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1.
Mar Drugs ; 17(7)2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31277207

RESUMO

The purpose of the present study is to improve the endothelial progenitor cells (EPC) activation, proliferation, and angiogenesis using enzyme-aided extraction of fucoidan by amyloglucosidase (EAEF-AMG). Enzyme-aided extraction of fucoidan by AMG (EAEF-AMG) significantly increased EPC proliferation by reducing the reactive oxygen species (ROS) and decreasing apoptosis. Notably, EAEF-AMG treated EPCs repressed the colocalization of TSC2/LAMP1 and promoted perinuclear localization of mTOR/LAMP1 and mTOR/Rheb. Moreover, EAEF-AMG enhanced EPC functionalities, including tube formation, cell migration, and wound healing via regulation of AKT/Rheb signaling. Our data provided cell priming protocols to enhance therapeutic applications of EPCs using bioactive compounds for the treatment of CVD.


Assuntos
Células Progenitoras Endoteliais/efeitos dos fármacos , Glucana 1,4-alfa-Glucosidase/metabolismo , Polissacarídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Progenitoras Endoteliais/metabolismo , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Cicatrização/efeitos dos fármacos
2.
Food Chem ; 300: 125192, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31362158

RESUMO

Dietary fiber has several benefits for humans, and the development of healthier rice with an improved dietary fiber composition has attracted increasing amounts of attention. Based on the method of AOAC 2002.02, we developed a simplified method to screen polished rice containing high total dietary fiber (TDF). Mutant cw with a high TDF content could be distinguished easily from R7954 (indica) and Nipponbare (japonica) by the digestion-resistant phenotype, which is characterized as an almost intact grain after hydrolysis by pepsin, pancreatic α-amylase and amyloglucosidase. The individuals identified from the F2 population showed digestion resistance all had TDF content higher than 5%, while those without a digestion-resistant phenotype had TDF content lower than 5%. The phenotype of digestion resistance could be a valuable index for identifying rice with higher TDF content, and the identification of this phenotype provides a simplified, economical and high throughput method for high TDF rice breeding.


Assuntos
Fibras na Dieta/análise , Análise de Alimentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Oryza/química , Culinária , Fibras na Dieta/metabolismo , Digestão , Glucana 1,4-alfa-Glucosidase/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Humanos , Hidrólise , Pepsina A/química , Pepsina A/metabolismo , Sementes/química , Amido/análise , Amido/química , alfa-Amilases/química , alfa-Amilases/metabolismo
3.
Biosci Biotechnol Biochem ; 83(8): 1385-1401, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31159661

RESUMO

The koji mold Aspergillus oryzae has been used in traditional Japanese food and beverage fermentation for over a thousand years. Amylolytic enzymes are important in sake fermentation, wherein production is induced by starch or malto-oligosaccharides. This inducible production requires at least two transcription activators, AmyR and MalR. Among amylolytic enzymes, glucoamylase GlaB is produced exclusively in solid-state culture and plays a critical role in sake fermentation owing to its contribution to glucose generation from starch. A recent study demonstrated that glaB gene expression is regulated by a novel transcription factor, FlbC, in addition to AmyR in solid-state culture. Amylolytic enzyme production is generally repressed by glucose due to carbon catabolite repression (CCR), which is mediated by the transcription factor CreA. Modifying CCR machinery, including CreA, can improve amylolytic enzyme production. This review focuses on the role of transcription factors in regulating A. oryzae amylolytic gene expression.


