biomiméticos/química , Glicemia/análise , Peróxido de Hidrogênio/análise , Estruturas Metalorgânicas/química , Aspergillus niger/enzimologia , Materiais Biomiméticos/síntese química , Técnicas Biossensoriais/métodos , Cobre/química , Fluorescência , Glucose Oxidase/química , Peroxidase do Rábano Silvestre/química , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Estruturas Metalorgânicas/síntese química , Espectrometria de Fluorescência/métodos , Tiamina/análogos & derivados , Tiamina/química
13.
Mikrochim Acta ; 186(9): 616, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31407097

RESUMO

Glucose oxidase was soaked into a porous carbon nanotube film coating on a platinum disk electrode, then trapped beneath a topcoat of electrodeposition paint. The resulting sensors, operated at a potential of +0.6 V (vs. Ag/AgCl), produced a glucose signal that was linear up to 40 mM, with a 50 µM detection limit. Signal stability over >100 h of continuous operation in a flow cell showed the remarkable functional durability of the biosensor, and confirmed that the electropaint coating effectively prevented loss of the enzyme. This performance is deemed to derive from the minimalistic immobilization layer design and the prevention of protein leakage. The immobilization method has a potentially wide scope, in that it may also be applicable in other types of enzymatic biosensor. Graphical abstract Illustration of an enzyme biosensor design that uses glucose oxidase in bare carbon nanotube electrode modifications with electropaint topcoat for amperometric glucose quantification. Immobilization matrix supplementation with extra functional (nano-) materials was unnecessary for high-quality and stable analysis performance.


Assuntos
Técnicas Biossensoriais/métodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Glucose/análise , Nanotubos de Carbono/química , Aspergillus niger/enzimologia , Técnicas Biossensoriais/instrumentação , Glicemia/análise , Calibragem , Eletroquímica , Eletrodos , Humanos , Limite de Detecção , Porosidade
14.
Bioprocess Biosyst Eng ; 42(11): 1843-1852, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31399865

RESUMO

With the advent of modern genetic engineering methods, microcultivation systems have become increasingly important tools for accelerated strain phenotyping and bioprocess engineering. While these systems offer sophisticated capabilities to screen batch processes, they lack the ability to realize fed-batch processes, which are used more frequently in industrial bioprocessing. In this study, a novel approach to realize a feedback-regulated enzyme-based slow-release system (FeedER), allowing exponential fed-batch for microscale cultivations, was realized by extending our existing Mini Pilot Plant technology with a customized process control system. By continuously comparing the experimental growth rates with predefined set points, the automated dosage of Amyloglucosidase enzyme for the cleavage of dextrin polymers into D-glucose monomers is triggered. As a prerequisite for stable fed-batch operation, a constant pH is maintained by automated addition of ammonium hydroxide. We show the successful application of FeedER to study fed-batch growth of different industrial model organisms including Corynebacterium glutamicum, Pichia pastoris, and Escherichia coli. Moreover, the comparative analysis of a C. glutamicum GFP producer strain, cultivated under microscale batch and fed-batch conditions, revealed two times higher product yields under slow growing fed-batch operation. In summary, FeedER enables to run 48 parallel fed-batch experiments in an automated and miniaturized manner, and thereby accelerates industrial bioprocess development at the screening stage.


Assuntos
Aspergillus niger/enzimologia , Corynebacterium glutamicum/crescimento & desenvolvimento , Dextrinas/química , Escherichia coli K12/crescimento & desenvolvimento , Proteínas Fúngicas/química , Glucana 1,4-alfa-Glucosidase/química , Glucose , Pichia/crescimento & desenvolvimento , Glucose/química , Glucose/metabolismo
15.
Int J Biol Macromol ; 139: 199-212, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31374272

RESUMO

In the pursuit of industrial aspartic proteases, aspergillopepsin A-like endopeptidase from the fungi Aspergillus niger, was identified and cultured by solid state fermentation. Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity. The enzyme was found to have single polypeptide chain with a molecular mass of 50 ±â€¯0.5 kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60 °C respectively. The enzyme was stable for 60 min at 50 °C. The purified enzyme had specific activity of 40,000 ±â€¯1800 U/mg. The enzyme had 85% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a Ki value of 0.045 µM. Based on homology modeling and predicted secondary structure, it was inferred that the aspartic protease is rich in ß-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. Degree of hydrolysis of commercial substrates indicated the order of cleaving ability of the enzyme to be hemoglobin > defatted soya flour > gluten > gelatin > skim milk powder. The enzyme also improved the functional characteristics of defatted soya flour. This aspartic protease was found to be an excellent candidate for genetic manipulation for biotechnological application in food and feed industries, due to its high catalytic turn over number and thermostability.


