Native Α-amylase Research Articles

The effect of chemical modification using citraconic anhydride on the stability of α-amylase from Aspergillus fumigatus

Abstract The purpose of this research is to improve the stability of α-amylase from Aspergillus fumigatus by chemical modification with citraconic anhydride. The α-amylase was isolated using a centrifugation technique, followed by purification using precipitation and dialysis of ammonium sulfate salt. The experimental results demonstrate that the purity of purified α-amylase is 13.41 times higher than that of the crude extract. A significant increase in the optimum temperature was also achieved, in which the optimum temperature of 50 °C was found for native α-amylase, while for modified α-amylase, the optimum temperature of 60 °C was found. Similarly, an increase in half-life was also evident, which is 38.72 min found for the native enzyme to 256.67–330.00 min for modified α-amylase, depending on the volume of citraconic anhydride used. Modification also resulted in increased free energy values (ΔG i) from 104.348 for the native enzyme to 109.585–110.281 kJ mol−1 for modified α-amylase, indicating that modified α-amylase is stiffer than native α-amylase. The results obtained in this work demonstrate that citric anhydride is a very promising modifying agent to improve the stability and performance of α-amylase enzyme isolated from A. fumigatus. The findings of this study also offer an opportunity for the application of citric anhydride for other enzymes.

The stability increase of α-amylase enzyme from Aspergillus fumigatus using dimethyladipimidate

Abstract This study’s purpose is to improve the α-amylase enzyme’s stability from Aspergillus fumigatus applying dimethyladipimidate (DMA). It was conducted in different stages, including production, isolation, purification, modification, and the characterization of native and modified enzymes by the DMA addition. The enzyme activity was specified using the Fuwa and Mandels methods, while the protein level was conducted by the Lowry method. The results indicated that the native enzyme contains a specific activity of 7010.42 U/mg, with an increase of 7.8 times than the crude extract, which contains 904.38 U/mg. Meanwhile, the native enzyme contains an optimum pH of 5 at 55 °C, with residual activity of 17.17% after 60 min of incubation at 55 °C and a half-life of 25.86 min. After the DMA addition with 0.5, 1, and 1.5% concentration, the enzymes had 5.5 optimum pH and 65 °C temperature. Meanwhile, after 60 min of incubation at 65 °C, the modified enzymes had 54.17, 46.18, and 34.44% of residual activity, and 85.55 58.25 and 37.46 min of half-lives, respectively. This showed that the addition of DMA to the native α-amylase from A. fumigatus increased the stability of the modified enzymes by 1.5–3.3 times than the native enzyme.

Cloning and characterization of alpha-amylase from a clinical isolate of staphylococcus aureus resistant to vancomycin

Staphylococcus aureus (S. aureus) is one of the prominent gram positive human pathogen secretes many surface and secretary proteins including various enzymes and pathogenic factors that favour the colonization and infection of host tissue. Alpha-amylase is one of the enzymes secreted by S.aureus which catalyses the breakdown of complex sugars to monosaccharides, which are required for colonization and survival of this pathogen. In the present study we have cloned, sequenced, expressed and characterized α-amylase from the clinical isolate of S.aureus resistant to vancomycin (VRSA). The 25kb plasma DNA was isolated from the clinical isolate of S.aureus and a 600bp PCR amplified product was obtained. The SDS-PAGE analysis of induced and un-induced clone SB3 indicates that α-amylase has a M.W. of 22KD. The rα-amylase was eluted from the gel and enzymes assay was performed. The Km was found to be close to Km of native α-amylase identified in the extracellular and in the cytosolic fraction of S.aureus resistant to vancomycin. The current study clearly indicate that there is clear distinction between the α-amylase of human and different prokaryotes, particularly S.aureus which made this enzyme an ideal target in the development of drugs against infections of tooth and oral cavities.

Increasing Stability of a-amylase Obtained from Bacillus subtilis ITBCCB148 by Immobilization with Chitosan

In this research, the immobilization of α-amylase from Bacillus subtilis ITBCCB148 by crosslinking method on chitosan matrix has been performed. This research aims to know the effect of immobilization on the thermal stability of α-amylase. The results showed that the native α-amylase has an optimum temperature of 65oC, KM = 1.6 mg mL-1 substrate, and Vmax = 39.7 µmol mL-1 min-1. The immobilized α-amylase has optimum temperature of 75oC, KM = 3.5 mg mL-1 substrate, and Vmax = 7.05 µmol mL-1 min-1. The residual activity of the native and immobilized enzyme on thermal stability test at 65oC for 80 minutes was 58% and 86.15%, respectively. The immobilized enzyme can be reused up to six repeated cycles.The thermodynamic data of native enzyme was t½ = 113.6 min, ki = 6.1x10-3 min-1, and ΔGi = 107.3 kJ mol-1, while the immobilized enzyme was t½ = 433.1 min, ki= 1.6x10-3 min-1, and ΔGi 111.1 kJ mol-1. Based on the decrease of ki, and the increase of ΔGi and half-life(t½) values, the immobilization of α-amylase with chitosan can increase the thermal stability of this enzyme.

