Showing papers on "Amylase published in 2014"

Anti-obesity action of gingerol: effect on lipid profile, insulin, leptin, amylase and lipase in male obese rats induced by a high-fat diet

Abstract: BACKGROUND Obesity represents a rapidly growing threat to the health of populations and diet intervention has been proposed as one of the strategies for weight loss Ginger and its constituents have been used for their anti-flatulent, expectorant and appetising properties and they are reported to possess gastro-protective and cholesterol-lowering properties The present study investigated the effects of gingerol on the changes in body weight, serum glucose, insulin, insulin resistance and lipid profile in plasma and liver as well as on the activity of amylase, lipase and leptin in high-fat diet (HFD)-induced obese rats RESULTS HFD-induced obese rats were treated orally with gingerol (25, 50 and 75 mg kg−1) once daily for 30 days A lorcaserin-treated group (10 mg kg−1) was included for comparison The levels of body weight, glucose, lipid profile and insulin, insulin resistance, leptin, amylase and lipase were increased significantly (P < 005) in HFD rats Rats treated with gingerol and fed a HFD showed significantly (P < 005) decreased glucose level, body weight, leptin, insulin, amylase, lipase plasma and tissue lipids when compared to normal control The effect at a dose of 75 mg kg−1 of gingerol was more pronounced than that of the dose 25 mg kg−1 and 50 mg kg−1 The lorcaserin-treated group also manifested similar effects to those of gingerol CONCLUSION These findings suggested that ginger supplementation suppresses obesity induced by a high fat diet and it might be a promising adjuvant therapy for the treatment of obesity and its complications © 2014 Society of Chemical Industry

Mechanism for starch granule ghost formation deduced from structural and enzyme digestion properties

Abstract: After heating in excess water under little or no shear, starch granules do not dissolve completely but persist as highly swollen fragile forms, commonly termed granule "ghosts". The macromolecular architecture of these ghosts has not been defined, despite their importance in determining characteristic properties of starches. In this study, amylase digestion of isolated granule ghosts from maize and potato starches is used as a probe to study the mechanism of ghost formation, through microstructural, mesoscopic, and molecular scale analyses of structure before and after digestion. Digestion profiles showed that neither integral nor surface proteins/lipids were crucial for control of either ghost digestion or integrity. On the basis of the molecular composition and conformation of enzyme-resistant fractions, it was concluded that the condensed polymeric surface structure of ghost particles is mainly composed of nonordered but entangled amylopectin (and some amylose) molecules, with limited reinforcement through partially ordered enzyme-resistant structures based on amylose (for maize starch; V-type order) or amylopectin (for potato starch; B-type order). The high level of branching and large molecular size of amylopectin is proposed to be the origin for the unusual stability of a solid structure based primarily on temporary entanglements.

PtrBAM1, a β-amylase-coding gene of Poncirus trifoliata, is a CBF regulon member with function in cold tolerance by modulating soluble sugar levels.

Abstract: β-Amylase (BAM) catalyses starch breakdown to generate maltose, which can be incorporated into sugar metabolism. However, the role of BAM genes in cold tolerance is less characterized. In this study, we report the isolation and functional characterization of a chloroplast-localizing BAM-encoding gene PtrBAM1 from Poncirus trifoliata. PtrBAM1 was induced by cold, dehydration and salt, but repressed by maltose. Overexpression of PtrBAM1 in tobacco (Nicotiana nudicaulis) increased BAM activity, promoted starch degradation and enhanced the contents of maltose and soluble sugars, whereas opposite changes were observed when PtrBAM1 homolog in lemon (Citrus lemon) was knocked down. The tobacco overexpressing lines exhibited enhanced tolerance to cold at chilling or freezing temperatures. Under cold stress, higher BAM activity and greater accumulation of maltose and soluble sugars were observed in the overexpressing lines when compared with the wild-type or empty vector transformants. Bioinformatics analysis demonstrated that PtrBAM1 promoter contained a CBF-recognizing element. Yeast one-hybrid assay demonstrated that PtrCBF could interact with the promoter fragment containing the element. Taken together, these results demonstrate that PtrBAM1 is a member of CBF regulon and plays an important role in cold tolerance by modulating the levels of soluble sugars acting as osmolytes or antioxidants.

