Acidic Α-amylase Research Articles

Nutrient Diagnostics and Biochemical Indicators of Oat Grain Quality Predicted by Leaf Juice Amino Acid Concentration

In order to find out the relationship between the level of nitrogen nutrition of oat plants of the Yakov variety and the content of amino acids in leaf juice, as well as the possibility of using this indicator to assess the content and composition of proteins, the activity of amylases, catalases and peroxidases in ripe grain and the activity of theseenzymes in germinating grain, a field experiment was carried out at the Field Experimental Station of Russian State Agrarian University – Moscow Timiryazev Agricultural Academy, on sod-podzolic medium loamy soil. In the field experiment, variants with different levels of nitrogen nutrition of oat plants that had passed post-harvest ripening were tudied. Ammonium nitrate, double superphosphate, and potassium chloride were applied in advance. As a result, it was found that as the level of nitrogen nutrition of plants increased, the concentration of amino acids in the juice of oat leaves at the stage of formation of the first stem node decreased, which was closely correlated with the nitrogen doses. These data indicate the possibility of using this indicator for the diagnosis of nitrogen nutrition in oat plants. The studies also revealed a reliable correlation between the content of amino acids in leaf juice and the concentration of proteins soluble in water and solutions of 10% KCl, 0.2% NaOH, as well as the activity of acid (pH 5.5), neutral (pH 7) and alkaline (pH 8) isoenzymes of α-amylase and acid isoforms of β-amylase in ripe grain. In addition, the amino acid content of leaf juice was found to be significantly correlated with the activity of acidic α-amylase, acidic and neutral β-amylase in germinated grain. The results obtained demonstrate the possibility of predicting the level of protein accumulation in oat grains. It is also possible to predict the protein composition, activity of α- and β-amylase, catalase, and peroxidase isoenzymes from the content of amino acids in leaf juice in the phase of the first stem node formation. A reliable correlation was also found for the activity of acid and neutral isoenzymes of α-amylase, catalase, and peroxidase in ripe and germinated oat grain.

Production, purification and applications of raw starch degrading and calcium-independent α-amylase from soil rich in extremophile

Calcium independent, raw starch hydrolyzing, acidic α-amylase (66 kDa) was synthesized by Bacillus subtilis S113 that is an aerobic, rod-shaped and Gram +ve bacteria. Purification of the enzyme was performed by HiTrap Capto Q (Ion-exchange chromatography; 19.28 fold; 22.41% yield). The purified enzyme was found stable at broad acidic pH (4–6.5) and high-temperature range (40–80 °C), that fulfilled the necessary criteria and laid the foundation to be utilized in starch saccharification industry. Kinetic studies of the enzyme revealed that Km and Vmax of the enzyme was 0.22% and 357.14 U/mg respectively. Scanning electron microscopy studies showed that the enzyme was capable of completely hydrolyzing raw wheat and potato starch, further confirming its role in the starch industry. It was found that only 7.93% of the activity was loss at 4 °C when kept for one year.

Catalytic and thermodynamic properties of an acidic α-amylase produced by the fungus Paecilomyces variotii ATHUM 8891.

An extracellular acid stable α-amylase from Paecilomyces variotii ATHUM 8891 (PV8891 α-amylase) was purified to homogeneity applying ammonium sulfate fractionation, ion exchange and gel filtration chromatography and exhibited a reduced molecular weight of 75kDa. The purified enzyme was optimally active at pH 5.0 and 60°C and stable in acidic pH (3.0-6.0). K m, v max and k cat for starch hydrolysis were found 1.1g L-1, 58.5 μmole min-1 (mg protein)-1, and 73.1s-1, respectively. Amylase activity was marginally enhanced by Ca2+ and Fe2+ ions while Cu2+ ions strongly inhibited it. Thermodynamic parameters determined for starch hydrolysis (Ε α, ΔH*, ΔG*, ΔS*, and ) suggests an effective capacity of PV8891 α-amylase towards starch hydrolysis. Thermal stability of PV8891 α-amylase was assessed at different temperatures (30-80οC). Thermodynamic parameters ( , ΔH*, ΔG*, ΔS*) as well as the integral activity of a continuous system for starch hydrolysis by the PV8891 α-amylase revealed satisfactory thermostability up to 60°C. The acidic nature and its satisfactory performance at temperatures lower than the industrially used amylases may represent potential applications of PV8891 α-amylase in starch processing industry.

