RESUMO
Starch, a crucial raw material, has been extensively investigated for biotechnological applications. However, its application in γ-polyglutamic acid (γ-PGA) production remains unexplored. Based on γ-PGA output of Bacillus subtilis SCP010-1, a novel asynchronous saccharification and fermentation process for γ-PGA synthesis was implemented. The results revealed that a starch concentration of 20%, α-amylase dosage of 75 U/g, liquefaction temperature of 72â, and γ-PGA yield of 36.31 g/L was achieved. At a glucoamylase dosage of 100 U/g, saccharification 38 h at 60â, the yield of γ-PGA increased to 48.88 g/L. The contents of total sugar, glucose, maltose and oligosaccharide in saccharified liquid were determined. Through batch fermentation of saccharified liquid in fermentor, the γ-PGA output was elevated to 116.08 g/L. This study can offer a potential cost reduction of 40%, which can be a promising advancement in industrial γ-PGA production. Moreover, our approach can be applied in other starch-based fermentation industries.
Assuntos
Bacillus subtilis , Fermentação , Glucana 1,4-alfa-Glucosidase , Ácido Poliglutâmico , Amido , Zea mays , alfa-Amilases , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/biossíntese , Ácido Poliglutâmico/metabolismo , Amido/metabolismo , Bacillus subtilis/metabolismo , alfa-Amilases/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , Zea mays/metabolismo , Zea mays/química , Temperatura , Maltose/metabolismo , Glucose/metabolismo , Reatores Biológicos/microbiologia , Oligossacarídeos/metabolismo , Microbiologia Industrial/métodosRESUMO
This study aimed at optimizing process protocols for development of low glycemic index (GI) rice flour (LGIRF) by employing enzymatic hydrolysis method using central composite rotatable design (CCRD). LGIRF was evaluated for pasting, farinographic, spectroscopic and microbiological attributes. Independent variables for optimization included concentrations of α-amylase (0.02-0.12 %), glucoamylase (0.02-0.24 %), as well as the incubation temperature (55-80°C). Resistant starch (RS), glycemic index (GI) and glycemic load (GL) were investigated as response variables. The optimum conditions for development of LGIRF with better quality were- α-amylase concentration of 0.040 %, glucoamylase concentration of 0.070 % and an incubation temperature of 60 °C. The results of mineral analysis revealed significantly (p < 0.05) lower levels of boron, potassium, zinc, phosphorus, magnesium, and manganese in LGIRF, while iron and copper were significantly higher. The viscosity profile as evident from pasting profile and farinographic characteristics of LGIRF were significantly (p < 0.05) lower than native rice flour. 1H NMR and 13C NMR spectroscopic studies showed an increase in flexible starch segments and a decrease in amorphous portion of starch LGIRF, along with chemical shift alterations in carbons 1 and 4. Free fatty acids and total plate count were significantly (p < 0.05) higher in LGIRF although was within limits.
Assuntos
Farinha , Glucana 1,4-alfa-Glucosidase , Índice Glicêmico , Oryza , Reologia , alfa-Amilases , Oryza/química , Hidrólise , Farinha/análise , alfa-Amilases/metabolismo , alfa-Amilases/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Glucana 1,4-alfa-Glucosidase/química , Amido/química , Amido/metabolismoRESUMO
Porous starch materials are environmentally friendly and renewable and exhibit high adsorption performances. Ultrasound and compound enzyme (α-amylase and glucoamylase) treatments were applied to prepare modified cassava starch. The granules, crystal morphology, crystal structure, and molecular structure of starch were investigated. The hydrolysis degree, solubility, swelling, and adsorption properties of cassava starch were analyzed. After the cassava starch was modified by ultrasound and enzyme treatments, the granule size of the starch decreased, and the surfaces were eroded to form pits, grooves and cavity structure. The starch spherulites weakened or even disappeared. The functional groups of starch did not change significantly, but the degree of crystal order decreased. The double-helix structure was reduced, and the crystal structure was composed of A + V-type crystals, with a decrease in crystallinity. The gelatinization temperature and thermal degradation temperatures enhanced. The enzymatic hydrolysis degree and solubility of the modified cassava starch increased. The swelling degree decreased, and oil adsorption, water adsorption improved. MB adsorption behavior of modified cassava starch closely followed a pseudo-second-order kinetics model and the Langmuir isotherm equation. These findings could help to understand the relationship between the structure and properties of modified starch, and guide its application in the field of adsorption.