Assuntos
Ácido Aspártico Proteases/química , Aspergillus niger/enzimologia , Pepstatinas/química , Inibidores de Proteases/química , Ácido Aspártico Proteases/antagonistas & inibidores , Ácido Aspártico Proteases/isolamento & purificação , Ácido Aspártico Proteases/metabolismo , Aspergillus niger/classificação , Aspergillus niger/genética , Catálise , Cromatografia Líquida , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Pepstatinas/farmacologia , Filogenia , Inibidores de Proteases/farmacologia , Ligação Proteica , Relação Estrutura-Atividade , Espectrometria de Massas em Tandem , Temperatura
16.
Sensors (Basel) ; 19(13)2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31277338

RESUMO

The inhibition effect of the selected heavy metals (Ag+, Cd2+, Cu2+, and Hg2+) on glucose oxidase (GOx) enzyme from Aspergillus niger (EC 1.1.3.4.) was studied using a new amperometric biosensor with an electrochemical transducer based on a glassy carbon electrode (GCE) covered with a thin layer of multi-wall carbon nanotubes (MWCNTs) incorporated with ruthenium(IV) oxide as a redox mediator. Direct adsorption of multi-wall carbon nanotubes (MWCNTs) and subsequent covering with Nafion® layer was used for immobilization of GOx. The analytical figures of merit of the developed glucose (Glc) biosensor are sufficient for determination of Glc in body fluids in clinical analysis. From all tested heavy metals, mercury(II) has the highest inhibition effect. However, it is necessary to remember that cadmium and silver ions also significantly inhibit the catalytic activity of GOx. Therefore, the development of GOx biosensors for selective indirect determination of each heavy metal still represents a challenge in the field of bioelectroanalysis. It can be concluded that amperometric biosensors, differing in the utilized enzyme, could find their application in the toxicity studies of various poisons.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas/métodos , Inibidores Enzimáticos/toxicidade , Glucose Oxidase/antagonistas & inibidores , Metais Pesados/toxicidade , Aspergillus niger/enzimologia , Calibragem , Técnicas Eletroquímicas/instrumentação , Eletrodos , Inibidores Enzimáticos/farmacologia , Glucose/análise , Glucose Oxidase/metabolismo , Peróxido de Hidrogênio/análise , Limite de Detecção , Metais Pesados/farmacologia , Nanotubos de Carbono , Compostos de Rutênio/química
17.
Bioelectrochemistry ; 130: 107325, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31295700

RESUMO

In order to understand how energy metabolism adapts to changes in neuronal activity it is imperative to perform direct measurements of the flux of glucose (and other metabolites) in brain tissue. Metabolic studies using brain slice preparations are attractive due to the controllability of recording conditions, absence of anesthetic interference and refined animal experimental protocols. In this work, taking advantage of the small size and versatility of carbon fiber microelectrodes (CFMs), we aimed to develop an amperometric glucose microbiosensor suitable for glucose measurement in brain slices. Potentiostatic- or galvanostatic-driven platinum electrodeposition was used to improve the analytical properties of CFMs towards detection of hydrogen peroxide. The platinized CFMs served as platform for the development of glucose microbiosensors through the immobilization of glucose-oxidase (GOx) by cross-linking with glutaraldehyde in the presence of BSA. Selective glucose measurements were attained by modifying the electrode with a permselective layer of meta-phenylenediamine and by integrating a null sensor. The in vitro characterization studies support the good analytical features of the CFM/Pt-based microbiosensors to reliably measure glucose in brain tissue. The ex vivo experiments in rodent hippocampal slices validated their suitability to measure evoked changes in extracellular glucose. This approach, encompassing the use of null sensor to cross-check the selectivity on a moment-to-moment basis, allowed us to provide the temporal and quantitative profile of extracellular glucose changes in hippocampal slices following a spreading depolarization event. Overall, these results support the potential of these microbiosensors to be used as a valuable tool to investigate the complex nature of glucose utilization in brain tissue linked to neuronal activation both in physiological and pathological conditions.


