Purification Of Amylase Research Articles

Purification and characterization of amylases from three freshwater fish species providing new insight application as enzyme molecular markers for zymography.

Purification of amylases from digestive tracts of three freshwater fish species with Q-Sepharose Fast Flow and Sephacryl S-200 columns displayed two isoforms of amylases from Osteochilus hasselti (O1, O2) and three isoforms of those from both Hampala dispar (UB, H1, H2) and Puntioplites proctozystron (P1, P2, P3). The optimum pH values displayed at 7.0 and 8.0, while the optimum temperatures revealed at 40 and 50°C. Almost isoenzyme activities were activated by NaCl and CaCl2, whereas EDTA and SDS strongly inhibited all enzymatic activities. Verification with an atomic absorption spectrophotometry exhibited the presence of Ca2+ ions in the range of 0.02-13.53ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25kDa validated with LC-MS/MS were selected. Three certain enzymes revealed high stability in a sample buffer after five cycles of freeze-thawing process upon storage at - 20°C for 12weeks. No protein degradation was observed on polyacrylamide gel, and the enzymes still displayed sharp and clear bands on zymograms. The result suggested that the purified fish amylases, which expressed high activities and stabilities, were potentially used as enzyme molecular weight markers for zymography.

Regulation and Characterization of Amylase Enzyme Secretion In the Digestive Tract of <i>Antheraea Assamensis</i> Helfer

Antheraea assamensis Helfer is an endemic non-mulberry lepidopteran species of North East India with high commercial demand for its golden hued silk. Antheraea assamensis suffers from a protozoan disease pebrine which lead to death of the silkworm. In this study, the effect of various factors (starvation, feeding, temperature, pH, Ca ion and larval stage) in the regulation of amylase secretion is compared in healthy and diseased muga silkworm. Moreover, enzyme zymography and purification of amylase from the midgut is also performed. Result shows that, the secretion of amylase in Antheraea assamensis in starved condition, significantly decreases. Refeeding experiments (after 2 days of starvation) suggest that amylase secretion sharply increases due to feeding stimulus. The purified enzyme molecular weight is confirmed as 56 kDa by SDS PAGE. The purification fold of purified enzyme is 34 times higher than the crude enzyme extract.

Isolation and Purification of Amylase from Serum of Patients with Pancreatitis and Comparing the Biochemical Properties with Amylase Purified from Healthy People

Back Ground: Amylases are a group of enzymes that hydrolyze starch into simple sugars. Amylase is secreted in the human body from saliva and the pancreas. Abnormal levels of the enzyme amylase indicate pancreatitis. Enzyme purification eliminates various proteins and other forms of biomolecules while restoring the majority of enzyme activity.
 Objectives: Isolation and purification of the alpha-amylase enzyme from the serum of a patient with pancreatitis and a healthy human, and estimation of the values ​​of Michalis constant Km as well as the maximum velocity Vmax to determine the affinity of the enzyme towards the substrate in both cases.
 Materials and Methods: The enzyme was purified by several steps, including precipitation, by adding ammonium sulfate at a concentration of 30-70%, then dialysis. The extract was transferred through the separation column by gel chromatography containing Sephadex G100 gel. 
 Results: The gel separation chromatography results indicated the appearance of four protein bands, one of which (the third peak) belongs to the amylase enzyme. The specific activity of the enzyme in the last step after concentration of the product was 38 units/gm for the patient and 11.62 U/gm for a healthy human. The yield was 44.67% and 50.53%, while the number of purification times was 9.11 and 9.3 for the patient and the healthy human, respectively. The kinetic constants (Km and Vmax) were estimated using the Leinweaver-Burke plot, Vmax for the patient and the healthy human was 149.3 and 83.3, respectively. the Km for the patient and the healthy human was 1.39 and 2.56, respectively. 
 Conclusion: It is inferred from the results that the affinity of the enzyme to bind to the substrate of patient with pancreatitis is higher than that of the healthy human.