Assuntos
Glucana 1,4-alfa-Glucosidase , Manihot , Solubilidade , Amido , alfa-Amilases , Manihot/química , Amido/química , Adsorção , Hidrólise , alfa-Amilases/química , alfa-Amilases/metabolismo , Glucana 1,4-alfa-Glucosidase/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Cinética , Ondas Ultrassônicas , Temperatura , Água/química , PorosidadeRESUMO
Ustilago maydis and Ustilago cynodontis are natural producers of a broad range of valuable molecules including itaconate, malate, glycolipids, and triacylglycerols. Both Ustilago species are insensitive toward medium impurities, and have previously been engineered for efficient itaconate production and stabilized yeast-like growth. Due to these features, these strains were already successfully used for the production of itaconate from different alternative feedstocks such as molasses, thick juice, and crude glycerol. Here, we analyzed the amylolytic capabilities of Ustilago species for metabolization of starch, a highly abundant and low-cost polymeric carbohydrate widely utilized as a substrate in several biotechnological processes. Ustilago cynodontis was found to utilize gelatinized potato starch for both growth and itaconate production, confirming the presence of extracellular amylolytic enzymes in Ustilago species. Starch was rapidly degraded by U. cynodontis, even though no α-amylase was detected. Further experiments indicate that starch hydrolysis is caused by the synergistic action of glucoamylase and α-glucosidase enzymes. The enzymes showed a maximum activity of around 0.5 U ml-1 at the fifth day after inoculation, and also released glucose from additional substrates, highlighting potential broader applications. In contrast to U. cynodontis, U. maydis showed no growth on starch accompanied with no detectable amylolytic activity.
Assuntos
Amido , Succinatos , Ustilago , Ustilago/metabolismo , Ustilago/genética , Ustilago/enzimologia , Ustilago/crescimento & desenvolvimento , Amido/metabolismo , Succinatos/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , HidróliseRESUMO
The survival rate of mesenchymal stem cells (MSC), a crucial factor in tissue engineering, is highly dependent on glucose supply. The purpose of this paper is to study the potential of starch foams as glucose suppliers. It is investigated through in vitro hydrolysis by amyloglucosidase in conditions that respect physiological constraints (37 °C and pH 7.4), including a duration of 21 days, and no stirring. Nine extruded starch foams with amylose contents ranging from 0 to 74 %, with various cell wall thicknesses (50 to 300 µm), and different crystallinities (0-30 %) were hydrolysed. These kinetics were fitted by a model which shows that the maximum rate of hydrolysis varies from 7 to 100 %, and which allows the rate of hydrolysis at 21 days to be calculated precisely. The results reveal the major role of amylose in glucose delivery kinetics, and the secondary roles of crystallinity and cell wall thickness of the foams. Additional hydrolysis of starch films revealed that thickness positively influences the amylose chain reorganisation during hydrolysis, which, in slows down and limits glucose delivery. A simple glucose delivery kinetics analysis procedure is proposed to select samples for testing as MSC glucose suppliers.
Assuntos
Amilose , Materiais Biocompatíveis , Glucose , Células-Tronco Mesenquimais , Amido , Hidrólise , Glucose/química , Amido/química , Materiais Biocompatíveis/química , Amilose/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Cinética , Glucana 1,4-alfa-Glucosidase/metabolismo , Glucana 1,4-alfa-Glucosidase/químicaRESUMO
In the presented study, natural rice containing high resistant starch content was used as a raw material to produce rice resistant starch (RRS) through enzymatic hydrolysis with heat-stable α-amylase and glucoamylase. The chemical composition, structural characteristics and in vitro glycemic index (GI) of RRS were evaluated. The effects of RRS at different doses on the body weight, serum biochemical levels, pathological indexes, production of short-chain fatty acids (SCFAs) in the gut and the intestinal microbial composition in T2DM mice were investigated. The results of physiochemical characterization indicated that, relative to rice flour, RRS mainly comprising resistant starch had higher crystallinity (25.85%) and a more stable structure, which contributed to its lower digestibility and decreased GI in vitro. Compared with the model control group, 1 g per kg BW and 2 g per kg BW oral gavage dosages of RRS effectively enhanced the SCFA productivity in the T2DM mouse gut, as well as alleviating T2DM symptoms, involving an increase in body weight, reduction in fasting blood glucose, total cholesterol, triglyceride, low-density lipoprotein cholesterol, alanine transaminase and aspartate aminotransferase, and an increase in serum insulin and high-density lipoprotein cholesterol. Besides, 1 g per kg BW and 2 g per kg BW dosages of RRS mitigated T2DM-induced pancreas damage. Furthermore, up-regulation in the abundance of probiotics (Lactobacillus, Ruminococcus, etc.) and down-regulation in the number of harmful bacteria (Desulfovibrio, Prevotella, etc.) were observed in all RRS-treated groups. In summary, this work suggested that RRS prepared using heat-stable α-amylase and glucoamylase could be a potential functional component for amelioration of T2DM applied in the fields of food and pharmaceutics.