Assuntos
Técnicas Biossensoriais/métodos , Fibra de Carbono/química , Glucose/análise , Platina/química , Animais , Aspergillus niger/enzimologia , Encéfalo/metabolismo , Química Encefálica , Galvanoplastia , Enzimas Imobilizadas/química , Glucose/metabolismo , Glucose Oxidase/química , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Microeletrodos , Ratos Wistar
18.
Nanoscale ; 11(39): 18081-18089, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31343649

RESUMO

Remote modulation of nanoscale biochemical processes in a living system using magnetic stimulation is appealing but is restricted by the lack of a highly efficient nanomediator which can deliver timely and effective response to biological molecules under an external magnetic field. Herein, we report the development of a novel nanocatalyst based on a ferrimagnetic vortex-domain nanoring (FVIO)-enzyme hybrid that enables real-time modulation of enzymatic catalysis under an alternating magnetic field (AMF). The role of the FVIO is to provide localized heating immediately upon exposure to an AMF, which efficiently and selectively promotes the activity of conjugated enzymes on the surface. The reaction rate of the as-fabricated FVIO-ß-Gal hybrid was shown to be boosted up to 180% of its initial value by localized heat generated under an AMF of 550 Oe in less than 2 s and without heating up the bulk solution. Moreover, the degree of activity acceleration was shown to be tunable by increasing the strength of the AMF. The concept of remote magnetic stimulation of enzymatic reactions has been further applied to other enzymes (e.g. FVIO-KPC and FVIO-GOx), demonstrating the general applicability of this strategy. Since almost all metabolic processes in cells rely on enzymatic catalysis to sustain life, the FVIO-enzyme system developed in this work provides a valuable nanoplatform for spatiotemporally manipulating biochemical reactions, which might pave the way for future remote manipulation of living organisms.


Assuntos
Aspergillus niger/enzimologia , Enzimas Imobilizadas/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas Fúngicas/química , Glucose Oxidase/química , Campos Magnéticos , Nanoestruturas/química , beta-Galactosidase/química , Catálise
19.
Int J Biol Macromol ; 138: 234-243, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31315021

RESUMO

Polygalacturonase (PG) from Aspergillus niger was immobilized using glyoxyl, vinylsulfone or glutaraldehyde-activated supports. The use of supports pre-activated with glutaraldehyde presented the best results. The immobilization of PG on glutaraldehyde-supports was studied under different conditions: at pH 5 for 24 h; at pH 5, 6.5 or 8 for 3 h and then incubated at pH 8 for 24 h; at pH 8 in the presence of 300 mM NaCl for 24 h, to prevent ion exchange. The immobilization under all conditions showed a significant increase in the enzyme thermal stability under inactivation conditions at pH 4-10. As a result, at temperatures over 70 °C or pH values over 7, the immobilized PG maintained significant levels of activity while the free PG was fully inactivated. The immobilization conditions presented a clear effect on enzyme activity, thermostability and operational stability, suggesting that the different conditions permitted to get immobilized PG having different orientations. Varying the immobilization protocol it is possible to achieve high activity or stability, and the optimal biocatalyst depends on the conditions where it will be utilized. The immobilized PG biocatalysts could be reused 10 times without a significant decrease in enzyme activity and offered very linear reaction courses.


Assuntos
Aspergillus niger/enzimologia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Poligalacturonase/química , Poligalacturonase/metabolismo , Aldeídos/química , Biocatálise , Celulose/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Glioxilatos/química , Concentração de Íons de Hidrogênio , Microesferas , Pectinas/metabolismo , Sefarose/química
20.
Nat Commun ; 10(1): 2826, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249381

RESUMO

Bio-catalytic micro- and nanomotors self-propel by the enzymatic conversion of substrates into products. Despite the advances in the field, the fundamental aspects underlying enzyme-powered self-propulsion have rarely been studied. In this work, we select four enzymes (urease, acetylcholinesterase, glucose oxidase, and aldolase) to be attached on silica microcapsules and study how their turnover number and conformational dynamics affect the self-propulsion, combining both an experimental and molecular dynamics simulations approach. Urease and acetylcholinesterase, the enzymes with higher catalytic rates, are the only enzymes capable of producing active motion. Molecular dynamics simulations reveal that urease and acetylcholinesterase display the highest degree of flexibility near the active site, which could play a role on the catalytic process. We experimentally assess this hypothesis for urease micromotors through competitive inhibition (acetohydroxamic acid) and increasing enzyme rigidity (ß-mercaptoethanol). We conclude that the conformational changes are a precondition of urease catalysis, which is essential to generate self-propulsion.


Assuntos
Acetilcolinesterase/química , Frutose-Bifosfato Aldolase/química , Glucose Oxidase/química , Nanoestruturas/química , Urease/química , Animais , Aspergillus niger/enzimologia , Biocatálise , Canavalia/enzimologia , Electrophorus , Enzimas Imobilizadas/química , Proteínas de Peixes/química , Proteínas Fúngicas/química , Cinética , Proteínas de Plantas/química , Conformação Proteica , Coelhos , Dióxido de Silício/química
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