Assuntos
Aspergillus oryzae/genética , Regulação Fúngica da Expressão Gênica , Glucana 1,4-alfa-Glucosidase/metabolismo , Proteínas Fúngicas/genética , Maltose/metabolismo , Fatores de Transcrição/metabolismo
4.
Acta Biochim Biophys Sin (Shanghai) ; 51(6): 638-644, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31081016

RESUMO

The purpose of this study was to obtain an engineered Aspergillus niger strain with high glucoamylase activity by overexpressing the glucoamylase gene glaA and α-amylase gene amyA in A. niger CICC2462. Three recombinant strains containing a single copy of amyA (1A), containing two copies of amyA (2A), and coexpressing amyA and glaA (AG), respectively, were constructed. The transcript levels of amyA in 1A and 2A were increased by 2.95 folds and 3.09 folds, respectively. The levels of amyA and glaA in AG were increased by 1.21 folds and 2.86 folds, but the maximum extracellular glucoamylase activities did not differ significantly. In addition, after 1% casein phosphopeptides (CPPs) was added to the fermentation medium, the maximum extracellular glucoamylase activities for strains 1A, 2A, and AG were 35,200, 37,300, and 40,710 U/ml, respectively, which were significantly higher than that of the parental strain CICC2462 (28,250 U/ml), while CPPs alone had no effect on the parental strain CICC2462. We demonstrate that overexpression of amyA and glaA substantially increases the expression and secretion of glucoamylase in A. niger, and CPPs effectively improves the yield of glucoamylase in recombinant A. niger strains overexpressing amyA and glaA. The newly developed strains and culture methods may have extensive industrial applications.


Assuntos
Aspergillus niger/genética , Proteínas Fúngicas/genética , Glucana 1,4-alfa-Glucosidase/genética , alfa-Amilases/genética , Aspergillus niger/enzimologia , Aspergillus niger/metabolismo , Caseínas/metabolismo , Caseínas/farmacologia , Fermentação/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Engenharia Genética/métodos , Glucana 1,4-alfa-Glucosidase/metabolismo , Fosfopeptídeos/metabolismo , Fosfopeptídeos/farmacologia , alfa-Amilases/metabolismo
5.
PLoS Pathog ; 15(4): e1007729, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31002734

RESUMO

The use of host nutrients to support pathogen growth is central to disease. We addressed the relationship between metabolism and trophic behavior by comparing metabolic gene expression during potato tuber colonization by two oomycetes, the hemibiotroph Phytophthora infestans and the necrotroph Pythium ultimum. Genes for several pathways including amino acid, nucleotide, and cofactor biosynthesis were expressed more by Ph. infestans during its biotrophic stage compared to Py. ultimum. In contrast, Py. ultimum had higher expression of genes for metabolizing compounds that are normally sequestered within plant cells but released to the pathogen upon plant cell lysis, such as starch and triacylglycerides. The transcription pattern of metabolic genes in Ph. infestans during late infection became more like that of Py. ultimum, consistent with the former's transition to necrotrophy. Interspecific variation in metabolic gene content was limited but included the presence of γ-amylase only in Py. ultimum. The pathogens were also found to employ strikingly distinct strategies for using nitrate. Measurements of mRNA, 15N labeling studies, enzyme assays, and immunoblotting indicated that the assimilation pathway in Ph. infestans was nitrate-insensitive but induced during amino acid and ammonium starvation. In contrast, the pathway was nitrate-induced but not amino acid-repressed in Py. ultimum. The lack of amino acid repression in Py. ultimum appears due to the absence of a transcription factor common to fungi and Phytophthora that acts as a nitrogen metabolite repressor. Evidence for functional diversification in nitrate reductase protein was also observed. Its temperature optimum was adapted to each organism's growth range, and its Km was much lower in Py. ultimum. In summary, we observed divergence in patterns of gene expression, gene content, and enzyme function which contribute to the fitness of each species in its niche.


Assuntos
Proteínas Fúngicas/genética , Glucana 1,4-alfa-Glucosidase/metabolismo , Nutrientes/metabolismo , Phytophthora/genética , Doenças das Plantas/parasitologia , Tubérculos/metabolismo , Solanum tuberosum/metabolismo , Adaptação Fisiológica , Evolução Molecular , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Interações Hospedeiro-Parasita/genética , Phytophthora/classificação , Phytophthora/fisiologia , Doenças das Plantas/genética , Tubérculos/crescimento & desenvolvimento , Tubérculos/parasitologia , Solanum tuberosum/crescimento & desenvolvimento , Solanum tuberosum/parasitologia
6.
Carbohydr Polym ; 212: 260-269, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30832856