Assuntos
Diabetes Mellitus Tipo 2 , Microbioma Gastrointestinal , Glucana 1,4-alfa-Glucosidase , Oryza , Amido , alfa-Amilases , Animais , Camundongos , alfa-Amilases/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Ácidos Graxos Voláteis/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Glucana 1,4-alfa-Glucosidase/metabolismo , Temperatura Alta , Oryza/química , Amido Resistente/farmacologia , Amido/química , Amido/metabolismo , Amido/farmacologiaRESUMO
Alpha-glucosidase (maltase, sucrase, isomaltase and glucoamylase) activities which are involved in carbohydrate metabolism are present in human intestinal maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). Hence, these proteins are important targets to identify drugs against postprandial hyperglycemia thereby for diabetes. To find natural-based drugs against MGAM and SI, Artocarpus heterophyllus leaf was explored for MGAM and SI inhibition in in vitro and in silico. A. heterophyllus leaf aqueous active fraction (AHL-AAF) was prepared using Soxhlet extraction followed by silica column chromatography. The phytoconstituents of AHL-AAF were determined using LC-ESI-MS/MS. AHL-AAF showed dose-dependent and mixed inhibition against maltase (IC50 = 460⯵g/ml; Ki = 300⯵g/ml), glucoamylase (IC50 = 780⯵g/ml; Ki = 480⯵g/ml), sucrase (IC50 = 900⯵g/ml, Ki = 504⯵g/ml) and isomaltase (IC50 = 860⯵g/ml, Ki = 400⯵g/ml). AHL-AAF phytoconstituents interaction with N-terminal (Nt) and C-terminal (Ct) subunits of human MGAM and SI was analyzed using induced-fit docking, molecular dynamics (MD), and binding free energy calculation. In docking studies, rhamnosyl hexosyl methyl quercetin (RHMQ), P-coumaryl-O-16-hydroxy palmitic acid (PCHP), and spirostanol interacted with active site amino acids of human MGAM and SI. Among these RHMQ stably interacted with all the subunits (Nt-MGAM, Ct-MGAM, Nt-SI and Ct-SI) whereas PCHP with Ct-MGAM and Nt-SI during MD analysis. In molecular docking, the docking score of RHMQ with NtMGAM, CtMGAM, NtSI and CtSI was -8.48, -12.88, -11.98 and -11.37â¯kcal/mol. The docking score of PCHP for CtMGAM and NtSI was -8.59 and -8.4â¯kcal/mol, respectively. After MD simulation, the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values further confirmed the stable protein-ligand interaction. The RMSD value of all the complexes were around 2.5â¯Å and the corresponding RMSF values were also quite low. In MM/GBSA analysis, the involvement of Van der Waals and lipophilic energy in the protein/ligand interactions are understood. Further binding free energy for Nt-MGAM-PCHP, Nt-MGAM-RHMQ, Nt-SI-PCHP, Nt-SI-RHMQ, Ct-MGAM-PCHP, Ct-MGAM-RHMQ and Ct-SI-RHMQ complexes was found to be -24.94, -46.60, -46.56, -44.48, -40.3, -41.86 and -19.39â¯kcal/mol, respectively. Altogether, AHL-AAF showed inhibition of α-glucosidase activities of MGAM and SI. AHL-AAF could be further studied for its effect on diabetes in in vivo.
