Glucose Releasing Research Articles

Exploration of unique starch physicochemical properties of novel buckwheat lines created by crossing Golden buckwheat and Tatary buckwheat

Buckwheat is considered as a healthy cereal food, and it is essential to cultivate new buckwheat lines with good starch physicochemical properties for both consumers and food producers. Six novel buckwheat (Duoku, Dk) were generated by crossing of Golden buckwheat and Tatary buckwheat, and their kernel appearance properties and starch physicochemical properties were analyzed together with one domestic line (Cimiqiao) and one wild line (Yeku). The results showed that Dk samples had better appearance properties than two control samples. The Dk samples showed lower amylose content, similar amylopectin molecular structure and chain length distributions, and larger starch granules compared with Cimiqiao. The digestion results showed that two Dk samples: Dk6 & Dk9 had high resistant starch content; while the other two Dk samples: Dk37 & Dk38 had a steady glucose releasing rate. The Dk samples also showed high gelatinization temperature, indicating they were good raw materials for producing glass noodle. This study proved that Dk buckwheat had unique starch physicochemical properties, and could be used as new food materials in the future.

A novel amylolytic enzyme from Palaeococcus ferrophilus with malto-oligosaccharide forming ability belonging to subfamily GH13_20

By heterologous expression of a gene from Palaeococcus ferrophilus, a novel recombinant enzyme designated AMPf was obtained and proved to be an amylolytic enzyme with unique catalytic characteristics. The optimal temperature and pH of AMPf were 50 °C and 7.0 respectively. Although sequence analysis and acarbose hydrolysis ability indicated that AMPf belongs to the subfamily GH13_20, interestingly, this enzyme hardly acts on cyclodextrins and pullulan distinguishing it from most enzymes in this subfamily. AMPf hydrolyzes starches to glucose, maltose, maltotriose, and maltotetrose as main products. AMPf mainly liberates glucose from starch with the concentration of 1% (w/v), while it shows malto-oligosaccharide forming ability with higher starch concentration of 4% (w/v). Also, the 4,6-ethylidene-4-nitrophenyl-maltoheptaose hydrolysis ability further indicates the unique combination endo-acting and glucose releasing exo-acting activtity of AMPf. AMPf could utilize maltose and maltotriose to produce malto-oligosaccharides by transglycosylation activity. It was proven AMPf has application protential in malto-oligosaccharides production.

A cell engineering approach to enzyme-based fed-batch fermentation

BackgroundA fundamental problem associated with E. coli fermentations is the difficulty in achieving high cell densities in batch cultures, attributed in large part to the production and accumulation of acetate through a phenomenon known as overflow metabolism when supplying enough glucose for the cell density desired. Although a fed-batch configuration is the standard method for reducing such issues, traditional fed-batch systems require components which become problematic when applying them at smaller scale. One alternative has been the development of a system whereby the enzymatic degradation of starch is used to release glucose at a controlled rate. However, to date, amylolytic enzymes have only been applied to the culture exogenously, whereas our goal is to design and construct a self-secreting amylolytic chassis capable of self-regulated enzyme-based fed-batch fermentation.ResultsA putative glucoamylase from C. violaceum has been cloned and expressed in E. coli BL21(DE3) and W3110, which exhibits significant glucose releasing amylolytic activity. Extracellular amylolytic activity was enhanced following a replacement of the enzymes native signal peptide with the DsbA signal sequence, contributing to a glucoamylase secreting strain capable of utilising starch as a sole carbon source in defined media. Introduction of PcstA, a glucose sensitive K12 compatible promoter, and the incorporation of this alongside C. violaceum glucoamylase in E. coli W3110, gave rise to increased cell densities in cultures grown on starch (OD600 ∼ 30) compared to those grown on an equivalent amount of glucose (OD600 ∼ 15). Lastly, a novel self-secreting enzyme-based fed-batch fermentation system was demonstrated via the simultaneous expression of the C. violaceum glucoamylase and a recombinant protein of interest (eGFP), resulting in a fourfold increase in yield when grown in media containing starch compared with the glucose equivalent.ConclusionsThis study has developed, through the secretion of a previously uncharacterised bacterial glucoamylase, a novel amylolytic E. coli strain capable of direct starch to glucose conversion. The ability of this strain to achieve increased cell densities as well as an associated increase in recombinant protein yield when grown on starch compared with an equivalent amount of glucose, demonstrates for the first time a cell engineering approach to enzyme-based fed-batch fermentation.