RESUMO

Glucoamylolysis of maize starch at 55 °C has been studied by means of scanning electron microscopy (SEM), wide-angle X-ray diffraction spectroscopy (WAXD), and differential scanning calorimetry (DSC). It was found that hydrolysis is accompanied by changes in thermodynamic parameters of diluted aqueous dispersions of partially hydrolyzed starches. Such changes are ensured by two processes directly from hydrolysis and accompanying annealing. At relatively low degrees of hydrolysis (less than 30%), changes in thermodynamic parameters are mainly controlled by annealing. At the same time, at high degrees of hydrolysis (more than 40%), the main contribution to changes in thermodynamic parameters of partially hydrolyzed starch granules is due to the hydrolysis itself. It has been established that the main controlling parameter is the thickness of crystalline lamellae Lcrl, which, when annealed, increases, but tends to decrease at deeper glucoamylolisis. It has been established that the thickness Lcrl of crystalline lamellae, which increases with annealing, but shows a tendency to decrease with deeper glucoamylolysis is the most representative parameter of changes in maize starch after hydrolysis.


Assuntos
Glucana 1,4-alfa-Glucosidase/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Amido/química , Amido/metabolismo , Termodinâmica , Zea mays , Fenômenos Químicos , Hidrólise , Amido/isolamento & purificação , Difração de Raios X/métodos
7.
Carbohydr Polym ; 212: 40-50, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30832873

RESUMO

The effects of amyloglucosidase digestion on the multi-scale supramolecular structural changes of native corn starch were examined by ultra-small angle neutron scattering (USANS), small angle X-ray scattering (SAXS), particle sizing, and scanning electron microscopy (SEM). Well-defined and spherical pores were formed upon amyloglucosidase digestion as revealed by SEM. The pore polydispersity was determined using USANS by assuming spherical pore morphology with log-normal distribution. Both USANS and SEM measurements demonstrated that the pores become larger and more polydisperse as the digestion time increased. Moreover, SAXS revealed that the lamellar peak area decreased gradually for both thermally and enzymatically treated starches, indicating partial loss of lamellar organisation. Overall, the results demonstrate structural changes occur on multiple length scales upon enzymatic digestion from granular to lamellar with small-angle scattering demonstrated to provide detailed characterization of the resultant microporous structures.


Assuntos
Glucana 1,4-alfa-Glucosidase/química , Difração de Nêutrons/métodos , Espalhamento a Baixo Ângulo , Amido/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Hidrólise , Estrutura Molecular , Tamanho da Partícula , Amido/metabolismo
8.
J Basic Microbiol ; 59(4): 375-384, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30681161

RESUMO

In the present study, glucoamylase produced from a soil bacterium Paenibacillus amylolyticus NEO03 was cultured under submerged fermentation conditions. The extracellular enzyme was purified by starch adsorption chromatography and further by gel filtration, with 2.73-fold and recovery of 40.02%. The protein exhibited molecular mass of ∼66,000 Da as estimated by SDS-PAGE and depicted to be a monomer. The enzyme demonstrated optimum activity at pH range 6.0-7.0 and temperature range 30-40 °C. Glucoamylase was mostly activated by Mn2+ metal ions and depicted no dependency on Ca2+ ions. The enzyme preferentially hydrolyzed all the starch substrates. High substrate specificity was demonstrated towards soluble starch and kinetic values Km and Vmax were 2.84 mg/ml and 239.2 U/ml, respectively. The products of hydrolysis of soluble starch were detected by thin layer chromatography which showed only D -glucose, indicating a true glucoamylase. The secreted glucoamylase from P. amylolyticus strain possesses properties suitable for saccharification processes such as biofuel production.