Assuntos
Artocarpus , Simulação de Acoplamento Molecular , Artocarpus/química , Humanos , alfa-Glucosidases/metabolismo , alfa-Glucosidases/química , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Simulação de Dinâmica Molecular , Glucana 1,4-alfa-Glucosidase/metabolismo , Glucana 1,4-alfa-Glucosidase/antagonistas & inibidores , Glucana 1,4-alfa-Glucosidase/química , Folhas de Planta/química , Complexo Sacarase-Isomaltase/antagonistas & inibidores , Complexo Sacarase-Isomaltase/metabolismo , Complexo Sacarase-Isomaltase/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Relação Estrutura-Atividade , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologiaRESUMO
Bimetallic nanoparticles (NPs) are considered superior in terms of stability and function with respect to its monometallic counterparts. Hence, in the present study Hibiscus rosa-sinensis flower extract was used to synthesis copper-iron bimetallic nanoparticles (HF-FCNPs). HF-FCNPs was characterized and its applications (biological and environmental) were determined. HF-FCNPs were spherical in shape with high percentage of copper inducted into the NPs. HF-FCNPs inhibited mammalian glucosidases [maltase (IC50: 548.71 ± 61.01 µg/mL), sucrase (IC50: 441.34 ± 36.03 µg/mL), isomaltase (IC50: 466.37 ± 27.09 µg/mL) and glucoamylase (IC50: 403.12 ± 14.03 µg/mL)], alpha-amylase (IC50: 16.27 ± 1.73 µg/mL) and acetylcholinesterase [AChE (IC50: 0.032 ± 0.004 µg/mL)] activities. HF-FCNPs showed competitive inhibition against AChE, maltase and sucrase activities; mixed inhibition against isomaltase and glucoamylase activities; whereas non-competitive inhibition against α-amylase activity. HF-FCNPs showed zone of inhibition of 16 ± 2 mm against S. mutans at 100 µg/mL concentration. HF-FCNPs inhibited biofilm formation of dental pathogen, S. mutans. SEM and confocal microscopy analysis revealed the disruption of network formation and bacterial cell death induced by HF-FCNPs treatment on tooth model of S. mutans biofilm. HF-FCNPs efficiently removed hexavalent chromium in pH-independent manner and followed first order kinetics. Through Langmuir isotherm fit the qmax (maximum adsorption capacity) was determined to be 62.5 mg/g. Further, HF-FCNPs removed both anionic and cationic dyes. Altogether, facile synthesis of HF-FCNPs was accomplished and its biological (enzyme inhibition and antibiofilm activity) and environmental (catalyst to remove pollutants) applications have been understood.
Assuntos
Hibiscus , Nanopartículas , Animais , alfa-Glucosidases/metabolismo , Glucana 1,4-alfa-Glucosidase , Corantes , Cobre , Hibiscus/metabolismo , Ferro , Acetilcolinesterase , Flores/metabolismo , Oligo-1,6-Glucosidase , Sacarase , Cromo , Biofilmes , alfa-Amilases , Mamíferos/metabolismoRESUMO
Porous starch can be applied as an adsorbent and encapsulant for bioactive substances in the food and pharmaceutical industries. By using appropriate modification methods (chemical, physical, enzymatic, or mixed), it is possible to create pores on the surface of the starch granules without disturbing their integrity. This paper aimed to analyze the possibility of obtaining a porous structure for native corn, potato, and pea starches using a combination of ultrasound, enzymatic digestion, and freeze-drying methods. The starch suspensions (30%, w/w) were treated with ultrasound (20 kHz, 30 min, 20 °C), then dried and hydrolyzed with amyloglucosidase (1000 U/g starch, 50 °C, 24 h, 2% starch suspension). After enzyme digestion, the granules were freeze-dried for 72 h. The structure of the native and modified starches were examined using VIS spectroscopy, SEM, ATR-FTIR, and LTNA (low-temperature nitrogen adsorption). Based on the electrophoretic mobility measurements of the starch granules using a laser Doppler velocimeter, zeta potentials were calculated to determine the surface charge level. Additionally, the selected properties such as the water and oil holding capacities, least gelling concentration (LGC), and paste clarity were determined. The results showed that the corn starch was the most susceptible to the combined modification methods and was therefore best suited for the production of porous starch.
Assuntos
Glucana 1,4-alfa-Glucosidase , Amido , Amido/química , Adsorção , PorosidadeRESUMO
Food waste is a lucrative source of complex nutrients, which can be transformed into a multitude of bioproducts by the aid of microbial cell factories. The current study emphasizes isolating Glucoamylase enzyme (GA) producing strains that can effectively break down mixed food waste (MW), which serves as a substrate for biomanufacturing. The screening procedure relied heavily on the growth of isolated fungi on starch agar media, to specifically identify the microbes with the highest starch hydrolysis potential. A strain displayed the highest GA activity of 2.9 ± 0.14 U/ml which was selected and identified as Aspergillus fumigatus via molecular methods of identification. Exposure of the A. fumigatus with 200 mM Ethyl methanesulphonate (EMS) led to a 23.79% increase compared to the wild-type GA. The growth conditions like cultivation temperature or the number of spores in the inoculum were investigated. Further, maximum GA activity was exhibited at pH 5, 55 °C, and at 5 mM Ca2+ concentration. The GA showed thermostability, retaining activity even after long periods of exposure to temperatures as high as 95 °C. The improvement of hydrolysis of MW was achieved by Taguchi design where a maximum yield of 0.57 g g-1 glucose was obtained in the hydrolysate. This study puts forth the possibility that mixed food waste, despite containing spices and other microbial growth-inhibitory substances, can be efficiently hydrolyzed to release glucose units, by robust fungal cell factories. The glucose released can then be utilized as a carbon source for the production of value-added products.