Production and Purification of Amylase from Bacillus subtilis Isolated from Soil

In spite of progress in biotechnology and enzymology, the enzymes have been industrialized in recent years for the mounting up the product development in various arena. The ultimate goal of this study comprises the production and purification the amylase enzyme from the bacterial strain. A powerful amylase producer, Bacillus subtilis ISOLATE-4 was isolated, screened and identified from the soil sample. In order to produce extracellular amylase, various physico-chemical parameters were optimized. During optimization, the maximal production of amylase by the isolate at 48 hrs of incubation in 100 rpm was found to be 6.93U/ml, 5.94U/ml, 6.0U/ml at 45ºC, pH 6 with 1% substrate concentration respectively. Ammonium sulphate fractionation was done for rapid precipitation of the amylase at a concentration of 60% and exposed to dialysis showed the 25% purification fold of an enzyme. The dialyzed product was further subjected to DEAE-Cellulose column chromatography resulted in an increase up to 75% purification fold than crude enzyme. The amylase enzyme might be suitable for the liquefaction of starch, detergent, textile and several additional industrial applications.

Partial-purification of Alpha-Amylase from MarineStreptomyces gancidicus-ASD_KT852565

The current study reveals the result of amylase producing actinomycetes isolated from marine sediments collected from south coastal areas of Tamilnadu, India which was found to be Streptomyces gancidicus_ASD by 16S rDNA sequencing with ABI 3730xl sequencer. Phylogeny identification was carried with the help of similar blast sequence from NCBI and online clustaw software for phylogenetic tree construction. Partial purification of amylase was done with 80% ammonium sulphate and overnight dialysis with 10ml of 100 mMTris buffer. The molecular weight of the potent strain by SDS-PAGE was found to be 44 KDa which was further proved by Zymogram for amylase production. Further, purification by HPLC(C-18) is under process.

Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases.

Bacillus licheniformis 9945a α-amylase is known as a potent enzyme for raw starch hydrolysis. In this paper, a mixed mode Nuvia cPrime™ resin is examined with the aim to improve the downstream processing of raw starch digesting amylases and exploit the hydrophobic patches on their surface. This resin combines hydrophobic interactions with cation exchange groups and as such the presence of salt facilitates hydrophobic interactions while the ion-exchange groups enable proper selectivity. α-Amylase was produced using an optimized fed-batch approach in a defined media and significant overexpression of 1.2 g L−1 was achieved. This single step procedure enables simultaneous concentration, pigment removal as well as purification of amylase with yields of 96% directly from the fermentation broth.

Purification and characterization of amylase from <i>Bacillus thuringiensis</i> subsp. <i>kurstaki</i>

In a biphasic fermentation strategy, Bacillus thuringiensis subsp. kurstaki ( Btk ) was cultivated in potato flour (10% w/v) supplemented Luria-Bertani medium for enhancing the production of δ- endotoxin. After 12 h fermentation, the supernatant was collected for the purification of amylase. The amylase secreted by Btk was purified to homogeneity (15 folds purified with 79% yield) through a series of steps including (NH 4 ) 2 SO 4 fractionation and sephadex G-100 gel filtration chromatography. From the profile on SDS-PAGE, the molecular weight of the purified amylase was judged as 51kDa. The maximum amylase activity (8088 U/gds) was obtained with 2.5% soluble starch at 60 o C in the presence of 3µM Ca 2+ (pH 6);however, SDS and EDTA retarded the activity. The amylase hydrolyzed starch with a Km of 2.9 mg/ml and Vmax of 0.053µmol/ml/min. This is the first report describing the purification and characterization of an amylase from a Bt strain ; since amylase can be purified as byproduct with no additional cost during the harvest of δ- endotoxin, recovery of valuable amylase from the effluent would be advantageous for the Bt bioindustry .

A Simple One Pot Purification of Bacterial Amylase From Fermented Broth Based on Affinity Toward Starch-Functionalized Magnetic Nanoparticle

Surface-functionalized adsorbant particles in combination with magnetic separation techniques have received considerable attention in recent years. Selective manipulation on such magnetic nanoparticles permits separation with high affinity in the presence of other suspended solids. Amylase is used extensively in food and allied industries. Purification of amylase from bacterial sources is a matter of concern because most of the industrial need for amylase is met by microbial sources. Here we report a simple, cost-effective, one-pot purification technique for bacterial amylase directly from fermented broth of Bacillus megaterium utilizing starch-coated superparamagnetic iron oxide nanoparticles (SPION). SPION was prepared by co-precipitation method and then functionalized by starch coating. The synthesized nanoparticles were characterized by transmission electron microscopy (TEM), a superconducting quantum interference device (SQUID, zeta potential, and ultraviolet–visible (UV-vis) and Fourier-transform infrared (FTIR) spectroscopy. The starch-coated nanoparticles efficiently purified amylase from bacterial fermented broth with 93.22% recovery and 12.57-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the purified amylase was 67 kD, and native gel showed the retention of amylase activity even after purification. Optimum pH and temperature of the purified amylase were 7 and 50°C, respectively, and it was stable over a range of 20°C to 50°C. Hence, an improved one-pot bacterial amylase purification method was developed using starch-coated SPION.