A Novel Amylolytic Enzyme from Palaeococcus Ferrophilus with Malto-Oligosaccharide Forming Ability Belonging to GH13_20 Subfamily

By heterologous expression of a gene from Palaeococcus ferrophilus , a novel recombinant enzyme designated AMPf was obtained and proved to be an amylolytic enzyme with unique catalytic characteristics. The optimal temperature and pH of AMPf were 50 °C and 7.0 respectively. Although sequence analysis and acarbose hydrolysis ability indicated that AMPf belongs to the GH13_20 subfamily, interestingly, this enzyme hardly acts on cyclodextrins and pullulan distinguishing it from most enzymes in this subfamily. AMPf hydrolyzes starches to glucose, maltose, maltotriose, and maltotetrose as main products. AMPf mainly liberates glucose from starch with the concentration of 1% (w/v), while it shows malto-oligosaccharide forming ability with higher starch concentration of 4% (w/v). Also, the 4,6-ethylidene-4-nitrophenyl-maltoheptaose hydrolysis ability further indicates the unique combination endo-acting and glucose releasing exo-acting activtity of AMPf. AMPf could utilize maltose and maltotriose to produce malto-oligosaccharides by transglucosylation activity. It was proven AMPf has application protential in malto-oligosaccharides production.

Molecular Dynamics Gives New Insights into the Glucose Tolerance and Inhibition Mechanisms on β-Glucosidases.

β-Glucosidases are enzymes with high importance for many industrial processes, catalyzing the last and limiting step of the conversion of lignocellulosic material into fermentable sugars for biofuel production. However, β-glucosidases are inhibited by high concentrations of the product (glucose), which limits the biofuel production on an industrial scale. For this reason, the structural mechanisms of tolerance to product inhibition have been the target of several studies. In this study, we performed in silico experiments, such as molecular dynamics (MD) simulations, free energy landscape (FEL) estimate, Poisson–Boltzmann surface area (PBSA), and grid inhomogeneous solvation theory (GIST) seeking a better understanding of the glucose tolerance and inhibition mechanisms of a representative GH1 β-glucosidase and a GH3 one. Our results suggest that the hydrophobic residues Y180, W350, and F349, as well the polar one D238 act in a mechanism for glucose releasing, herein called “slingshot mechanism”, dependent also on an allosteric channel (AC). In addition, water activity modulation and the protein loop motions suggest that GH1 β-Glucosidases present an active site more adapted to glucose withdrawal than GH3, in consonance with the GH1s lower product inhibition. The results presented here provide directions on the understanding of the molecular mechanisms governing inhibition and tolerance to the product in β-glucosidases and can be useful for the rational design of optimized enzymes for industrial interests.

Detection of local Bacillus subtilis producing Endo-β-1,4-glucanase Of Thi Qar province

The study included isolating and diagnosing of Bacillus subtilis different soils at Thi Qar province. The ability of bacterial isolates to produce Endo-β-1,4-glucanase was determined after growing on carboxymethyl cellulose CMC medium, Using Congo red and NaCl. Enzyme Endo-β-1,4-glucanase isolates were identified using biochemical tests and VITEK2 as B. subtilis. Isolates were identified using 16S rRNA tests after extraction of DNA from isolates and amplified by PCR using 27F primers (Forward) and 1492R (Reverse) . All isolates in the current study were positive for this gene and the size of the gene for all isolates was at 1500 pb. After identifying the gene sequences and comparing them with the data available in the Gen Bank, NCBI data showed that they were new strains of B. subtilis strain m1 (MF449304) and B. subtilis strain M2 (MF449461) bacteria. Isolates were recorded in NCBI GenBank and were design for each evolutionary tree isolation by Software MEGA6. After the diagnosis of bacterial isolates, the ideal conditions for the production of Endo-β-1,4-glucanase were changed for incubation period, temperature, pH , Incubator Shake, carbon and nitrogen sources. Enzymatic efficacy was determined using of dinitro salicylic acid DNS detector to detect the glucose releasing glucose molecules Endo-β-1,4-glucanase. The enzyme was produced after growing of bacterial isolates of plant culture containing plant and cardboard residues as natural sources of carbon and alternative to costly industrial sources as a source of carbon at a concentration of 1% at 45 ° C for 48 hours .