Assuntos
Glucana 1,4-alfa-Glucosidase/isolamento & purificação , Glucana 1,4-alfa-Glucosidase/metabolismo , Paenibacillus/enzimologia , Meios de Cultura , Glucana 1,4-alfa-Glucosidase/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Peso Molecular , Amido/metabolismo , Especificidade por Substrato , Temperatura Ambiente
9.
Appl Microbiol Biotechnol ; 103(5): 2367-2379, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30675636

RESUMO

High-throughput function-based screening techniques remain the major bottleneck in the novel biocatalysts development pipeline. In the present study, we customized protocols for amylolytic activity determination (Somogyi-Nelson and starch-iodine tests) to micro-volume thermalcycler-based assays (linearity range 60-600 µM of reducing sugar, R2 = 0.9855; 0-2 mg/mL of starch, R2 = 0.9921, respectively). Exploitation of a thermalcycler enabled rapid and accurate temperature control, further reduction of reagents and samples volumes, and limited evaporation of the reaction mixtures, meeting several crucial requirements of an adequate enzymatic assay. In the optimized micro-volume Somogyi-Nelson protocol, we were able to reduce the time required for high-temperature heating sixfold (down to 5 min) and further increase sensitivity of the assay (tenfold), when compared to the previous MTP-based protocol. The optimized microassays have complementary scope of specificities: micro-starch-iodine test for endoglucanases, micro-Somogyi-Nelson test for exoglucanases. Due to rapid, micro-volume and high-throughput character, the methods can complement toolbox assisting development of novel biocatalysts and analysis of saccharides-containing samples.


Assuntos
Ensaios Enzimáticos/métodos , Ensaios de Triagem em Larga Escala/métodos , alfa-Amilases/metabolismo , Celulase/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , Temperatura Alta , Amido/análogos & derivados , Amido/metabolismo
10.
Ultrason Sonochem ; 53: 77-82, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30642800

RESUMO

Recovering valuable materials from food waste by applying the concept of a bio-refinery is attracting considerable interest. To this effect, we investigated the possibility of enhancing the enzymatic hydrolysis of food waste using ultrasonic technology. The effect of pre-treating blended food waste with high-intensity ultrasound (20 kHz) on subsequent hydrolysis by glucoamylase was investigated as a function of sonication time and temperature. Particle sizing by laser diffraction, and imaging via scanning electronic microscopy showed that ultrasonic pre-treatment could reduce the particle size of the blended food waste significantly, resulting in a better interaction with the enzyme. As a consequence, the glucose yield of enzymatic hydrolysis was ∼10% higher for food waste pre-sonicated using the most intensive ultrasonication conditions studied (5 min sonication at a power of 0.8 W/mL at 20 °C) than for the untreated control. In addition, the time required to achieve high yields of glucose could be more than halved using ultrasonic pre-treatment. This could enable the hydrolysis reactor size or the enzyme usage to be reduced by more than 50%. Therefore, an ultrasound-assisted bioconversion process from food waste into a value-added product has been demonstrated.


Assuntos
Alimentos , Glucana 1,4-alfa-Glucosidase/metabolismo , Glucose/química , Ondas Ultrassônicas , Resíduos , Hidrólise , Cinética , Tamanho da Partícula , Temperatura Ambiente
11.
Spectrochim Acta A Mol Biomol Spectrosc ; 206: 613-621, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30098884

RESUMO

1-Deoxynojirimycin (DNJ), a representative polyhydroxylated alkaloids, is widely used in the field of antidiabetic, antitumor, and anti-HIV. The present study tried to clarify the interaction mechanism of DNJ with glucoamylase by multi-spectroscopic techniques, dynamic light scattering in combination with molecular modeling strategies from biophysics point of view. Fluorescence and UV-vis data indicated that fluorescence quenching mechanism of glucoamylase and DNJ was a dynamic manner. The association constant, binding site and thermodynamic parameters were also obtained from fluorescence spectrum at different temperatures. Synchronous fluorescence, circular dichroism and dynamic light scattering methods demonstrated that their interaction induced microenvironment changes around tryptophan residue and protein conformational alteration. The main driving force was hydrophobic interaction and hydrogen bonding. In addition, molecular docking study indicated that 1-deoxynojirimycin could bind in the catalytic domain of glucoamylase and interact with amino acid residues Arg78, Asp79, Glu203 and Glu424 by forming hydrogen bonds. Molecular dynamics simulation demonstrated that profiles of atomic fluctuation remained the rigidity of ligand binding site. This study elucidated the detailed interaction mechanism of DNJ with glucoamylase, which will be helpful for pharmaceutical companies to design new α-glucosidase inhibitor drugs based on polyhydroxylated alkaloids compound like DNJ.