Secretory production of N-glycan-deleted glycoprotein in Aspergillus oryzae

The pharmaceutical industry has a high demand for glycoprotein production. The glycoform of glycoproteins is crucial for pharmacological activity. However, in general, cells produce glycoproteins with a heterologous glycoform, which is unfavorable for making uniform, efficacious therapeutic proteins. Here, to produce more glycoproteins with N-glycan uniformity, we applied the GlycoDelete strategy, in which endo-β-N-acetylglucosaminidase (ENGase) from the fungus Hypocrea jecorina (EndoT) is expressed at the Golgi membrane to cleave N-glycan from secretory glycoproteins, to Aspergillus oryzae cells. First, we selected candidate transmembrane domains to target EndoT to the Golgi membrane in A. oryzae cells, generated constructs for expressing the transmembrane-fused EndoT proteins and produced four potential AoGlycoDelete strains. We then confirmed that these strains produced α-amylase with a molecular weight lower than that of native α-amylase without an effect on growth. To test whether the A.oryzae α-amylase proteins had been cleaved by EndoT, we expressed and purified HA-tagged α-amylase AmyB and glucoamylase GlaA proteins from the AoGlycoDelete strain. MS and N-glycan analyses of the intact proteins confirmed neither AmyB-HA nor GlaA-HA produced from the AoGlycoDelete strain contained N-glycan. Lastly, we determined the enzymatic activities of the amylases produced by the AoGlycoDelete strain, which showed that the lack of N-glycan did not affect their activity under the conditions tested. Collectively, our findings demonstrate successful generation of an AoGlycoDelete strain that might be a good candidate for producing pharmaceutical glycoproteins with a uniform N-glycan structure.

Effect of differential processing of the native and recombinant α-amylase from Bacillus amyloliquefaciens JJC33M on specificity and enzyme properties

AmyJ33, an α-amylase isolated from Bacillus amyloliquefaciens JJC33M, has been characterized as a non-metalloenzyme that hydrolyzes raw starch. In this work, the gene that codifies for AmyJ33 was isolated and cloned. The recombinant α-amylase (AmyJ33r) produced had a molecular weight of 72kDa, 25kDa higher than the native α-amylase (AmyJ33). Our results suggest that the C-terminal was processed in a different way in the native and the recombinant enzyme causing the difference observed in the molecular weight. Additionally, the enzyme activity, specificity and biochemical behavior were affected by this larger C-terminal extra region in AmyJ33r, since the enzyme lost the ability to hydrolyze raw starch compared to the native but increased its thermal stability and pH stability, and modified the profile of activity toward alkaline pH. It is suggested that the catalytic domain in recombinant enzyme, AmyJ33r, could be interfered or blocked by the amino acids involved in the C-terminal additional region producing changes in the enzyme properties.

Random and direct mutagenesis to enhance protein secretion in Ashbya gossypii

To improve the general secretion ability of the biotechnologically relevant fungus Ashbya gossypii, random mutagenesis with ethyl methane sulfonate (EMS) was performed. The selection and screening strategy followed revealed mutants with improved secretion of heterologous Trichoderma reesei endoglucanase I (EGI), native α-amylase and/or native β-glucosidase. One mutant, S436, presented 1.4- to 2-fold increases in all extracellular enzymatic activities measured, when compared with the parent strain, pointing to a global improvement in protein secretion. Three other mutants exhibited 2- to 3-fold improvements in only one (S397, B390) or two (S466) of the measured activities. A targeted genetic approach was also followed. Two homologs of the Saccharomyces cerevisiae GAS1, AgGAS1A (AGL351W) and AgGAS1B (AGL352W), were deleted from the A. gossypii genome. For both copies deletion, a new antibiotic marker cassette conferring resistance to phleomycin, BLE3, was constructed. GAS1 encodes an β-1,3-glucanosyltransglycosylase involved in cell wall assembly. Higher permeability of the cell wall was expected to increase the protein secretion capacity. However, total protein secreted to culture supernatants and secreted EGI activity did not increase in the Aggas1AΔ mutants. Deletion of the AgGAS1B copy affected cellular morphology and resulted in severe retardation of growth, similarly to what has been reported for GAS1-defficient yeast. Thus, secretion could not be tested in these mutants.