Kluyveromyces marxianus yeast enables the production of low FODMAP whole wheat breads

There is evidence that a diet low in Fermentable Oligo-, Di-, Monosaccharides And Polyols (FODMAPs) alleviates symptoms in approx. 70% of the patients suffering from irritable bowel syndrome. Through fructans, wheat containing products are a major source of FODMAPs in the western diet. Although fructans are partially degraded during dough fermentation by Saccharomyces cerevisiae invertase, wheat bread contains notable fructan levels. In this study, it was shown that Kluyveromyces marxianus strain CBS6014 can degrade more than 90% of the fructans initially present in wheat whole meal during bread making, which can be attributed to its high inulinase activity. As K. marxianus CBS6014 was not able to consume maltose during fermentation, alternative sugars (sucrose) or glucose releasing enzymes (amyloglucosidase) had to be included in the bread making recipe to ensure sufficient production of CO2 and high bread quality. Five volatile flavor compounds were produced in significantly different levels when K. marxianus CBS6014 was used as starter culture compared with the conventional S. cerevisiae bakery strain. These differences were, however, not detected when sensory analysis of the crumb was performed. This study demonstrates the potential of inulinase-producing K. marxianus strains for the production of (whole meal) breads low in FODMAPs.

Biochemical characterization and synergism of cellulolytic enzyme system from Chaetomium globosum on rice straw saccharification.

BackgroundEfficient hydrolysis of lignocellulosic materials to sugars for conversion to biofuels and chemicals is a key step in biorefinery. Designing an active saccharifying enzyme system with synergy among their components is considered a promising approach.ResultsIn this study, a lignocellulose-degrading enzyme system of Chaetomium globosum BCC5776 (CG-Cel) was characterized for its activity and proteomic profiles, and synergism with accessory enzymes. The highest cellulase productivity of 0.40 FPU/mL was found for CG-Cel under the optimized submerged fermentation conditions on 1% (w/v) EPFB (empty palm fruit bunch), 2% microcrystalline cellulose (Avicel®) and 1% soybean meal (SBM) at 30 °C, pH 5.8 for 6 d. CG-Cel worked optimally at 50–60 °C in an acidic pH range. Proteomics analysis by LC/MS/MS revealed a complex enzyme system composed of core cellulases and accessory hydrolytic/non-hydrolytic enzymes attacking plant biopolymers. A synergistic enzyme system comprising the CG-Cel, a β-glucosidase (Novozyme® 188) and a hemicellulase Accellerase® XY was optimized on saccharification of alkaline-pretreated rice straw by a mixture design approach. Applying a full cubic model, the optimal ratio of ternary enzyme mixture containing CG-Cel: Novozyme® 188: Accellerase® XY of 44.4:20.6:35.0 showed synergistic enhancement on reducing sugar yield with a glucose releasing efficiency of 256.4 mg/FPU, equivalent to a 2.9 times compared with that from CG-Cel alone.ConclusionsThe work showed an approach for developing an active synergistic enzyme system based on the newly characterized C. globosum for lignocellulose saccharification and modification in bio-industries.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0312-7) contains supplementary material, which is available to authorized users.

Synergistic action of recombinant accessory hemicellulolytic and pectinolytic enzymes to Trichoderma reesei cellulase on rice straw degradation

Synergism between core cellulases and accessory hydrolytic/non-hydrolytic enzymes is the basis of efficient hydrolysis of lignocelluloses. In this study, the synergistic action of three recombinant accessory enzymes, namely GH62 α-l-arabinofuranosidase (ARA), CE8 pectin esterase (PET), and GH10 endo-1,4-beta-xylanase (XYL) from Aspergillus aculeatus expressed in Pichia pastoris to a commercial Trichoderma reesei cellulase (Accellerase® 1500; ACR) on hydrolysis of alkaline pretreated rice straw was studied using a mixture design approach. Applying the full cubic model, the optimal ratio of quaternary enzyme mixture was predicted to be ACR:ARA:PET:XYL of 0.171:0.079:0.100:0.150, which showed a glucose releasing efficiency of 0.173gglc/FPU, higher than the binary ACR:XYL mixture (0.122gglc/FPU) and ACR alone (0.081gglc/FPU) leading to a 47.3% increase in glucose yield compared with that from ACR at the same cellulase dosage. The result demonstrates the varying degree of synergism of accessory enzymes to cellulases useful for developing tailor-made enzyme systems for bio-industry.