Assuntos
Glucana 1,4-alfa-Glucosidase , Eliminação de Resíduos , Glucana 1,4-alfa-Glucosidase/química , Perda e Desperdício de Alimentos , Alimentos , Fungos , Hidrólise , Amido , GlucoseRESUMO
In multinuclear and multicellular filamentous fungi little is known about how mRNAs encoding secreted enzymes are transcribed and localized spatiotemporally. To better understand this process we analyzed mRNA encoding GlaA, a glucoamylase secreted in large amounts by the industrial filamentous fungus Aspergillus oryzae, by the MS2 system, in which mRNA can be visualized in living cells. We found that glaA mRNA was significantly transcribed and localized near the hyphal tip and septum, which are the sites of protein secretion, in polarity-dependent expression and localization manners. We also revealed that glaA mRNA exhibits long-range dynamics in the vicinity of the endoplasmic reticulum (ER) in a manner that is dependent on the microtubule motor proteins kinesin-1 and kinesin-3, but independent of early endosomes. Moreover, we elucidated that although glaA mRNA localized to stress granules (SGs) and processing bodies (PBs) under high temperature, glaA mRNA was not seen under ER stress, suggesting that there are different regulatory mechanisms of glaA mRNA by SG and PB under high temperature and ER stress. Collectively, this study uncovers a dynamic regulatory mechanism of mRNA encoding a secretory enzyme in filamentous fungi.
Assuntos
Glucana 1,4-alfa-Glucosidase , Cinesinas , Glucana 1,4-alfa-Glucosidase/genética , Glucana 1,4-alfa-Glucosidase/metabolismo , Cinesinas/metabolismo , Retículo Endoplasmático/metabolismo , Transporte Proteico , Fungos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
Knowledge of the metabolism of functional enzymes is the key to accelerate the transformation and utilization of raw materials during high temperature Daqu (HTD) manufacturing. However, the metabolic contribution of raw materials-wheat is always neglected. In this research, the relationship between the metabolism of wheat and microorganisms was investigated using physicochemical and sequencing analysis method. Results showed that the process of Daqu generation was divided into three stages based on temperature. In the early stage, a positive correlation was found between Monascus, Rhizopus and glucoamylase metabolism (r > 0.8, p < 0.05). Meanwhile, the glucoamylase metabolism in wheat occupied 63.8 % of the total matrix at the day 4. In the middle to later stages, the wheat metabolism of proteases, α-amylases and lipases in gradually reached their peak. Additionally, Lactobacillus and α-amylases presented a positive correlation (r > 0.7, p < 0.05), and the α-amylases metabolism in wheat occupied 22.18 % of the total matrix during the same time period. More importantly, the changes of enzyme activity metabolic pathway in wheat and microorganism were reflected by respiratory entropy (RQ). Overall, these results guide the choice of substrate during Daqu production.
Assuntos
Bactérias , Microbiota , Fermentação , Bactérias/genética , Bactérias/metabolismo , Triticum/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , Temperatura , alfa-Amilases/metabolismo , Bebidas AlcoólicasRESUMO
Aspergillus species have been highlighted in enzyme production looking for industrial applications, notably, amylases are one of the most interesting enzymes. They are capable of hydrolyzing α-glycosidic linkages of starch and widely used in industrial processes to produce ethanol, glucose, and fructose syrup as well as in the textiles, detergents, and paper industries applications. In this context, this work aimed at the biochemical characterization of the glucoamylase from Aspergillus japonicus and its application in the bio-bleaching process of recycled paper. The optimum temperature and pH for the glucoamylase assay were standardized as 50°C and 5.5. After 1 h of incubation, glucoamylase retained 90% of its activity at 30-50°C. It also kept 70% of its activity in the pH range of 4.0-6.5 after an hour of incubation. The enzyme led to an increase of 30% in the relative whiteness of 10 dry grams of sulfite paper and magazine paper when applied along with commercial cellulase and 10 mM MnCl2 . In addition, after the treatments, the glucoamylase recovered activity was 30%-32%, which indicates a prolonged availability of the enzyme and can considerably curtail the redundant downstream process of the recycled paper bio-bleaching. Thus, the glucoamylase from A. japonicus has a significant role in bio-bleaching recycled paper, reducing the necessity of hard chemicals, and improving the industrial process in an interesting economic and ecological mode.