Agrowaste-based Polyhydroxyalkanoate (PHA) production using hydrolytic potential of Bacillus thuringiensis IAM 12077

Abstract: The study identified the innate enzymatic potential (amylase) of the PHB producing strain B.thuringiensis IAM 12077 and explored the same for cost-effective production of PHB using agrowastes, eliminating the need for pretreatment (acid hydrolysis and/or commercial enzyme). Comparative polyhydroxyalkanoate (PHA) production by B. thuringiensis IAM 12077 in biphasic growth conditions using glucose and starch showed appreciable levels of growth (5.7 and 6.8 g/L) and PHA production (58.5 and 41.5%) with a PHA yield of 3.3 and 2.8 g/L, respectively. Nitrogen deficiency supported maximum PHA yield (2.46 g/L) and accumulation (53.3%). Maximum growth (3.6 g/L), PHB yield (2.6 g/L) and PHA accumulation (72.8%) was obtained with C:N ratio of 8:1 using starch as the carbon source (10 g/L). Nine substrates (agro and food wastes) viz. rice husk, wheat bran, ragi husk, jowar husk, jackfruit seed powder, mango peel, potato peel, bagasse and straw were subjected to two treatments- acid hydrolysis and hydrolysis by innate enzymes, and the reducing sugars released thereby were utilized for polymer production. All the substrates tested supported comparable PHB production with acid hydrolysis (0.96 g/L-8.03 g/L) and enzyme hydrolysis (0.96 g/L -5.16 g/L). Mango peel yielded the highest PHB (4.03 g/L; 51.3%), followed by jackfruit seed powder (3.93 g/L; 29.32%). Varied levels of amylase activity (0.25U-10U) in all the substrates suggested the enzymatic hydrolysis of agrowastes.

Saccharification and liquefaction of cassava starch: An alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process

Abstract: Cassava starch is considered as a potential source for the commercial production of bioethanol because of its availability and low market price. It can be used as a basic source to support large-scale biological production of bioethanol using microbial amylases. With the progression and advancement in enzymology, starch liquefying and saccharifying enzymes are preferred for the conversion of complex starch polymer into various valuable metabolites. These hydrolytic enzymes can selectively cleave the internal linkages of starch molecule to produce free glucose which can be utilized to produce bioethanol by microbial fermentation. In the present study, several filamentous fungi were screened for production of amylases and among them Aspergillus fumigatus KIBGE-IB33 was selected based on maximum enzyme yield. Maximum α-amylase, amyloglucosidase and glucose formation was achieved after 03 days of fermentation using cassava starch. After salt precipitation, fold purification of α-amylase and amyloglucosidase increased up to 4.1 and 4.2 times with specific activity of 9.2 kUmg-1 and 393 kUmg-1, respectively. Concentrated amylolytic enzyme mixture was incorporated in cassava starch slurry to give maximum glucose formation (40.0 gL-1), which was further fermented using Saccharomyces cerevisiae into bioethanol with 84.0% yield. The distillate originated after recovery of bioethanol gave 53.0% yield. An improved and effective dual enzymatic starch degradation method is designed for the production of bioethanol using cassava starch. The technique developed is more profitable due to its fast liquefaction and saccharification approach that was employed for the formation of glucose and ultimately resulted in higher yields of alcohol production.

In vitro α-amylase inhibitory activity and in vivo hypoglycemic effect of methanol extract of Citrus macroptera Montr. fruit.

Abstract: Objective To investigate the therapeutic effects of methanol extract of Citrus macroptera Montr.fruit in α-amylase inhibitory activity (in vitro) and hypoglycemic activity in normal and glucose induced hyperglycemic rats (in vivo).

Salivary proteins of plant-feeding hemipteroids - implication in phytophagy.

