Thermostability Of Α-amylase Research Articles

Exploring the inhibitory mechanism of p-coumaric acid on α-amylase via multi-spectroscopic analysis, enzymatic inhibition assay and molecular docking

The impact of p-coumaric acid (p-CA) on α-amylase activity was investigated through multi-spectroscopic methods, enzymatic assays and molecular docking. UV–vis analysis suggests that the α-amylase-p-CA complex is stabilised by non-covalent bonds, with molecular docking suggesting that hydrogen bonding, π-π stacking interactions and Van Der Waals forces are mainly responsible for ligand stabilisation within the active site of α-amylase. Fourier transform infrared (FTIR) and circular dichroism (CD) spectra showed that complex formation induced a reduction of α-helix and β-sheet components in α-amylase, while enhancing disordered structures. Fluorescence quenching and Job plot results argue for significant interactions between α-amylase and p-CA, yielding a binding affinity of 2.57 × 104 M−1 and a 1:1 binding stoichiometry. Thermostability of α-amylase was also impacted upon complexation, with increasing concentrations of p-CA reducing the thermal stability of α-amylase. p-CA showed a competitive inhibitory action on α-amylase activity, with the IC50 value calculated to be 3.09 mM, which is comparable to the 2.03 mM of the acarbose positive control. The findings provide a theoretical basis for potential application of p-CA in functional foods or as a nutraceutical.

Hunt for α-amylase from metagenome and strategies to improve its thermostability: a systematic review.

With the advent of green chemistry, the use of enzymes in industrial processes serves as an alternative to the conventional chemical catalysts. A high demand for sustainable processes for catalysis has brought a significant attention to hunt for novel enzymes. Among various hydrolases, the α-amylase has a gamut of biotechnological applications owing to its pivotal role in starch-hydrolysis. Industrial demand requires enzymes with thermostability and to ameliorate this crucial property, various methods such as protein engineering, directed evolution and enzyme immobilisation strategies are devised. Besides the traditional culture-dependent approach, metagenome from uncultured bacteria serves as a bountiful resource for novel genes/biocatalysts. Exploring the extreme-niches metagenome, advancements in protein engineering and biotechnology tools encourage the mining of novel α-amylase and its stable variants to tap its robust biotechnological and industrial potential. This review outlines α-amylase and its genetics, its catalytic domain architecture and mechanism of action, and various molecular methods to ameliorate its production. It aims to impart understanding on mechanisms involved in thermostability of α-amylase, cover strategies to screen novel genes from futile habitats and some molecular methods to ameliorate its properties.

Influence of Calcium Ions on the Thermal Characteristics of α-amylase from Thermophilic Anoxybacillus sp. GXS-BL.

Background: α-Amylases are starch-degrading enzymes and used widely, the study on thermostability of α-amylase is a central requirement for its application in life science and biotech-nology.Objective:In this article, our motivation is to study how the effect of Ca2+ ions on the structure and thermal characterization of α-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL.Methods: α-Amylase activity was assayed with soluble starch as the substrate, and the amount of sugar released was determined by DNS method. For AGXA with calcium ions and without calcium ions, optimum temperature (Topt), half-inactivation temperature (T50) and thermal inactivation (half-life, t1/2) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD methods. 3D structure of AGXA was homology modeled with α-amylase (5A2A) as the template.Results:With calcium ions, the values of Topt, T50, t1/2, Tm and ΔH in AGXA were significantly high-er than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on α-amylase structure with the increased temperature. Based on DSC measurements AGXA underwent thermal denaturation by adopting two-state irreversible unfolding processes. Based on the CD spectra, AGXA without calcium ions exhibited two transition states upon unfolding, including α-helical contents increasing, and the transition from α-helices to β-sheet structures, which was obviously dif-ferent in AGXA with Ca2+ ions, and up to 4 Ca2+ ions were located on the inter-domain or intra-domain regions according to the modeling structure.Conclusion: These results reveal that Ca2+ ions have pronounced influences on the thermostability of AGXA structure.

Comparisons of Barley Malt Amylolytic Enzyme Thermostabilities to Wort Osmolyte Concentrations, Malt Extract, ASBC Measures of Malt Quality, and Initial Enzyme Activities