Assuntos
Aspergillus , Glucana 1,4-alfa-Glucosidase , Glucana 1,4-alfa-Glucosidase/química , Temperatura , Amido , Concentração de Íons de HidrogênioRESUMO
BACKGROUND: Baijiu brewing adopts the solid-state fermentation method, using starchy raw materials, Jiuqu as saccharifying fermenting agent, and distilled spirits made by digestion, saccharification, fermentation and distillation. In the late stages of solid-state fermentation of Baijiu, the reduced activity of glucoamylase leads to higher residual starch content in the Jiupei, which affects the liquor yield. The direct addition of exogenous glucoamylase leads to problems such as the temperature of the fermentation environment rising too quickly, seriously affecting the growth of microorganisms. RESULTS: To solve the problem of reduced activity of glucoamylase in the late stage of solid-state fermentation of Baijiu, microcapsule beads (M-B) based on microcapsule emulsion were prepared and the effect of M-B on solid-state fermentation of Baijiu was investigated. The results showed that the release of M-B before and after drying was 53.27% and 25.77% in the liquid state (120 h) and 29.84% and 22.62% in the solid state (15 days), respectively. Adding M-B improved the alcohol by 0.33 %vol and reducing sugar content by 0.51%, reduced the residual starch content by 1.21% of the Jiupei, and had an insignificant effect on the moisture and acidity of the Jiupei. CONCLUSION: M-B have excellent sustained-release properties. The addition of M-B in solid-state fermentation significantly increased the alcohol content, reduced the residual starch content of Jiupei, ultimately improving the starch utilization rate and liquor yield of Baijiu brewing. The preparation of M-B provides methods and approaches for applying other active substances and microorganisms in the brewing of Baijiu. © 2023 Society of Chemical Industry.
Assuntos
Bebidas Alcoólicas , Glucana 1,4-alfa-Glucosidase , Fermentação , Cápsulas , Bebidas Alcoólicas/análise , Amido/metabolismoRESUMO
Targeting multiple factors such as oxidative stress, alpha glucosidase and acetylcholinesterase (AChE) are considered advantageous for the treatment of diabetes and diabetes associated-cognitive dysfunction. In the present study, Hibiscus rosa-sinensis flowers anthocyanin-rich extract (HRA) was prepared. Phytochemical analysis of HRA using LC-ESI/MS/MS revealed the presence of various phenolic acids, flavonoids and anthocyanins. HRA showed in vitro antioxidant activity at low concentrations. HRA inhibited all the activities of mammalian glucosidases and AChE activity. The IC50 value of HRA for the inhibition of maltase, sucrase, isomaltase, glucoamylase and AChE was found to be 308.02 ± 34.25⯵g/ml, 287.8 ± 19.49⯵g/ml, 424.58 ± 34.75⯵g/ml, 408.94 ± 64.82⯵g/ml and 264.13 ± 30.84⯵g/ml, respectively. Kinetic analysis revealed mixed-type inhibition against all the activities except for glucoamylase (competitive) activity. In silico analysis confirmed the interaction of two active constituents cyanidin 3-sophoroside (CS) and quercetin 3-O-sophoroside (QS) with four subunits, n-terminal and c-terminal subunits of human maltase-glucoamylase and sucrase-isomaltase as well as with AChE. Molecular dynamics simulation, binding free energy calculation, DCCM, PCA, PCA-based free energy surface analysis ascertained the stable binding of CS and QS with target proteins studied. HRA could be used as complementary therapy for diabetes and cognitive improvement.