Assuntos
1-Desoxinojirimicina/química , Glucana 1,4-alfa-Glucosidase/química , Inibidores de Glicosídeo Hidrolases/química , Análise Espectral/métodos , 1-Desoxinojirimicina/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica
12.
Food Chem ; 271: 182-186, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30236665

RESUMO

Starch granules contain an internal channel structure that can be used to encapsulate and deliver active ingredients such as nutrients, drugs, chemicals and microorganisms. Nature creates a broad range of starch granule sizes and compositions in different plants and this range of encapsulation matrices provides a great deal of commercial opportunity. Starch granules can be utilised for encapsulation in their native form or treated with amylases/amyloglucosidase to facilitate the entry of active ingredients into the granules. Interactions within the granules can be relatively passive or more structured in the form of complexes (such as amylose-lipid complexes). This mini-review evaluates this application of starch and potential commercial product opportunities.


Assuntos
Sistemas de Liberação de Medicamentos , Amido/química , Amilases , Amilose , Suplementos Nutricionais , Glucana 1,4-alfa-Glucosidase/metabolismo , Micronutrientes/administração & dosagem , Probióticos/administração & dosagem
13.
Bioresour Technol ; 272: 398-406, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30388577

RESUMO

The aim of this study was to establish a simplified operational process for lactic acid (LA) production by Bacillus coagulans IPE22 from inedible starchy biomass with open fermentation method. First, 29.47 mU/mg specific amylase activity was detected in direct batch fermentation from soluble starch, but the activity of the produced amylase was too low for effective production of LA. Then seven batches from 72 g/L soluble starch were conducted without sterilization. It was found that one step simultaneous liquefaction, saccharification and fermentation (SLSF) with the addition of mesothermal α-amylase and glucoamylase was the optimal mode with LA concentration, yield and productivity of 68.72 g/L, 0.99 g/g and 1.72 g/L h respectively. Finally, inedible starchy biomass, cassava and sorghum flours, were proved to be alternatives to refined soluble starch. For the first time, one step open SLSF of inedible starchy biomass was reported for LA production by B. coagulans.


Assuntos
Bacillus coagulans/metabolismo , Biomassa , Fermentação , Ácido Láctico/biossíntese , Glucana 1,4-alfa-Glucosidase/metabolismo , Amido/metabolismo
14.
Int J Biol Macromol ; 126: 1125-1132, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30594622

RESUMO

The aim of the present work was to prepare high loading capacity carriers for immobilizing glucoamylase. Different sizes of chitosan based particles were successfully prepared by different methods to evaluate the performance in immobilization. Chitosan particles on millimeter size were prepared by dripping granulation method, chitosan covered magnetic nanoparticles and chitosan mixted graphene oxide nanosheets covered magnetic nanoparticles were synthesized by one-step method, chitosan-glucoamylase nanoparticles were synthesized by ionic cross linking method with Sodium tripolyphosphate. These particles were characterized by SEM, TEM, FTIR and DLS analysis. The performance of the immobilized enzyme was also investigated. The results showed that the loading capacity was greatly increased on chitosan based nanoparticles. The reaction conditions of immobilized enzyme were optimized, the reusability and storage stability was also investigated. The results showed the pH durance and storage stability of the immobilized enzyme on nanosize particles were enhanced.