The hypothesis that wort osmolyte concentration (OC) would correlate much better than malt extract (ME) with barley amylolytic enzyme thermostabilities of malts produced over several days of germination was tested. Seeds of four two-row and four six-row North American barley cultivars were malted in a micromalter and sampled every 24 hr throughout 6 days of germination. α- and β-amylases and limit dextrinase were assayed before and after mashing at 70°C for 30 min to determine thermostabilities. Wort OC, ME, and ASBC measures of malt quality were determined for each day of germination. For all cultivars combined, over all days of germination, wort β-amylase thermostabilities correlated negatively and highly significantly with both wort OC and ME, although more strongly with OC (r = −0.62, P < 0.0001; r = −0.46, P < 0.001, respectively). Correlations of limit dextrinase thermostabilities with wort OC were also much stronger than with ME (r = −0.87, P < 0.0001; r = −0.68, P < 0.0001, respectively). α-Amylase thermostability was either unaffected by or increased after mashing at 70°C. These data suggest that β-amylase and limit dextrinase thermostabilities become more limiting to starch degradation as reflected by OC than as reflected by ME as germination proceeds. β-Amylase intron III allelic variation had no effect on OC or ME in these North American barley cultivars. For all cultivars combined over 6 days of germination, the correlations for β-amylase and limit dextrinase thermostabilities versus the initial activities of α- and β-amylase and limit dextrinase were significant and negative ([β-amylase thermostabilities versus initial activities of α-amylases: r = −0.63, P < 0.0001, β-amylases: r = −0.68, P < 0.0001, and limit dextrinases: r = −0.54, P < 0.0001] and [dextrinase thermostabilities versus initial activities of α-amylase: r = −0.63, P < 0.0001, β-amylase: r = −0.68, P < 0.0001, and limit dextrinase: r = −0.54, P < 0.0001]). These data suggest that selection for high initial activity of any of these amylolytic enzymes would also select for high thermostability of β-amylase and limit dextrinase.

A Comparison of Barley Malt Amylolytic Enzyme Thermostabilities and Wort Sugars Produced during Mashing

This study was conducted primarily to determine the relationship between barley amylolytic enzyme thermolability of malts produced over several days of germination and wort sugar concentrations produced during mashing. Seeds of 4 two-row and 4 six-row North American elite barley cultivars were steeped and germinated in a micromalter. At 24 hour intervals throughout 6 days of germination, green malt was removed and kilned. Malts were assayed for activities of α- and β-amylase and limit dextrinase before and after mashing at 70°C for 30 min to determine thermostabilities. Wort sugars were assayed after mashing. α-Amylase thermostability was either unaffected or higher after the 70°C mashing indicating that it is not thermolabile for 30 min at 70°C. In contrast, malt β-amylase and limit dextrinase thermostabilities greatly decreased between days 1 and 2, indicating a strong developmental influence on this parameter. For all cultivars combined, over all days of germination, malt β-amylase and limit dextrinase thermostabilities correlated negatively and highly significantly with total wort sugars (r = −0.656, P < 0.0001; r = −0.767, P < 0.0001, respectively) and with fermentable wort sugars. Six-row cultivar β-amylase and limit dextrinase thermostabilities correlated slightly better than two-row cultivar thermostabilities with total and individual sugar concentrations (e.g., total sugars: two-row β-amylase and limit dextrinase, r = −0.645, P < 0.0001, r = −0.770, P < 0.0001, respectively; six-row β-amylase and limit dextrinase, r = −0.686, P < 0.0001, r = −0.808, P < 0.0001, respectively). In contrast, the non-fermentable maltodextins, maltotetraose through maltoheptaose, correlated positively and highly significantly with β-amylase and limit dextrinase thermostabilities. These data suggest that β-amylase and limit dextrinase thermostabilities become more limiting to starch degradation as germination proceeds. Also, these data are in direct contrast to correlations of initial activities of these enzymes and wort sugars produced by malts over 6 days of germination. β-Amylase intron III allelic variation had no effect on thermostability or activity on the day of optimal malt modification (day 5) or any other day where LSD analysis was significant, indicating that, as in past studies, North American barley germplasm, β-amylase thermostability, and activity are not influenced by intron III allelic variation.

Thermostable mutant variants of Bacillus sp. 406 α-amylase generated by site-directed mutagenesis

AbstractSeveral mutations are known to increase the thermostability of α-amylase of B. licheniformis and other α-amylases. Site-directed mutagenesis was used to introduce similar mutations into the sequence of the α-amylase gene from mesophilic Bacillus sp. 406. The influence of the mutations on thermostability of the enzyme was studied. It was shown that the Gly211Val and Asn192Phe substitutions increased the half-inactivation temperature (Tm) of the enzyme from 51.94±0.45 to 55.51±0.59 and 58.84±0.68°C respectively, in comparison to the wild-type enzyme. The deletion of Arg178-Gly179 (dRG) resulted in an increase of Tm of the α-amylase to 71.7±1.73°C. The stabilising effect of mutations was additive. When combined they increase the Tm of the wild-type amylase by more than 26°C. Thermostability rates of the triple mutant are close to the values which are typical for industrial heat-stable α-amylases, and its ability to degrade starch at 75°C was considerably increased. The present research confirmed that the Gly211Val, Asn192Phe and dRG mutations could play a significant role in thermostabilization of both mesophilic and thermophilic α-amylases.