Abstract: Many hemipteroids are major pests and vectors of microbial pathogens, infecting crops. Saliva of the hemipteroids is critical in enabling them to be voracious feeders on plants, including the economically important ones. A plethora of hemipteroid salivary enzymes is known to inflict stress in plants, either by degrading the plant tissue or by affecting their normal metabolism. Hemipteroids utilize one of the following three strategies of feeding behaviour: salivary sheath feeding, osmotic-pump feeding and cell-rupture feeding. The last strategy also includes several different tactics such as lacerate-and-flush, lacerate-and-sip and macerate-and-flush. Understanding hemipteroid feeding mechanisms is critical, since feeding behaviour directs salivary composition. Saliva of the Heteroptera that are specialized as fruit and seed feeders, includes cell-degrading enzymes, auchenorrhynchan salivary composition also predominantly consists of cell-degrading enzymes such as amylase and protease, whereas that of the Sternorhyncha includes a variety of allelochemical-detoxifying enzymes. Little is known about the salivary composition of the Thysanoptera. Cell-degrading proteins such as amylase, pectinase, cellulase and pectinesterase enable stylet entry into the plant tissue. In contrast, enzymes such as glutathione peroxidase, laccase and trehalase detoxify plant chemicals, enabling the circumvention of plant-defence mechanisms. Salivary enzymes such as M1-zinc metalloprotease and CLIP-domain serine protease as in Acyrthosiphon pisum (Aphididae), and non-enzymatic proteins such as apolipophorin, ficolin-3-like protein and 'lava-lamp' protein as in Diuraphis noxia (Aphididae) have the capacity to alter host-plant-defence mechanisms. A majority of the hemipteroids feed on phloem, hence Ca++-binding proteins such as C002 protein, calreticulin-like isoform 1 and calmodulin (critical for preventing sieve-plate occlusion) are increasingly being recognized in hemipteroid-plant interactions. Determination of a staggering variety of proteins shows the complexity of hemipteroid saliva: effector proteins localized in hemipteran saliva suggest a similarity to the physiology of pathogen-plant interactions.

Changes in Biochemical Characteristics and Activities of Ripening Associated Enzymes in Mango Fruit during the Storage at Different Temperatures

Abstract: As a part of the study to explore the possible strategy for enhancing the shelf life of mango fruits, we investigated the changes in biochemical parameters and activities of ripening associated enzymes of Ashwina hybrid mangoes at 4-day regular intervals during storage at −10°C, 4°C, and °C. Titratable acidity, vitamin C, starch content, and reducing sugar were higher at unripe state and gradually decreased with the increasing of storage time at all storage temperatures while phenol content, total soluble solid, total sugar, and nonreducing sugar contents gradually increased. The activities of amylase, -mannosidase, -glucosidase, and invertase increased sharply within first few days and decreased significantly in the later stage of ripening at °C. Meanwhile polyphenol oxidase, -galactosidase, and -hexosaminidase predominantly increased significantly with the increasing days of storage till later stage of ripening. At −10°C and 4°C, the enzymes as well as carbohydrate contents of storage mango changed slightly up to 4 days and thereafter the enzyme became fully dormant. The results indicated that increase in storage temperature and time correlated with changes in biochemical parameters and activities of glycosidases suggested the suppression of -galactosidase and -hexosaminidase might enhance the shelf life of mango fruits.

Production and Partial Purification of Alpha Amylase from Bacillus subtilis (MTCC 121) Using Solid State Fermentation

Abstract: Amylase is an enzyme that catalyzes the breakdown of starch into sugars and plays a pivotal role in a variety of areas like use as digestives, for the production of ethanol and high fructose corn syrup, detergents, desiring of textiles, modified starches, hydrolysis of oil-field drilling fluids, and paper recycling. In the present work, solid state fermentation (SSF) for α-amylase production has been used in lieu of submerged fermentation (SmF) due to its simple technique, low capital investment, lower levels of catabolite repression, and better product recovery. Bacillus subtilis has been well known as producer of alpha amylase and was tested using solid state fermentation for 48 hours at 37°C with wheat bran as substrate. Comparison between different fermentation hours demonstrated high yield of alpha amylase after 48 hours. This alpha amylase has optimum pH and temperature at 7.1 and 40°C, respectively. With the goal to purify alpha amylase, 30–70% (NH4)2SO4 cut concentrated the amylase activity threefold with respect to crude fermented extract. This was verified in quantitative DNS assay method as well as in zymogram gel profile. The exact molecular weight of the amylase is yet to be determined with the aid of other protein purification techniques.

Ultrasound irradiation in the production of ethanol from biomass

Abstract: Ethanol produced from renewable biomass, such as lignocellulosic feedstock, is one of the alternative energy resources that can be environmentally friendly. However, physical and chemical barriers caused by the close association of the main components of lignocellulosic biomass, as well as starch, hinder the hydrolysis of cellulose and hemicellulose in lignocellulose as well as amylase and amylopectin in starch to fermentable sugars. One of the main goals of pretreatment for enzymatic hydrolysis is to increase the enzyme accessibility for improving digestibility of cellulose and starch. Ultrasound irradiation applied to cellulosic materials and starch-based feedstock was found to enhance the efficiency of hydrolysis and subsequently increase the sugar yield. Prior research conducted on applying ultrasonic technology for cellulose and starch pretreatment has considered a variety of effects on physical and chemical characteristics, hydrolysis efficiency and ethanol yield. This paper reviews the application of ultrasound irradiation to cellulose and starch prior to and during hydrolysis in terms of sugar and ethanol yields. It also addresses characteristics such as accessibility, crystallinity, degree of polymerization, morphological structure, swelling power, particle size and viscosity as influenced by ultrasonic treatment.