Stepwise Adaptations to Low Temperature as Revealed by Multiple Mutants of Psychrophilic α-Amylase from Antarctic Bacterium

The mutants Mut5 and Mut5CC from a psychrophilic α-amylase bear representative stabilizing interactions found in the heat-stable porcine pancreatic α-amylase but lacking in the cold-active enzyme from an Antarctic bacterium. From an evolutionary perspective, these mutants can be regarded as structural intermediates between the psychrophilic and the mesophilic enzymes. We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: compactness; kinetically driven stability; thermodynamic stability; resistance toward chemical denaturation, and the kinetics of unfolding/refolding. Concomitantly to this improved stability, both mutants have lost the kinetic optimization to low temperature activity displayed by the parent psychrophilic enzyme. These results provide strong experimental support to the hypothesis assuming that the disappearance of stabilizing interactions in psychrophilic enzymes increases the amplitude of concerted motions required by catalysis and the dynamics of active site residues at low temperature, leading to a higher activity.

Kinetic analysis, structural studies and prediction of p Ka values of Bacillus KR-8104 α-amylase: The determinants of pH-activity profile

A native α-amylase capable of activity at low pH was isolated from Bacillus sp. KR-8104 (KRA) naturally occurred in rhizoplane zone of potato cultured soils. An investigation was set out to account some evidence for its specific properties. Kinetic parameters of KRA with respect to Bacillus licheniformis α-amylase (BLA) and B. amyloliquefaciens α-amylase (BAA) showed a shift in acidic limb for KRA pH-profile. After isolation of KRA gene and determination of its nucleotide and protein sequences, the three-dimensional structure of KRA was simulated based on BLA as template. Using PROPKA program the p K a values of titrable groups in KRA model together with other few members of this family were predicted either from simulated model or available structure at protein data bank. A correlation between catalytic residue p K a values and the optimal pH-activity profile was seen. Both our experimental data and prediction studies indicated a shift in p K a value of catalytic residues to acidic pH. Moreover alignment studies raised amino acid substitutions in KRA compare to BLA which may affect the KRA putative active site leading to the formation of an extra hydrogen bond between Glu261 and Arg229 (regarding to BLA numbering) and in turn shifts the pH-activity profile to lower amount. Also a common intermediate with characteristics of molten globular state was detected at a pH value lower than other members of α-amylase family using far UV circular dichroism, intrinsic and ANS fluorescence and quenching experiments.

Are sonochemically prepared α-amylase protein microspheres biologically active?

Using high-intensity ultrasound, we have synthesized α-amylase microspheres. The paper presented characterization as well as catalytic experiments of the sonochemically-produced microspheres. It also provided an estimate of the efficiency of the sonochemical process in converting the native protein to microspheres. These microspheres showed a very good enzymatic activity compared with the native α-amylase. The enzymatic activity of the amylase microspheres was 27% of that of the native protein after a short reaction time (3 min), while over a longer reaction time (1 h) it reached 56% of the activity of the native protein.

Bacillus subtilis α-amylase: interactions of a partially folded conformer with small unilamellar vesicles

We studied the interactions between conformers of exocellular α-amylase and small unilamellar vesicles (SUV) composed of the major membrane lipids of Bacillus subtilis under physiological conditions of pH, temperature and ionic strength. Using fluorescence spectroscopy, surface plasmon resonance (SPR) and phase separation, we show that the native α-amylase has no affinity for the SUV, whereas a partially folded form, displaying structural properties in common with the competent state for secretion, binds to the vesicles (KA≈105 M−1). This association prevented its subsequent folding. The complex was destabilized in the presence of PrsA, a major peripheric lipoprotein of B. subtilis which displays a strong affinity for SUV (KA≈1.5×108 M−1). Vesicles coated with PrsA lost their ability to bind the partially folded conformer.The approach in vitro, in which our aim was to mimic the last stage of α-amylase translocation, indicates that PrsA possibly helps, in vivo, the secreted protein to acquire its native conformation by modulating the interaction between the latter and the lipid polar heads on the trans side of the cytoplasmic membrane.