Assuntos
Quitosana/química , Enzimas Imobilizadas/metabolismo , Nanopartículas/química , Difusão Dinâmica da Luz , Glucana 1,4-alfa-Glucosidase/metabolismo , Nanopartículas/ultraestrutura , Tamanho da Partícula , Espectroscopia de Infravermelho com Transformada de Fourier
15.
Curr Top Med Chem ; 18(27): 2327-2337, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30499400

RESUMO

Diabetes mellitus type 2 (DMT2) is a metabolic disease characterized by a chronic increase in glycemia that promotes several long-term complications and high mortality. Some enzymes involved in glycaemic control, such as α -(1,4)-glucosidase, have now been established as novel pharmacological targets. Coumarins have shown benefits in attenuating signs and complications of DMT2, including inhibition of this enzyme. In this work, new synthetic coumarins (bearing different amide and aryl substituents) were studied in vitro as inhibitors of α-(1,4)-glucosidase. Among them, five molecules proved to be excellent α-(1,4)-glucosidase inhibitors, being compound 7 (IC50 = 2.19 µM) about 200 times more potent than acarbose, a drug currently used for the treatment of DMT2. In addition, most of the coumarins presented uncompetitive inhibition for the α-(1,4)-glucosidase. Molecular docking studies revealed that coumarins bind to the active site of the enzyme in a more external area comparing to the substrate, without interfering with it, and displaying aromatic and hydrophobic interactions, as well as some hydrogen bonds. According to the results, aromatic interactions with two phenylalanine residues, 157 and 177, were the most common among the studied coumarins. This study is a step forward for the understanding of coumarins as potential anti-diabetic compounds displaying α-(1,4)-glucosidase inhibition.


Assuntos
Cumarínicos/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Glucana 1,4-alfa-Glucosidase/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/farmacologia , Cumarínicos/química , Relação Dose-Resposta a Droga , Glucana 1,4-alfa-Glucosidase/metabolismo , Inibidores de Glicosídeo Hidrolases/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
16.
Ultrason Sonochem ; 49: 128-136, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30100267

RESUMO

The present report studied the role of ultrasound (US) energy in the amyloglucosidase-based starch hydrolysis using two complementary approaches: (i) in the activity of six commercially-available amyloglucosidases (using soluble starch as substrate), and (ii) in the hydrolysis of four pure starches from different botanical sources. This corresponds to the first systematic evaluation of the role of US in starch hydrolysis mediated by amyloglucosidase, being a consequence of our previous report that assessed the effect of US in the activity of alpha-amylase (LWT - Food Science and Technology 84 (2017) 674-685). Regarding amlyloglucosidases, three enzymes obtained from Aspergillus niger (AN1-AN3), and Spirizyme Achieve (SPA), Spirizyme Fuel (SPF) and Spirizyme Ultra (SPU) were submitted to a Box-Behnken experimental design in order to establish the optimum conditions for their maximum activity. In the presence of US, we found both inactivation and activation, ranging from -88% (AN3) to 699% (SPA). The US promoted the enzyme activity when combined with lower temperatures (40-60 °C), with a marked effect in Spirizyme enzymes. Based on the optimum conditions established by the experimental design, we also evaluated the role of US in the glucose yield resulting from the hydrolysis of pure starches (corn, rice, potato, wheat). In this case, US led to higher glucose yields in all conditions tested. The enhancement factors observed ranged from 1.2 (AN1, rice starch) to 65 (SPA, potato starch) times. We compared these findings with previous reports, which highlighted the role of US in intensifying amyloglucosidase-based saccharification in mild conditions, by simultaneously influencing both enzyme and substrate. Hence, US power has to be fine-tuned for each particular enzyme in order to maximize process intensification.


Assuntos
Glucana 1,4-alfa-Glucosidase/metabolismo , Amido/química , Ondas Ultrassônicas , Aspergillus niger/enzimologia , Biocatálise , Glucose/química , Hidrólise
17.
FEMS Yeast Res ; 18(7)2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30085077