Production and Partial Purification of Amylase By Aspergillus niger Isolated from Cassava Peel

Aspergillus niger strains 1, 2 and 3 isolated from cassava dumpsites were used for the production of amylase enzyme. The Aspergillus niger strains 1, 2 and 3 had diameter (mm) zone of clearance of 17.0, 23.0 and 8.0 respectively using Potato dextrose agar plates fortified with starch. Studies on the amylase enzyme activity (mg/ml) of Aspergillus niger strains 1 and 2 showed 19,340 and 16,510 respectively. These values were higher than the commercially available amylase enzyme that had an activity of 5,722.2. The protein (mg/ml) and specific activity (units/mg) for amylase from Aspergillus niger strain 1 was 28.39 and 681.23 while 21.76 and 758.73 from Aspergillus niger 2 respectively. Purification using ammonium sulphate (% w/v) at 60, 80 and 100 on amylase enzyme from Aspergillus niger strain 1 for enzyme activity, protein and specific activity was 44405.49, 17.01 and 2610.55, 28949.76, 23.62 and 1225.65, 36220.25, 16.67, and 2172.787 respectively. The microbial production of Amylase enzyme in Nigeria from Cassava peel will reduce cost of production, convert cassava peel from waste condition to wealth, and will boost economy through indigenous industrialization.

Partial Purification of Amylase from the Culture Filtrates of Aspergillus flavus Grown on Cassava Peels

Amylase from a Cassava peel culture of Aspergillus flavus was partially purified by Ammonium Sulphate precipitation as well as dialysis. The dialysed 60% (NH4)2 SO4 precipitated enzyme had an activity of 1.087 mg T.R.S released/ min/mg protein which was two folds of the activity of the crude culture filtrate. Hydrogen ion concentration as well as temperature had profound influence on enzyme activity of the partially purified enzyme while Amylase activity increased progressively as pH was increased from 3 to 7 reaching a maximum of 1.68 mg T.R.S released/min/mg/protein at PH 7.0. A rapid decrease in amylase activity was observed as pH was increased from 7 to 9 while the amylase activity increased with increase in temperature from 300C to 450C and reached a maximum of 1.15 mg T.R.S. released/min/mg/protein at 450C. Subsequent increase in temperature resulted into decrease in activity of the amylase enzyme.

PURIFICATION OF AMYLASE FROM TILAPIA BY MAGNETIC PARTICLE

Recent development in magnetic carrier technology involves the use of nonmagnetic substrates attached to superparamagnetic particles forming functionally modified magnetic support to isolate a particular enzyme in a controllable magnetic field. In this study, the superparamagnetic particles were modified by epichlorohydrin and other agents to cross-link with starch to form the purification support. This affinity support was able to bind the amylase and was used in the purification of amylase from Taiwan tilapia. After ammonium sulfate precipitation of amylase from Taiwan tilapia, the modified superparamagnetic particles were able to purify the crude amylase by 20.78-fold with recovery of activity of 75.6%. The molecular weight of the amylase was estimated to be 66.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both crude and purified amylase reached an optimum at a pH of 8.0 and temperature of 50C, and the enzyme was stable between 20 and 50C. PRACTICAL APPLICATIONS Because of the rapid development of high technology such as carrier supports for enzyme purification, the development, research and application of magnetic carriers are timely needed. The present study demonstrated that the affinity superparamagnetic particles could be used as a carrier support to absorb and purify the amylase and that technology of affinity purification can be widely used in protein purification. Compared with the traditional chromatography used in the purification of proteins, this novel affinity superparamagnetic particle technology is rapid, has low operation cost, requires simple facilities, and involves easy separation and recovery of the enzymes.

Purification of Amylase from Honey

ABSTRACT:The major amylase in honey was concentrated by ultrafiltration, isolated by ultracentrifugation and gel filtration, and purified by ion‐exchange chromatography. The amylase activity was in the flow‐through fraction of the anion‐exchange column, suggesting a high isoelectric point (>7.4) for the enzyme. The enzyme fraction from the anion‐exchange chromatography was loaded onto a cation‐exchange column, and the amylase activity was eluted as a single band at 50 mM NaCl. The purification factor after this step was 531‐fold. The purified enzyme was an a‐amylase, as determined by thin‐layer chromatography, with a molecular weight of 57000 Da according to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The results supported the concept that amylase in honey had a high degree of similarity with bee amylase.