Interaction of α-amylase with n-alkylammonium bromides

The interaction of α-amylase with n-alkylammonium bromides above and below their critical micellar concentrations (cmc) has been studied in buffer at pH 7 and 10 by UV spectrophotometry, photon correlation spectroscopy and Doppler microelectrophoresis. This interaction produces a complex that is dependent on pH of the medium. This complex appears at surfactant concentrations below the cmc, which means that individual surfactant molecules can bind tightly to native α-amylase. The complex maintains its aggregation state when the concentration of surfactants with a hydrocarbon chain of 16 carbons increases, but not for surfactants of 12 and 14 carbons. Measurements of ζ-potential indicate the influence of electrostatic and hydrophobic forces. When the size of the aggregate is maximal, proteins are at their point of zero charge. In such conditions, Van der Waals forces and contacts between the alkyl chain and the hydrophobic core of the protein favour the formation of a larger aggregate.

Detection of binding of denatured salivary α-amylase to Streptococcus sanguis

Native α-amylase, either solubilized, or immobilized and tested with an overlay immuno-technique, was bound in a species-specific manner to Streptococcus mitis and to one of the Streptococcus gordonii strains. However, only insignificant amounts of α-amylase were bound to Streptococcus sanguis and all other strains tested. When α-amylase was denatured before immobilization, Streptococcus sanguis bound strongly to the protein while binding of other strains was insignificant.

Use of spherical tannin resin as a support for immobilized enzyme

Abstractα‐Amylase (α‐1, 4‐glucan 4‐glucanohydrolase 3.2.1.1) was used to investigate whether the spherical tannin resins made by the reaction of Mimosa (Acacia Mollissima, Wattle) tannin (condensed‐type tannin) (MT) with formaldehyde could be used as a support for the immobilization of enzymes. It was found that the optimum pH range of the immobilized α‐amylase was wider than that of native α‐amylase, and the shelf life of the enzyme was increased by immobilization. Furthermore, the immobilized enzyme could be used repeatedly, and in a continuous enzyme reaction increased a further advantage of the technique. On the basis of these data, it was concluded that this resin could be used as a support for the immobilization of enzymes. © 1992 John Wiley & Sons, Inc.

粒状固定化酵素カラムによる可溶性でんぷんの加水分解

Guucoamylase was immobilized by amidination reaction on the granular polyacrilonitrile resin which had been preliminarily treated with dry HCl in methanol. The granular polyacrilonitrile resin was made by drop-wise addition of the dimethylf ormamide solution of polyacrilonitrile to methanol. Continuous hydrolysis of soluble starch was operated in a column which was packed with the immobilized glucoamylase (granularity : 1 mmφ). Estimated values of apparent MICHAELIS constant (Km) with the immobilized glucoamylase depended on flow rates in the column and were found to be higher than that with the native glucoamylase in the batch system. The immobilized glucoamylase column was connected in a series with a column of the immobilized a-amylase (granularity : 30-200 μmφ). And, a soluble starch solution was firstly passed through the immobilized α-amylase column and then through the immobilized glucoamylase column. The effect of the pretreatment of soluble starch in the immobilized α-amylase column on the rate of hydrolysis was recognized with regard to the balance between amount of immobilized α-amylase and the degree of the hydrolysis in terms of reducing sugar unit. A similar effect was observed in the pretreatment of soluble starch with native α-amylase in a batch system. The molecular weight distribution of the reaction product of α-amylase processing was analyzed by the gel filtration (Sephadex G-75)method for the higher molecular weight fraction and by the thin layer chromatographic method for the lower molecular weight fraction up to DP 12. On the basis of these results, the pretreatment with α-amylase was thought to be significant particularly in that the effective concentration of the substrate was increased through the fission of the glucoside chains. The endurance test of the immobilized glucoamylase column was pursued by the continuous operation for two weeks with a flow rate of 0.5 ml/min at room temperature and 40°C. Similar situations were referred to in the previous study was obtained.

Preparation and properties of α-amylase chemically coupled to microcrystalline cellulose

Methods are reported for the preparation of water-insoluble derivatives of α-amylase, in which the enzyme is covalently linked to microcrystalline cellulose. Coupling was effected via diazotised 3-( p-aminophenoxy)-2-hydroxypropyl ethers and 2-hydroxy-3-( p-isothiocyanatophenoxy)propyl ethers of cellulose having different degrees of substitution; low substitution results in higher retention of α-amylase activity. The water-insoluble enzyme shows greater heat stability than the native α-amylase, 20% of the original activity remaining after 7 days at 45°.