RESUMO

Cost-effective consolidated bioprocessing (CBP) of raw starch for biofuel production requires recombinant Saccharomyces cerevisiae strains expressing α-amylases and glucoamylases. Native Aureobasidium pullulans apuA, Aspergillus terreus ateA, Cryptococcus sp. S-2 cryA and Saccharomycopsis fibuligera sfiA genes encoding raw-starch α-amylases were cloned and expressed in the S. cerevisiae Y294 laboratory strain. Recombinant S. cerevisiae Y294[ApuA] and Y294[AteA] strains produced the highest extracellular α-amylase activities (2.17 U mL-1 and 2.98 U mL-1, respectively). Both the ApuA and AteA α-amylases displayed a preference for pH 4 to 5 and retained more than 75% activity after 5 days at 30°C. When ateA was co-expressed with the previously reported Aspergillus. tubingensis glucoamylase gene (glaA), the amylolytic S. cerevisiae Y294[AteA-GlaA] strain produced 45.77 g L-1 ethanol after 6 days. Ethanol production by this strain was improved with the addition of either 2.83 µL STARGEN 002 (54.54 g L-1 ethanol and 70.44% carbon conversion) or 20 µL commercial glucoamylase from Sigma-Aldrich (73.80 g L-1 ethanol and 90.19% carbon conversion). This is the first report of an engineered yeast strain that can replace up to 90% of the enzymes required for raw starch hydrolysis, and thus contributes to the realisation of a CBP yeast for starch-based biofuel production.


Assuntos
Proteínas Fúngicas/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , Saccharomyces cerevisiae/metabolismo , Amido/metabolismo , alfa-Amilases/metabolismo , Biocombustíveis , Etanol/metabolismo , Proteínas Fúngicas/genética , Fungos/enzimologia , Fungos/genética , Glucana 1,4-alfa-Glucosidase/genética , Hidrólise , Microbiologia Industrial , Engenharia Metabólica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , alfa-Amilases/genética
18.
J Agric Food Chem ; 66(34): 9121-9126, 2018 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-30084254

RESUMO

Dry-hopping, the addition of hops to beer during or after fermentation, is a common practice in brewing to impart hoppy flavor to beer. Previously assumed to be inert ingredients, recent evidence suggests that hops contain biologically active compounds that may also extract into beer and complicate the brewing process by altering the final composition of beer. Experiments described herein provide evidence of microbial and/or plant-derived enzymes associated with hops ( Humulus lupulus) which can impact beer quality by influencing the composition of fermentable and nonfermentable carbohydrates in dry-hopped beer. Fully attenuated and packaged commercial lager beer was dry-hopped at a rate of 10 g hops/L beer with pelletized Cascade hops, dosed with 106 cells/mL of ale yeast, and incubated at 20 °C. Real extract of the treated beer declined significantly within several days with a reduction of 1 °P (% w/w) after 5 days and then slowly to a total reduction of approximately 2 °P after 40 days. When fully fermented, this was equivalent to the production of an additional 4.75% (v/v) of CO2 and an additional 1.3% (v/v) of alcohol. The refermentation of beer driven by dry-hopping was attributed to the low but persistent activities of several starch degrading enzymes present in Cascade hops including amyloglucosidase, α-amylase, ß-amylase, and limit dextrinase. The effect of hop-derived enzymes on beer was time, temperature, and dose-dependent. Characterizing bioactive enzymes in hops will help hop suppliers and brewers to address the unexpected quality and safety issues surrounding hopping practices in beer.


Assuntos
Cerveja/análise , Humulus/enzimologia , Proteínas de Plantas/metabolismo , Amilases/química , Amilases/metabolismo , Cerveja/microbiologia , Cromatografia Líquida de Alta Pressão , Dextrinas/metabolismo , Manipulação de Alimentos , Glucana 1,4-alfa-Glucosidase/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Humulus/química , Humulus/microbiologia , Hidrólise , Cinética , Proteínas de Plantas/química , Leveduras/metabolismo
19.
J Biotechnol ; 287: 28-40, 2018 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-30134150