Assuntos
Flores , Glucosidases , Hibiscus , Animais , Humanos , Acetilcolinesterase/metabolismo , alfa-Glucosidases/metabolismo , Antocianinas/farmacologia , Diabetes Mellitus , Flores/química , Glucana 1,4-alfa-Glucosidase/antagonistas & inibidores , Glucana 1,4-alfa-Glucosidase/metabolismo , Glucosidases/antagonistas & inibidores , Hibiscus/química , Cinética , Oligo-1,6-Glucosidase/antagonistas & inibidores , Extratos Vegetais/farmacologia , Sacarase/antagonistas & inibidores , Espectrometria de Massas em Tandem , Inibidores de Glicosídeo Hidrolases/farmacologia , Compostos Fitoquímicos/farmacologiaRESUMO
Wheat Qu plays the role of saccharification fermentation, providing microorganisms and flavor in the fermentation of huangjiu, and the use of functional microorganisms to fortify wheat Qu is becoming increasingly popular. Yet, the mechanisms promoting microbial successions of wheat Qu remain unclear. In this study, we first correlated microbial community succession with physicochemical factors (moisture, temperature, acidity, glucoamylase and amylase) in inoculated raw wheat Qu (IRWQ) with Saccharopolyspora rosea. The Mantel test was performed to investigate the significance and found that temperature (r = 0.759, P = 0.001), moisture (r = 0.732, P = 0.006), and acidity (r = 0.712, P = 0.017) correlated significantly with the bacterial community in phase 1 (0-40 h). Meanwhile, temperature correlated significantly with the fungal community in phases 1 and 2 (40-120 h). To confirm the effect of temperature on microbial communities, the artificial reduction of bio-heat (37°C) in IRWQ also reduced the relative abundance of heat-resistant microorganisms including Bacillus and Saccharopolyspora. A higher abundance of Saccharopolyspora (87%) in IRWQ was observed following biofortified inoculation of S. rosea, in which glucoamylase activity increased by 40% compared to non-inoculated raw wheat Qu (NIRWQ) (1086 U/g vs 776 U/g). Finally, the IRWQ was employed to mechanized huangjiu fermentation and it was found to reduce the bitter amino acid and higher alcohol content by 27% and 8%, respectively, improving the drinking comfort and quality of huangjiu.
Assuntos
Bacillus , Microbiota , Glucana 1,4-alfa-Glucosidase/metabolismo , Bactérias/metabolismo , Bacillus/genética , Bacillus/metabolismo , Fermentação , ChinaRESUMO
Glucoamylases (GAs) are one of the principal groups of enzymes involved in starch hydrolysis and belong to the glycosylhydrolase family. They are classified as exo-amylases due to their ability to hydrolyze α-1,4 glycosidic bonds from the non-reducing end of starch, maltooligosaccharides, and related substrates, releasing ß-D-glucose. Structurally, GAs possess a characteristic catalytic domain (CD) with an (α/α)6 fold and exhibit five conserved regions within this domain. The CD may or may not be linked to a non-catalytic domain with variable functions depending on its origin. GAs are versatile enzymes with diverse applications in food, biofuel, bioplastic and other chemical industries. Although fungal GAs are commonly employed for these purposes, they have limitations such as their low thermostability and an acidic pH requirement. Alternatively, GAs derived from prokaryotic organisms are a good option to save costs as they exhibit greater thermostability compared to fungal GAs. Moreover, a group of cold-adapted GAs from psychrophilic organisms demonstrates intriguing properties that make them suitable for application in various industries. This review provides a comprehensive overview of the structural and sequential properties as well as biotechnological applications of GAs in different industrial processes.
Assuntos
Amilases , Glucana 1,4-alfa-Glucosidase , Biocombustíveis , Biotecnologia , AmidoRESUMO
BACKGROUND: Glucoamylase is an important enzyme for starch saccharification in the food and biofuel industries and mainly produced from mesophilic fungi such as Aspergillus and Rhizopus species. Enzymes produced from thermophilic fungi can save the fermentation energy and reduce costs as compared to the fermentation system using mesophiles. Thermophilic fungus Myceliophthora thermophila is industrially deployed fungus to produce enzymes and biobased chemicals from biomass during optimal growth at 45 °C. This study aimed to construct the M. thermophila platform for glucoamylase hyper-production by broadening genomic targeting range of the AsCas12a variants, identifying key candidate genes and strain engineering. RESULTS: In this study, to increase the genome targeting range, we upgraded the CRISPR-Cas12a-mediated technique by engineering two AsCas12a variants carrying the mutations S542R/K607R and S542R/K548V/N552R. Using the engineered AsCas12a variants, we deleted identified key factors involved in the glucoamylase expression and secretion in M. thermophila, including Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2. Deletion of four targets led to more than 1.87- and 1.85-fold higher levels of secretion and glucoamylases activity compared to wild-type strain MtWT. Transcript level of the major amylolytic genes showed significantly increased in deletion mutants. The glucoamylase hyper-production strain MtGM12 was generated from our previously strain MtYM6 via genetically engineering these targets Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2 and overexpressing Mtamy1 and Mtpga3. Total secreted protein and activities of amylolytic enzymes in the MtGM12 were about 35.6-fold and 51.9â55.5-fold higher than in MtWT. Transcriptional profiling analyses revealed that the amylolytic gene expression levels were significantly up-regulated in the MtGM12 than in MtWT. More interestingly, the MtGM12 showed predominantly short and highly bulging hyphae with proliferation of rough ER and abundant mitochondria, secretion vesicles and vacuoles when culturing on starch. CONCLUSIONS: Our results showed that these AsCas12a variants worked well for gene deletions in M. thermophila. We successfully constructed the glucoamylase hyper-production strain of M. thermophila by the rational redesigning and engineering the transcriptional regulatory and secretion pathway. This targeted engineering strategy will be very helpful to improve industrial fungal strains and promote the morphology engineering for enhanced enzyme production.