Review: cyclodextrins and their interaction with amylolytic enzymes.

This review is concerned with inhibition of amylases by cyclodextrins (cyclic maltooligosaccharides), the interaction that occurs between amylases and cyclodextrins and the application of cyclodextrin affinity chromatography in the purification of amylases. In many cases, amylases that are competitively inhibited by cyclodextrins can be purified by cyclodextrin affinity chromatography with the cyclodextrins interacting with the active site on such enzymes. Interestingly amylases that are not competitively inhibited by cyclodextrins may also be purified by cyclodextrin affinity chromatography. Therefore, cyclodextrin affinity chromatography can function in the purification of such amylolytic enzymes with the interaction occurring at a site removed from the active site. In such cases it appears that the cyclodextrin is interacting with an affinity site or binding site that is present on some amylolytic enzymes. It seems that certain similarities occur among the binding sites of such enzymes. Literature concerning amylases, and their subsequent purification using cyclodextrin affinity chromatography is reviewed and the fundamental basis of the interaction of the cyclodextrin with amylolytic enzymes is discussed here.

Purification of amylases and other enzymes by a forced-affinity chromatography method.

An affinity matrix of soluble starch gel was prepared by cross-linking catalyzed by epichlorohydrin. The elution pattern of Taka-amylase A (TAA) indicated that the amount of enzyme bound to the starch gel column increased with increases in the ammonium sulfate (AmS) concentration in the equilibrating buffer. TAA had an affinity for the gels with a starch structure, and desorbed from the column with the buffer containing no AmS. Bound TAA was also eluted with starch and cyclodextrin solution. The AmS stimulative effect was partially replaced by polyethylene glycol and surfactants. Besides TAA, various, other amylases bound satisfactorily to the starch gel. Moreover, affinity purifications of dextranase, cellulase, and pectinase were done by gels with dextran, cellulose, and pectin structures, respectively. By the aid of forced effects of AmS, various carbohydrases could be purified by the affinity gels of polysaccharide linked by epichlorohydrin.

Partial Purification of Amylases and Starch Phosphorylases from Poplar Wood

Summary The three main fractions of amylase activity and both starch phosphorylase isoforms from xylem parenchyma cells of poplar twigs ( Populus x canadensis Moench «robusta» ) were separated by Q Sepharose anion exchange chromatography and gel filtration through Sephacryl S-300-HR. Analysis of the substrate specificity of the starch hydrolases showed that the chromogenic substrates, blocked PNP-linked G5 and G6 maltooligosaccharides (Testomar) and starch azure, were highly specific for assaying β-amylase (EC 3.2.1.2) and α-amylase (EC 3.2.1.1) activity, respectively. In this way, five bands of the complex electrophoretic amylase isozyme pattern in protein extracts from poplar wood were identified as α-amylases (A1–A5), and three bands (B1–B3) as β-amylases. Product analysis by HPLC supported these results. Exoamylase released almost exclusively maltose from soluble starch, while several maltooligosaccharides, G6–G10 being the main products, were released by α-amylase. Another amylase band of unknown classification was able to degrade starch granules and polysaccharides immobilized in acrylamide gels, but was inactive with soluble polysaccharide substrates. Poplar wood extracts contained two bands of starch phosphorylase (EC 2.4.1.1) activity, which exerted typical properties of the Type I (cytoplasmic) and Type II (plastidic) isoforms.

Precepitation and purification of amylase enzyme produced byStreptomyces aureofaciens 77

Precipitation and purification of amylase secreted byStreptomyces aureofaciens 77 in liquid inorganic salts-starch medium under the optimum conditions were carried out. Ammonium sulphate fractionation was used to precipitate amylase in cell free culture filtrate. (NH4)2SO4 at a concentration of 50–70% saturation gave the highest enzyme yield. The obtained precipitates were redissolved in phosphate buffer (pH 7.0) and subjected to dialysis. The dialyzed enzyme preparation was applied to DEAE-cellulose column chromatography which resulted in an increase of purification up to 59.48 fold. A further step of purification was done by applying the obtained puified sample to Sephadex-G200 column chromatography which resulted in an increase of purification up to 73.92 fold. The results clearly indicated that the isolated amylase from S.aureofaciens 77 was only on type.