The influence of moisture content on the thermostability of Aspergillus oryzae α-amylase

The thermal stability of Aspergillus oryzae α-amylase in systems of different moisture content was investigated. To obtain systems of reduced moisture content the enzyme was freeze-dried with addition of maltodextrin and equilibrated above saturated salt solutions. Other systems applied were aqueous solutions of maltodextrin. The thermal inactivation kinetics could be accurately described by a first-order model in all systems studied except the system at moisture content 0.029 g H 2O/g dry basis, which showed a biphasic inactivation pattern. Reduced moisture content had a significant effect on α-amylase thermostability, the enzyme being far more thermostable in maltodextrin systems at reduced moisture content than in aqueous solutions. When the moisture content decreased from 3.5 g H 2O/g dry basis to 0.029 g H 2O/g dry basis, the temperature range of inactivation increased from 70–75 °C to 100–115 °C. The activation energy ( z-value) of thermal inactivation was also affected by the moisture content in the system.

Modification ofα-Amylase by Sodium Alginate

Sodium alginate, activated by periodate oxidation, was covalently linked to porcine pancreatic α-amylase via reductive alkylation with NaBH4. The enzyme-polymer conjugate, purified by gel filtration on Fractogel EMD BioSEC (S), retained about 50% of the native specific amylolytic activity. The sugar content was estimated to be 712 mol of monosaccharides per mol of enzyme protein. An average of 11 amino groups out of 21 groups from α-amylase were modified with the polysaccharide. The functional stability was improved for α-amylase after conjugation with sodium alginate. In comparison with the native enzyme, the thermostability of α-amylase was increased by this modification. In addition, the stability in the range of pH 5.0–11.0 was improved for the modified enzyme. The conjugate was also more resistant to denaturation by 0.3% sodium dodecylsulphate, retaining about 10% of its initial activity after 120 min of incubation. The formation of stabilizing salt bridges in the protein surface of the α-amylase-polysaccharide complex was confirmed by FT-IR spectrometry. Attending to the results obtained, we conclude that the covalent attachment of the anionic polysac-charide sodium alginate to the enzymes might be a useful and non-expensive method for improving the stabilization of these biocatalysts under various denaturing conditions.

Improved Thermostability of aBacillusα-Amylase by Deletion of an Arginine-Glycine Residue Is Caused by Enhanced Calcium Binding

α-Amylase from alkaliphilicBacillusKSM-1378 (LAMY) is a novel semi-alkaline enzyme which has a high specific activity, a value 5-fold higher than that of aBacillus licheniformisenzyme at alkaline pH. Thermostability of this enzyme could be improved by deletion of the Arg181-Gly182 residue by means of site-directed mutagenesis. The wild-type and engineered LAMYs were very similar with respect to specific activity, pH-activity curve, temperature-activity curve, susceptibility to inhibitors, and pattern of hydrolysis products from soluble starch and maltooligosaccharides. However, the engineered enzyme also acquired increased pH stability and resistance to sodium dodecyl sulfate and especially chelating reagents, such as ethylenediaminetetraacetate and ethyleneglycol-bis (β-aminoethylether)tetraacetate. This is the first report that thermostability of α-amylase is improved by enhanced calcium binding to the enzyme molecule.

Modification of α-amylase from Bacillus licheniformis by the polyaldehyde derived from β-cyclodextrine and α-amylase thermostability

The cleavage of β-cyclodextrine by sodium periodate at the seven 2–3 diols of the glucose unit gives rise to the polyaldehyde 1, used to modify α-amylase. The reductive modification of α-amylase from Bacillus licheniformis reduced the number of reactive lysine groups from 8 to 3.5 per mol of enzyme with an activity loss of 25% and increased the half-life at 80°C from 4.7 to 7.0 minutes.

Increased thermal stability of Bacillus licheniformis α-amylase in the presence of various additives

The influence of various additives such as polyhydric alcohols, sugars, polyethylene glycol, and substrates on the thermostability of Bacillus licheniformis α-amylase has been studied. Results obtained showed a thermostabilizing effect of most of the additives tested (ethylene glycol, glycerol, sorbitol, glucose, maltose, maltotriose, and polyethylene glycol). The stabilizing efficiency appears to be strongly dependent on additive concentration. α-Amylase thermostability has also been assayed in organic solvents. In these media, particularly in octane, α-amylase acquired a remarkable stability since no deactivation occurred at 75°C for at least 90 min.

Role of calcium ion in the thermostability of alpha-amylase produced from Bacillus stearothermophilus.

In 1961, Campbell and Manning provided one clue to elucidate the molecular basis for thermostability of the enzyme from thermophile(1–3). They reported that the α-amylase of B. stearothermophilus in the native state exists in a semi-random or random coiled and well hydrated molecule with slight extent of secondary structure formed by disulfide bonds (2). This less-ordered structure was postulated as the reason for thermostability of the enzyme.