Effects of Glucose Releasing Rate on Cell Growth and Performance of Simultaneous Saccharification and Fermentation (SSF) in Sweet Potato Medium for Fuel Ethanol Production Using Saccharomyces cerevisiae

Abstract Sweet potato medium containing 230.0 g•kg-1 liquefied starch was used for simultaneous saccharification and fermentation (SSF) for fuel ethanol production. Glucose releasing rate was controlled by the initial addition of incremental glucoamylase. The increasing rates of glucose concentration display a positive relationship when glucoamylase is added in early stage (0 to 8 hours (h)). Serious cells growth inhibition occurred in the early stage when 1.0 g•kg-1 glucoamylase is added, whereas glucose providing limitation occurred in the batch with 0.2 g•kg-1 glucoamylase added in later stage (64 to 80 h). The optimum dosage of glucoamylase was 0.8 g•kg-1, where a final ethanol concentration of 118.2 g•kg-1 was attained within 72 h. The results of our study suggest cell growth inhibition and substrate providing limitation can be avoided simultaneously by adding a proper dosage of glucoamylase. It is indicated further that cells growth inhibition in early stage in the batch with 1.0 g•kg-1 glucoamylase added was due to the high increasing rate of initial glucose concentration, but not the high overall glucose concentration.

Metabolic syndrome and growth hormone deficiency in adult survivors of childhood acute lymphoblastic leukemia

The purpose of the study was to determine the prevalence of metabolic syndrome, growth hormone deficiency, and cardiovascular risk factors among adult survivors of childhood acute lymphoblastic leukemia (ALL) treated with or without cranial irradiation. Follow-up was undertaken of 75 randomly selected long-term childhood ALL survivors. Testing included fasting insulin, glucose, lipids, and growth hormone (GH) releasing hormone plus arginine stimulation test. The prevalence of metabolic syndrome was compared with population norms from 1999-2002 National Health and Nutrition Examination Study (NHANES) data, and internally between those with and without past cranial irradiation and those with normal (>16.5 microg/L) versus insufficient (9-16.5 microg/L) versus deficient (<9 microg/L) peak GH secretion. The mean subject age was 30 years and the mean time since ALL diagnosis was 25 years. The prevalence of metabolic syndrome did not differ statistically (P = .87) between study subjects (16.6%) and same-age, same-sex population norms (17.5%). However, 60% of subjects treated with cranial irradiation, compared with 20% of those who were not, had 2 or more of the 5 components of metabolic syndrome. Untreated abnormally low GH was present in 64% of subjects overall and 85% of those who received past cranial irradiation. Cranial irradiation was strongly related to GH deficiency, and in turn lower insulin-like growth factor 1 (IGF-1), higher fasting insulin, abdominal obesity, and dyslipidemia, particularly in women. Hematologists who treat childhood ALL patients, and particularly those who provide primary care to adult survivors, should be aware of the potential for long-term GH deficiency and adverse cardiovascular and diabetes risk profiles as a consequence of leukemia treatment.

Aspects of the neuroendocrine control of somatotropic function in calorically restricted dogs and patients with eating disorders: studies with cholinergic drugs

A series of studies was devised in both an experimental model of food deprivation, i.e., beagle dogs undergoing a progressive reduction of calorie intake and adolescent females with anorexia nervosa (AN) in the acute and recovery phase, and in patients with atypical eating disorders. The studies were aimed at ascertaining whether the alleged function of the hypothalamic system inhibitory to growth hormone (GH) secretion, i.e., the somatostatinergic, may account for at least some of the abnormalities of GH secretion present in AN patients (e.g., elevated basal GH levels, paradoxical GH rise after glucose or thyrotropin releasing hormone, etc). Caloric restricted dogs or patients with eating disorders were given an intravenous injection of the physiologic GH-releasing peptide GHRH alone or preceded by pirenzepine, a muscarinic cholinergic antagonist reportedly capable of eliciting hypothalamic release of somatostatin (SS), or pyridostigmine, a muscarinic cholinergic agonist which, conversely, would restrain hypothalamic release of SS. In addition, dogs were challenged with acute administration of glucose or thyrot-ropin-releasing hormone, compounds also thought to act via somatostatinergic influences. Data obtained in dogs under caloric restriction or in AN patients in the acute phase of the disease with drugs affecting cholinergic transmission suggest that the latter is increased in both conditions (only partial suppression of the GHRH-induced GH rise with pirenzepine, failure of pyridostigmine to further enhance the GH response to GHRH). Instead, in the same AN patients in the acute phase tested during recovery, in AN patients during the recovery phase, and in patients with atypical eating disorders, pirenzepine completely suppressed the GH response to GHRH, as it did in controls. Finally, data obtained on basal and GHRH-stimulated GH release in dogs given glucose or thyrot-ropin-releasing hormone and in AN patients given arginine, another compound thought to act via inhibition of somatostatinergic influences, do not support the view that somatostatinergic function is impaired in states of food deprivation.