A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda : molecular cloning, heterologous expression and biochemical characterization

Abstract: A novel gene (amyZ) encoding a cold-active and salt-tolerant α-amylase (AmyZ) was cloned from marine bacterium Zunongwangia profunda (MCCC 1A01486) and the protein was expressed in Escherichia coli. The gene has a length of 1785 bp and encodes an α-amylase of 594 amino acids with an estimated molecular mass of 66 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 13 and shows the highest identity (25%) to the characterized α-amylase TVA II from thermoactinomyces vulgaris R-47. The recombinant α-amylase showed the maximum activity at 35 °C and pH 7.0, and retained about 39% activity at 0 °C. AmyZ displayed extreme salt tolerance, with the highest activity at 1.5 M NaCl and 93% activity even at 4 M NaCl. The catalytic efficiency (k cat/K m) of AmyZ increased from 115.51 (with 0 M NaCl) to 143.30 ml mg(-1) s(-1) (with 1.5 M NaCl) at 35 °C and pH 7.0, using soluble starch as substrate. Besides, the thermostability of the enzyme was significantly improved in the presence of 1.5 M NaCl or 1 mM CaCl2. AmyZ is one of the very few α-amylases that tolerate both high salinity and low temperatures, making it a potential candidate for research in basic and applied biology.

Immobilization of thermostable α-amylase from Bacillus licheniformis by cross-linked enzyme aggregates method using calcium and sodium ions as additives

Abstract: Cross-linked enzyme aggregates (CLEAs) of thermostable α-amylase from Bacillus licheniformis have been prepared for starch liquefaction. Among the six different precipitants, tert -butanol performed the best with an aggregation efficiency of 99.54% for starch hydrolysis. The optimal conditions for the immobilization process required 5 mM glutaraldehyde, 0.96 mg/mL enzyme, 1:2 ratio (0.5 ratio) of enzyme/bovine serum albumin (BSA), and 12 h crosslinking at 2–3 °C. Starch was used as the main substrate in enzyme assay. Immobilization did not affect pH (5.5) and temperature (95 °C) optima of free α-amylase thus, these values remained constant for produced CLEAs. Different concentrations of calcium and sodium ions were added to the enzyme during the aggregation process. It was observed that simultaneous addition of both calcium and sodium (1200 ppm of calcium and 400 ppm of sodium ions, in a ratio of 3:1) significantly improved the catalytic efficiency ( k cat / K m ), of CLEAs–BSA–CN from 3.91 × 10 5 to 4.57 × 10 5 (1.2-folds) compared with free enzyme. Although immobilization did not significantly affect V max (5.35–5.25), substrate affinity of the enzyme increased (1.34–1.12) after addition of mixed ions to the prepared CLEAs. Moreover, compared with free α-amylase, enzyme half-life ( t 1/2 ) of CLEAs–BSA–CN increased from 43.31 to 115 min (about 3-folds) at 110 °C. The CLEAs–BSA–CN retained 76% residual activity even after 10 cycles of reuse.

In vitro investigation of the potential health benefits of wild Mediterranean dietary plants as anti-obesity agents with α-amylase and pancreatic lipase inhibitory activities.

Abstract: BACKGROUND Inhibition of digestive enzymes is one of the most widely studied mechanisms used to determine the potential efficacy of natural products as anti-obesity agents. In vitro studies reported here were performed to evaluate the inhibitory activity of formulations of edible plants from Italy on amylase and lipase by monitoring the hydrolysis of nitrophenyl caprilate and the hydrolysis of glycoside bonds in digestible carbohydrate foods. RESULTS The formulation obtained from Capparis sicula exhibited the strongest inhibitory effect on pancreatic lipase (IC50 = 0.53 mg mL−1) while the Borago officinalis formulation exhibited the strongest inhibitory effect on α-amylase (IC50 = 31.61 µg mL−1). In order to characterise the extracts, high-performance thin-layer chromatography analysis of the formulations was performed, revealing the predominance of (±)-catechin in Mentha aquatica formulation, rutin in C. sicula, and caffeic acid and chlorogenic acid in Echium vulgare. CONCLUSION The results obtained indicated that the extracts of C. sicula and B. officinalis could be good candidates for further studies to isolate pancreatic lipase and α-amylase inhibitors, respectively. © 2013 Society of Chemical Industry