Production, purification and characterization of raw starch hydrolyzing thermostable acidic α-amylase from hot springs, India

Alpha-amylase is an important hydrolytic enzyme used for various industrial processes. In the present study, Geobacillus bacterium (K1C), producing a thermostable α-amylase was isolated from Manikaran hot springs, India. We have purified and characterized the biochemical properties of α-amylase. The optimum temperature and pH for α-amylase activity was 80 °C and pH 6.0 respectively. The far-UV CD spectra of the enzyme indicated the presence of random coil conformation and showed an intermediate phase during temperature-induced unfolding. In the presence of substrate, thermostability of the α-amylase was increased as 50% initial activity was retained at 70 °C for 6 h and at 80 °C for 2 h. Moreover, the enzyme also showed remarkable pH stability as 90% of the initial activity was retained even after 48 h of incubation at pH 5.0, 6.0 and 7.0. Interestingly, amylase activity of the purified enzyme was Ca2+independent, whereas the complete inhibition of activity was observed in the presence of Cu2+, Pb2+, and Hg2+. The purified α-amylase was stable in the presence of detergents, organic solvents and Proteinase K. Furthermore, it exhibited the ability to hydrolyze raw starches (e.g. rice, wheat, corn, potato) efficiently; thus this enzyme has the potential to be used for industrial applications.

Acidic α-amylase of Bacillus acidicola: Attempts in Ameliorating Catalytic Efficiency and Production

Acidic α-amylase of Bacillus acidicola: Attempts in Ameliorating Catalytic Efficiency and Production

Production of Chimeric Acidic α-Amylase by the Recombinant Pichia pastoris and Its Applications

Recombinant chimeric α-amylase (Ba-Gt-amy) has been produced extracellularly in Pichia pastoris under AOX promoter. Clones of P. pastoris with multiple gene copies have been generated by multiple transformations and post-transformational vector amplification, which led to 10.7-fold enhancement in α-amylase titre as compared to a clone with a copy of the gene. The recombinant P. pastoris integrated eight copies of Ba-Gt-amy in the genome of P. pastoris, as revealed by real time PCR data analysis. Heterologous protein expression as well as mRNA level of Ba-Gt-amy was higher in multi-copy clone than that with single copy. The pure Ba-Gt-amy expressed in P. pastoris is a glycoprotein of 75 kDa, which is optimally active at pH 4.0 and 60°C with T1/2 of 40 min at 70°C. The Kinetic parameters and end product analysis suggested that glycosylation has no effect on catalytic properties of Ba-Gt-amy. The enzyme saccharifies soluble as well as raw starches efficiently and generates maltose and maltooligosaccharides, thus, useful in baking and sugar syrup industries. The strategy for generating multi-copy clones is being reported for the first time, which could be useful in enhancing the production of other recombinant proteins.

Single-site mutation of C363G or N463T strengthens thermostability improvement of IG181–182 deleted acidic α-amylase from deep-sea thermophile Geobacillus sp.

ABSTRACTThe acidic α-amylase Gs4j-AmyA from deep-sea thermophile Geobacillus sp. is more acid-resistant than the commercial sources as it displays more than 50% of the optimum at a range of pH 4.5–7. This may allow the removal of the step of pH adjustment in starch processing and save costs. Unfortunately, this amylase is not very stable at 90–95°C. Therefore, to develop a new commercial acidic α-amylase targeted to the food industry, it is necessary to further improve the thermostability of the α-amylase Gs4j-AmyA. In this study, 11 different α-amylase mutants were obtained by site-mutagenesis. Among them, the mutant IG181–182* (IG181–182 deletion) showed significant improvement in thermostability, whose half-life at 70°C was 63.4 times longer than the wild type. Interestingly, single-site mutants C363G and N463T showed no enhancement on thermostability, while the half-lives of the combination mutants IG181–182*/C363G and IG181–182*/N463T at 70°C were further extended by 16.8 and 38.7%, respectively. Unfortunately, the catalytic constants (kcat) of the mutants C363G, N463T, IG181–182*/C363G and IG181–182*/N463T declined by 59, 49, 37 and 16%, respectively. The optimum temperature (65–70°C) and pH (5.5–5.6) of the mutants was unchanged. The thermostability improvement by IG181–182 deletion could be strengthened by synchronous C363G or N463T mutation.

Production of Ca2+-Independent and Acidstable Recombinant α-Amylase of Bacillus acidicola Extracellularly and its Applicability in Generating Maltooligosaccharides.