Differential growth hormone responses to glucose and growth hormone releasing hormone in lactating dairy cows

Dairy cows in early lactation were reported to secrete growth hormone in response to declining glucose concentrations at day 5 but not day 30 post-partum, whereas GH responses to TRH were reported to be enhanced after day 30 post partum. The present study examined GH response following glucose infusion and the effect of GHRH as well as effects of SRIH on GHRH-stimulated GH release. Declining plasma glucose concentrations after glucose infusion stimulated GH release at day 5 post partum but not in nonpregnant, nonlactating cows or in cows at days 30 and 90 post partum. GHRH stimulated GH release on all days tested, but the response was highest at day 30 post partum when compared to other days. Somatostatin infusion inhibited GHRH effects on GH concentrations only at day 30 post partum and in nonpregnant, nonlactating cows. Thus, a differential response of the GH regulatory system could be demonstrated between days 5 and 30 post partum utilizing different stimuli. Evaluation of plasma glucose and free fatty acid concentrations on days 5, 10, 20, and 30 post partum revealed a progressive decrease in FFA but not glucose as lactation progressed. Decreased plasma FFA concentrations were paralleled by a decrease in basal GH, somatomedin-C and epinephrine. Thus, a decline in FFA may be responsible for the disparity between effects of GHRH and glucose on GH release between days 5 and 30 post partum.

Serum growth hormone in patients with carcinoid tumours; basal levels and response to glucose and thyrotrophin releasing hormone.

The regulation of serum growth hormone was studied in 33 consecutive patients with carcinoid tumours; both diurnal serum GH and GH responses to an i.v. glucose load and TRH were assessed. Seventeen of the patients (52%) showed disturbed regulation of serum GH and 10 had at least two abnormal tests. Four patients had clinically overt acromegaly. The diurnal mean serum GH levels were higher (P less than 0.001) in patients with carcinoid tumours than in control subjects and more than one third (41%) had levels in a range similar to that of acromegalic patients without carcinoid tumours. The disturbance in serum GH regulation might have been caused in some patient by tumour secreted growth hormone releasing factor(s) which act directly on pituitary somatotrophs, but other tumour-related non-specific stimulation must be considered. Carcinoid tumours should be considered in the aetiology of acromegaly.

The Influence of Induced Hypoxaemia on the Carbohydrate Metabolism of the Liver with Special Reference to General and Hepatic Circulations : CLINICAL PATHO-PHYSIOLOGICAL STUDIES BY THE HEPATIC VENOUS CATHETERIZATION METHOD

Carbohydrate metabolism of the liver in a resting and fasting condition (Part I), and during induced hypoxaemia by 10% O2 gas inhalation (Part II), was studied in man by the hepatic venous catheterization method.1) Studies in Part I revealed that in the patients with hepatic diseases, there were a functional decrease in the hepatic mechanism for the blood sugar regulation, reduction in the glucose releasing ability of the liver and impairment of the hepatic lactate and pyruvate metabolism. About half of the patients with non-severe types of pulmonary tuberculosis showed abnormalities in carbohydrate metabolism of the liver, suggesting hepatic disturbance.Studies under induced hypoxaemia in Part II revealed that, in the normal subjects, the amount of hepatic glucose release was increased during the induced hypoxaemia, and the amounts of hepatic uptake of lactate and pyruvate were increased. In patients with hepatic disturbances, including those with pulmonary tuberculosis, the studies with hypoxaemia made the various abnormalities of the carbohydrate metabolism noted above more apparent and easy to understand.2) It was found that the amount of glucose supply to the liver regulates the amount of glucose release out of the liver. During the hypoxaemia the organism must cope with an increased extrahepatic demand for glucose as the result of increased anaerobic glycolysis, and adapt to the changed cardiac output. To this end the following sequence of events seem to take place ; an elevation of the hepatic inhibition-threshold of the glucose supply (as irritation) to the glucose release (as reaction) → increased glucose release out of the liver → increased arterial blood glucose concentration → increased glucose supply to the whole body, especially to main glucose consuming organs.In the cases with hepatic disturbances, this hepatic regulation of the blood sugar was reduced.