Starch bioengineering affects cereal grain germination and seedling establishment

Abstract: Cereal grain germination is central for plant early development, and efficient germination has a major role in crop propagation and malting. Endosperm starch is the prime energy reserve in germination and seedling establishment. In this study, it was hypothesized that optimized starch granule structure, and not only the endosperm starch content per se, is important for germination and seedling establishment. For that purpose, wild-type (WT), and specifically engineered degradable hyperphosphorylated (HP) starch and more resistant amylose-only (AO) starch barley lines were used. The transgenics showed no severe phenotypes and the WT and HP lines degraded the starch similarly, having 30% residual starch after 12 d of germination. However, the AO line showed significant resistance to degradation, having 57% residual starch. Interestingly, protein and β-glucan (BG) degradation was stimulated for both HP and AO lines as compared with the WT. At late seedling establishment stages, specific sugars were rapidly consumed in the AO line. α-Amylase activity was distinctly suppressed in both the HP and the AO lines. Pre-germination β-amylase deposition was low in the AO grains and β-amylase was generally suppressed in both HP and AO lines throughout germination. As further supported by scanning electron microscopy and histochemical analyses on grain and seedlings, it was concluded that inadequate starch granule deposition in combination with the suppressed hydrolase activity leads to temporal and compensating re-direction of starch, sugar, and protein catabolism important to maintain metabolic dynamics during grain germination and seedling establishment.

High-resolution α-amylase assay combined with high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy for expedited identification of α-amylase inhibitors: proof of concept and α-amylase inhibitor in cinnamon.

Abstract: Type 2 diabetes affects millions of people worldwide, and new improved drugs or functional foods containing selective α-amylase inhibitors are needed for improved management of blood glucose. In this article the development of a microplate-based high-resolution α-amylase inhibition assay with direct photometric measurement of α-amylase activity is described. The inhibition assay is based on porcine pancreatic α-amylase with 2-chloro-4-nitrophenyl-α-d-maltotriose as substrate, which this gives a stable, sensitive, and cheap inhibition assay as requested for high-resolution purposes. In combination with HPLC–HRMS–SPE–NMR, this provides an analytical platform that allows simultaneous chemical and biological profiling of α-amylase inhibitors in plant extracts. Proof-of-concept with an artificial mixture of six compounds—of which three are known α-amylase inhibitors—showed that the high-resolution α-amylase inhibition profiles allowed detection of sub-microgram amounts of the α-amylase inhibitors. Furthermore,...

Production of Fungal Amylases Using Cheap, Readily Available Agriresidues, for Potential Application in Textile Industry

Abstract: The study aimed at isolation and screening of fungal amylase producer, optimization of solid state fermentation conditions for maximum amylase production by the best amylase producer, and characterization of the crude amylases, so produced. Aspergillus fumigatus NTCC1222 showed the highest amylase activity (164.1 U/mL) in secondary screening under SSF conditions and was selected for further studies. The test strain showed maximum amylase production (341.7 U/mL) and supernatant protein concentration (9.7 mg/mL) for incubation period (6 days), temperature (35°C), initial pH (6.0), nutrient salt solution as moistening agent, and beef extract as nitrogen source. Pomegranate peel produced maximum amylase activity, but wheat bran (only slightly lesser amylase activity as compared to that of pomegranate peel) was chosen for further studies, keeping in mind the seasonal availability of pomegranate peel. TLC confirmed the amylase produced to be α-type and 60 kDa was the molecular weight of the partially purified amylase. The enzyme showed maximum enzyme activity at pH 6.0, temperature of 55°C, and incubation time of 60 minutes. UV (616.0 U/mL) and chemical (814.2 U/mL) mutation enhanced amylase activity as compared to wild test strain. The study indicates that Aspergillus fumigatus NTCC1222 can be an important source of amylase and the crude enzyme, hence obtained, can be cost effectively applied in multiple sections of textile wet processing.