The recombinant acidstable α-amylase (Ba-amy) of acidophilic bacterium Bacillus acidicola TSAS1 has been produced extracellularly using a combination of cloning (E. coli and P. pastoris) and physico-chemical treatment strategies. A total of 150,000U/L of Ba-amy were attained under constitutive promoter in P. pastoris, which is 15-fold higher than that of the wild strain B. acidicola (10,000U/L). The recombinant P. pastoris integrated two copies of Ba-amy under GAP promoter. The pure Ba-amy expressed in P. pastoris is a glycoprotein of 66kDa, which is optimally active at pH 4.0 and 60°C with a T 1/2 of 25min at 70°C. The K m, V max and K cat values of the recombinant Ba-amy are 1.66mg/mL, 53.6µmol/mg/min and 106.8/s, respectively. The enzyme generates maltose (30%), maltotriose (20%) and other higher maltooligosaccharides from starch, thus, useful in baking as an antistale. This is the first report on the optimization of extracellular production of recombinant acidic α-amylase of an acidophilic bacterium.

Enhancing the production of recombinant acidic α-amylase and phytase in Pichia pastoris under dual promoters [constitutive (GAP) and inducible (AOX)] in mixed fed batch high cell density cultivation

The methylotrophic yeast Pichia pastoris is widely used as an expression host for heterologous protein production using methanol as a sole carbon source. Slow growth rate and low specific productivity in methanol are, however, bottlenecks for its utility at industrial scale production. We have, therefore, developed a strategy for overcoming the bottlenecks and improving production of heterologous proteins. Four recombinant P. pastoris strains (Amy-AOX and Phy-AOX that produce α-amylase and phytase under only AOX promoter, and Amy-GAP-AOX and Phy-GAP-AOX that produce α-amylase and phytase under both AOX and GAP promoters) were generated. This has led to 1.8- and 1.3-fold improvement in α-amylase and phytase production in mixed fed batch cultivation as compared to that of Amy-AOX and Phy-AOX. The recombinant strains Phy-GAP-AOX integrated five copies of Phy (3 copies under AOX promoter and two copies under GAP promoter), while Phy-AOX integrated 3 copies of Phy under AOX promoter. On the other hand Amy-GAP-AOX integrated a total of 3 copies of Amy (one copy under AOX promoter and two copies under GAP promoter), while Amy-AOX integrated one copy under AOX promoter. The data suggest that this strategy would be useful in producing high titres of other heterologous proteins too in P. pastoris.

Purification and Partial Characterization of an Acidic α-Amylase from a Newly Isolated Bacillus subtilis ZJ-1 that may be Applied to Feed Enzyme

An amylase-producing strain was isolated from soy sauce and designated as Bacillus subtilis ZJ-1. Purification of α-amylase from B. subtilis ZJ-1 to homogeneity by ethanol fractionation, ultrafiltration, and Sephadex G-100 gel filtration resulted in recovery of 8.9% and a specific activity of 542.7 U/mg protein. The molecular mass was estimated to be 58 kD by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme reached its maximum activity at a pH of 5.0 and a temperature of 50°C. The enzyme remained at 89.4 ± 3.0% of its activity at 40°C. The enzyme retained 87.7 ± 3.7% and 63.4 ± 2.9% of its original activity at 40°C after a 60-min incubation in the presence of 5 mM CaCl2 at a pH of 5.0 and 4.0, respectively. These properties indicate that the novel enzyme has a theoretically high survival rate and excellent starch catalytic efficiency in the typical chicken gastrointestinal-tract environment (pH 3.5–7.0, 40°C). In addition, the enzyme remained at 78.4 ± 3.6% of its activity after a 5-min incubation at 80°C, which demonstrates that the enzyme could maintain a high survival rate in the pelleting process of feed production. The characteristics just described make this enzyme a good candidate for use as a chicken feed enzyme.

Characteristics of a High Maltose-Forming, Acid-Stable, and Ca2+-Independent α-amylase of the Acidophilic Bacillus acidicola

The purified acidic α-amylase of Bacillus acidicola is a monomer of 66.0 kDa, optimally active at pH 4.0 and 60 °C. The enzyme is Ca2+ independent with T1/2 for 18 min at 80 °C. The Km, Vmax, and catalytic efficiency (kcat/Km) of the enzyme are 1.6 mg mL(−1), 23.8 μmol mg(−1) min(−1), and 981 μmol s(−1), respectively. Among detergents, Tween 20, 40, and 80 stimulated enzyme activity, whereas sodium dodecyl sulfate and Triton X-100 inhibited even at low concentration. EGTA has not affected the activity, whereas EDTA β-mercaptoethanol, iodoacetic acid, and Dithiothreitol exhibited a slight inhibitory action. Phenylmethanesulfonyl fluoride, N-bromosuccinimide, and Hg2+ strongly inhibited enzyme activity. The experimental activation energy and temperature quotient are 50.12 kJ mol(−1) and 1.37. When thermodynamic parameters (ΔH and ΔS) of the enzyme have been determined at different temperatures, ΔG is positive suggesting that the enzyme is thermostable. The enzyme hydrolyzes raw starches, and therefore, the enzyme finds application in raw starch saccharification at sub-gelatinization temperatures that saves energy needed for gelatinization of raw starch at 105 °C.