RESUMO

Environmental gradient is an important common issue during scale-up process for protein production. To address the dynamic regulatory mechanism of Aspergillus niger being exposed to inhomogeneous glucose concentrations, glucose perturbation were experimented on the steady state of A. niger chemostat culture, and dynamic profiles of the intracellular metabolites in central carbon metabolism were tracked in a time scale of seconds. The upper glycolysis and pentose phosphate pathway showed sharp variations after glucose perturbation, while the lower glycolysis, TCA cycle and amino acid pools represented a moderate and prolonged response due to the allosteric regulation of enzymes and buffering function of metabolites with large pool sizes. Improved glucose-6-phosphate enhanced the metabolic flux to PP pathway remarkably, which provided not only more redox cofactors (NADPH) for protein synthesis but also more precursors (phosphoribosyl pyrophosphate and ribose-5-phosphate) for cell growth. Moreover, reduction of the total adenine nucleotides and major precursor amino acids indicated the upregulated RNA synthesis was required to produce stress proteins, and partially explained the drop of glucoamylase production when A. niger experienced a fluctuated glucose concentration environment. These findings would be valuable for improving bioreactor operation, design, and scale-up from engineering or genetic aspects.


Assuntos
Aspergillus niger , Proteínas Fúngicas/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , Glucose/metabolismo , Aspergillus niger/enzimologia , Aspergillus niger/metabolismo , Reatores Biológicos/microbiologia , Glicólise/fisiologia , Cinética , Redes e Vias Metabólicas
20.
Appl Environ Microbiol ; 84(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29980558

RESUMO

Soil fungi produce a wide range of chemical compounds and enzymes with potential for applications in medicine and biotechnology. Cellular processes in soil fungi are highly dependent on the regulation under environmentally induced stress, but most of the underlying mechanisms remain unclear. Previous work identified a key GATA-type transcription factor, Penicillium oxalicum NsdD (PoxNsdD; also called POX08415), that regulates the expression of cellulase and xylanase genes in P. oxalicum PoxNsdD shares 57 to 64% identity with the key activator NsdD, involved in asexual development in Aspergillus In the present study, the regulatory roles of PoxNsdD in P. oxalicum were further explored. Comparative transcriptomic profiling revealed that PoxNsdD regulates major genes involved in starch, cellulose, and hemicellulose degradation, as well as conidiation and pigment biosynthesis. Subsequent experiments confirmed that a ΔPoxNsdD strain lost 43.9 to 78.8% of starch-digesting enzyme activity when grown on soluble corn starch, and it produced 54.9 to 146.0% more conidia than the ΔPoxKu70 parental strain. During cultivation, ΔPoxNsdD cultures changed color, from pale orange to brick red, while the ΔPoxKu70 cultures remained bluish white. Real-time quantitative reverse transcription-PCR showed that PoxNsdD dynamically regulated the expression of a glucoamylase gene (POX01356/Amy15A), an α-amylase gene (POX09352/Amy13A), and a regulatory gene (POX03890/amyR), as well as a polyketide synthase gene (POX01430/alb1/wA) for yellow pigment biosynthesis and a conidiation-regulated gene (POX06534/brlA). Moreover, in vitro binding experiments showed that PoxNsdD bound the promoter regions of the above-described genes. This work provides novel insights into the regulatory mechanisms of fungal cellular processes and may assist in genetic engineering of Poxalicum for potential industrial and medical applications.IMPORTANCE Most filamentous fungi produce a vast number of extracellular enzymes that are used commercially for biorefineries of plant biomass to produce biofuels and value-added chemicals, which might promote the transition to a more environmentally friendly economy. The expression of these extracellular enzyme genes is tightly controlled at the transcriptional level, which limits their yields. Hitherto our understanding of the regulation of expression of plant biomass-degrading enzyme genes in filamentous fungi has been rather limited. In the present study, regulatory roles of a key regulator, PoxNsdD, were further explored in the soil fungus Penicillium oxalicum, contributing to the understanding of gene regulation in filamentous fungi and revealing the biotechnological potential of Poxalicum via genetic engineering.


Assuntos
Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Penicillium/metabolismo , Pigmentos Biológicos/biossíntese , Esporos Fúngicos/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Biodegradação Ambiental , Celulase/genética , Celulase/metabolismo , Celulose/metabolismo , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Glucana 1,4-alfa-Glucosidase/genética , Glucana 1,4-alfa-Glucosidase/metabolismo , Penicillium/enzimologia , Penicillium/genética , Penicillium/crescimento & desenvolvimento , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Fatores de Transcrição/genética , alfa-Amilases/genética , alfa-Amilases/metabolismo
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