Assuntos
Glucana 1,4-alfa-Glucosidase , Engenharia Metabólica , Glucana 1,4-alfa-Glucosidase/genética , Glucana 1,4-alfa-Glucosidase/metabolismo , Fungos/metabolismo , Amido/metabolismoRESUMO
The best amylolytic activity production by Aspergillus clavatus UEM 04 occurred in submersed culture, with starch, for 72 h, at 25 °C, and 100 rpm. Exclusion chromatography partially purified two enzymes, which ran as unique bands in SDS-PAGE with approximately 84 kDa. LC-MS/MS identified a glucoamylase (GH15) and an α-amylase (GH13_1) as the predominant proteins and other co-purified proteins. Zn2+, Cu2+, and Mn2+ activated the glucoamylase, and SDS, Zn2+, Fe3+, and Cu2+ inhibited the α-amylase. The α-amylase optimum pH was 6.5. The optimal temperatures for the glucoamylase and α-amylase were 50 °C and 40 °C, and the Tm was 53.1 °C and 56.3 °C, respectively. Both enzymes remained almost fully active for 28-32 h at 40 °C, but the α-amylase thermal stability was calcium-dependent. Furthermore, the glucoamylase and α-amylase KM for starch were 2.95 and 1.0 mg/mL, respectively. Still, the Vmax was 0.28 µmol/min of released glucose for glucoamylase and 0.1 mg/min of consumed starch for α-amylase. Moreover, the glucoamylase showed greater affinity for amylopectin and α-amylase for maltodextrin. Additionally, both enzymes efficiently degraded raw starch. At last, glucose was the main product of glucoamylase, and α-amylase produced mainly maltose from gelatinized soluble starch hydrolysis.
Assuntos
Glucana 1,4-alfa-Glucosidase , alfa-Amilases , alfa-Amilases/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , Amido/metabolismo , Cromatografia Líquida , Espectrometria de Massas em Tandem , Glucose , Concentração de Íons de HidrogênioRESUMO
BACKGROUND AND OBJECTIVES: Pompe disease (PD) results from a deficiency of lysosomal acid α-glucosidase that leads to glycogen accumulation in lysosomes in multiple tissues. There are two phenotypes: infantile-onset Pompe disease (IOPD) and late-onset Pompe disease (LOPD). The objective was to evaluate the diagnostic and follow-up outcomes of children identified with PD through newborn screening (NBS) in the state of Minnesota over a 4-year period. METHODS: This study is a retrospective analysis of infants born in Minnesota between August 1, 2017, and July 31, 2021, by the Minnesota Department of Health NBS Program for Pompe disease. Newborn screening and clinical diagnostic data are summarized for all newborns with positive newborn screens for Pompe disease. RESULTS: Children with IOPD had abnormal biomarkers necessitating immediate initiation of treatment. Children with LOPD are asymptomatic to date (1.25-4.58 years) with normal biomarkers including creatine kinase, urine glucotetrasaccharides, liver function tests, and echocardiogram. The estimated birth prevalence of PD is 1:15,160. The positive predictive value for PD was 81% with a false positive rate of 1.9 per 10 positive screens. 32% of the children with LOPD were lost to follow up among which 66% were from minority ethnic groups. CONCLUSION: This emphasizes the disparity in access to health care among specific demographics, as well as the importance of a primary care provider's early involvement in educating these families. To accomplish this, and ensure equality in follow-up care, the Minnesota Pompe Disease Consortium has been formed.