Structural and biochemical features of acidic α-amylase of Bacillus acidicola

The investigation is aimed at understanding structure–function aspect of α-amylase of an acidophilic bacterium Bacillus acidicola (BAamy), which is Ca2+-independent and active at acidic pH of native starch, and thus, suits better in starch saccharification process. The CD spectroscopic data analysis revealed that the enzyme has 30% α-helices, 14.2% β-sheets, and 55.8% random coils at 60°C and pH 4.0. Using Bacillus stearothermophilus α-amylase (BStA) as the template, 3-D structure of rBAamy has been proposed. A complete loss in α-amylase activity was recorded when the amino acid residues (D231, E261 and D328) were substituted that confirmed their role in catalysis. The CD studies indicated a decrease in the α-helices content below and beyond the optimum pH and temperature that suggests a critical role of α-helix in maintaining the structural conformation of the enzyme. Fluorescence-quenching by N-bromosuccinimide (NBS) suggested the role of tryptophan in maintaining structural integrity of α-amylase and that by acrylamide indicated interaction by simple collision process.

Identification of an acidic α-amylase from Alicyclobacillus sp. A4 and assessment of its application in the starch industry

An acidic α-amylase was purified from thermoacidophilic Alicyclobacillus sp. A4 by ion exchange chromatography with 22% recovery, and showed a molecular mass of 64kDa by SDS–PAGE. Its amino acid sequence was determined by sequencing three internal peptides and the complete genome of strain A4, and shared highest identity (64%) with Alicyclobacillusacidocaldarius α-amylase. Compared with other reported α-amylases, the purified enzyme had some distinct characteristics. The optimal activity was found to occur at 75°C and pH 4.2, similar to the glucamylase widely used in the starch industry. The enzyme was Ca2+ independent, and had strong ability to digest raw starch (96.71%) with commercial glucamylase in one step. These properties of the purified enzyme make up the deficiency of the commercial α-amylases currently used and avoid repeated adjustment of pH and temperature in double-enzymatic sugar-making process. The purified enzyme will be commercially valuable in the starch industry.

Optimization of medium components and cultural variables for enhanced production of acidic high maltose-forming and Ca2+-independent α-amylase by Bacillus acidicola

The production of acidic α-amylase by a novel acidophilic bacterium Bacillus acidicola TSAS1 was optimized in submerged fermentation using statistical approaches. The process parameters that significantly affected α-amylase production (starch, K(2)HPO(4), inoculum size and temperature) were identified by Plackett and Burman design. The optimum levels of the significant variables as determined using central composite design of response surface methodology are starch (2.75%), K(2)HPO(4) (0.01%), inoculum size [2% (v/v) containing 1.9×10(8) CFU ml(-1)], and temperature (33°C). An overall 2.4 and 2.9-fold increase in enzyme production has been attained in batch and fed-batch fermentations in the laboratory fermentor, respectively.

Enzyme activity for monitoring the stability in a thermophilic anaerobic digestion of wastewater containing methanol

To maintain good conditions in a thermophilic methane bioreactor treating methanol as the main substrate, microbial enzyme activity in the reactor was investigated. In a preliminary study, seven enzymes were tested for their suitability as indicators using an acid-former, 22a originating from a digester with low efficiency, Methanosarcina sp. (CHTI-55) and Desulfotomaculum nigrificans (Delft 74). Among the tested strains, the activities of seven enzymes were the highest in 22a. Acidic phosphatase (ACP), glutamic-pyruvic transaminase (GPT) and α-amylase (AMY) were chosen as hopeful indicators for lab-scale tests and their activities were measured at the optimum temperature of 55°C. In the lab-scale test, reactor failure was induced by nitrogen deficiency or addition of dimethyl disulfide (DMDS) as an inhibitor. ACP, GPT and AMY outperformed the conventional parameters as indicators of any instability in the process.