Characterization Of Α-galactosidase Research Articles

Purification, characterization of α-galactosidase from a novel Bacillus megaterium VHM1, and its applications in the food industry

An extracellular thermostable α-galactosidase from Bacillus megaterium VHM1 was purified 94.26-fold by precipitation with ethanol, followed by sequential column chromatography with DEAE-Sephacel and G75 column. The purified enzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). It was found to be a monomeric protein with a molecular weight of about 66 kDa. The purified enzyme showed optimum activity in p-nitrophenyl α-D-galactopyranoside (PNPG) at pH 7.0 and a temperature of 60??C. The enzyme was thermostable, showing complete activity even after heating at 55??C for 60 minutes. The substrate specificity of α-galactosidase on PNPG, ortho-Nitrophenyl-β-galactoside, and raffinose was investigated and Km was found to be 0.508, 0.529, and 5.0 mM and Vmax was 3.492, 4.287, and 14.20 μ mol/ml/minute, respectively. Among the metal ions and reagents tested, Hg2+, Cu2+, and Ag2+ strongly inhibited the α-galactosidase activity, and ethylenediaminetetraacetic acid showed no effect on enzymes. The present study supports the application of α-galactosidase from B. megaterium VHM1 as a potent enzyme in the food processing industry.

Bioaffinity immobilization and characterization of α-galactosidase on aminophenylboronicacid derivatized chitosan and Sepabeads EC-EA

Enzyme immobilization with affinity binding which is based on the specific affinity interactions have an important advantage as; high selectivity. In the present study, chitosan and Sepabeads EC-EA were derivatized with aminophenylboronicacid(APBA) for the affinity immobilization of α-galactosidase. The influence of various process parameters on immobilization of the enzyme is investigated to get high immobilization yields. Under optimized immobilization conditions, the chitosan and Sepabeads EC-EA immobilized enzymes exhibited activity yield of 89.5% and 72%, respectively. The maximum activities were detected at 40 °C for free and Sepabeads EC-EA immobilized enzyme and 55 °C for chitosan immobilized enzyme. The optimum pH was found as pH 5.0 for free and Sepabeads EC-EA immobilized enzyme and pH 5.5 for chitosan immobilized enzyme. Both immobilized enzymes were very stable at temperature ranged from 4 to 55 °C and also in a pH range of 2.6–7.0. The immobilized α-galactosidases were also used in the hydrolysis of raffinose. The chitosan and Sepabeads EC-EA immobilized enzymes hydrolysed 55% and 42% of raffinose in 32 h at 50 °C, respectively. The obtained results shed light for the useability of these immobilized enzymes in the hydrolysis of raffinose in food industry and make these immobilized enzymes good candidates for their various biotechnological applications.

Purification and Characterization of α-Galactosidase from White Chickpea (Cicer arietinum)

Glycosylated α-galactosidase (melibiase) has been purified from white chickpea ( Cicer arietinum ) to 340-fold with a specific activity of 61 units/mg. Cicer α-galactosidase showed a M(r) of 45 kDa on SDS-PAGE and by MALDI-TOF. The optimum pH and temperature with pNPGal were 4.5 and 50 °C, respectively. The K(m) for hydrolysis of pNPGal was 0.70 mM. Besides hydrolyzing the pNPGal, Cicer α-galactosidase also hydrolyzed natural substrates such as melibiose, raffinose, and stachyose very effectively; hence, it can be exploited commercially for improving the nutritional value of soy milk. Galactose was found to be a competitive inhibitor. The property of this enzyme to cleave the terminal galactose residues can be utilized for converting the group B erythrocytes to group O erythrocytes.

Purification and characterization of a novel protease-resistant α-galactosidase from Rhizopus sp. F78 ACCC 30795

A novel extracellular α-galactosidase, named Aga-F78, from Rhizopus sp. F78 ACCC 30795 was induced, purified and characterized in this study. This soybean-inducible α-galactosidase was purified to homogeneity by ammonium sulfate precipitation and fast protein liquid chromatography (FPLC), with a yield of 14.6% and a final specific activity of 74.6 U mg −1. Aga-F78 has an estimated relative molecular mass of 78 kDa from SDS-PAGE while native mass of 210 kDa and 480 kDa from non-denaturing gradient PAGE. This α-galactosidase had no N- or O-glycosylated. Amino acid sequences of three internal fragments were determined, and fragment 1, NQLVLDLTR, shared high homology with bacterial and fungal GH-36 α-galactosidases. The optimum pH and temperature on activity of Aga-F78 were 4.8 and 50 °C, respectively. The properties of pH and temperature stability, effect of ions and chemicals were also studied. Furthermore, the resistant to neutral and alkaline proteases and substrate specificity of natural substrates (melibiose, raffinose, stachyose and guar gum) were also studied to enlarged the application of Aga-F78 in more fields. Kinetic studies revealed a K m and V max of 2.9 mmol l −1 and 246.1 μmol (mg min) −1, respectively, using pNPG as substrate. To our knowledge, this is the first report of purification and characterization of α-galactosidase from Rhizopus with some special properties, which may aid its utilization in the food and feed industries.

Purification and Characterization of α-Galactosidase from Lactobacillus salivarius subsp. salivarius Nam27

Lactobacillus salivarius subsp. salivarius CNU27 possessed a high level of <TEX>${\alpha}$</TEX>-galactosidase activity. Purified <TEX>${\alpha}$</TEX>-galactosidase was obtained after sonication of harvested cell pellet followed by DEAE-Sephadex A-50 and Mono Q anion exchange chromatography. The specific activity of the purified enzyme was 8,994 units/mg protein which is 17.09 times higher than that in crude extract. The native enzyme was a monomer with a molecular mass of 56,397.1 dalton. The optimum temperature and pH for the enzyme were <TEX>$40^{\circ}C$</TEX> and 6.0, respectively. The enzyme was stable between 25 and <TEX>$50^{\circ}C$</TEX>. However, <TEX>${\alpha}$</TEX>-galactosidase activity was lost rapidly below pH 4.5 and above pH 8.5. The enzyme activity decreased to 6.73% and 4.30% of the original activity by addition of <TEX>$Cu^{2+}$</TEX> and <TEX>$Hg^{2+}$</TEX>, respectively. Other metal compounds did not affect the enzyme activity significantly. The enzyme liberated galactose from melibiose, raffinose, and stachyose. The rate of substrates hydrolysis was measured by HPLC. Raffinose, stachyose and melibiose were completely decomposed after 24 hr at <TEX>$40^{\circ}C$</TEX>.

Purification and characterization of α-galactosidase from a thermophilic fungus Thermomyces lanuginosus

An extracellular α-galactosidase was purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of a mannanolytic strain of the thermophilic fungus Thermomyces lanuginosus. Molecular mass of the enzyme is 57 kDa. The pure enzyme which has a glycoprotein nature, afforded several forms on IEF, indicating its microheterogeneity. Isoelectric point of the major form was 5.2. Enzyme is the most active against aryl α- d-galactosides but efficiently hydrolyzed α-glycosidically linked non-reducing terminal galactopyranosyl residues occurring in natural substrates such as melibiose, raffinose, stachyose, and fragments of galactomannan. In addition, the enzyme is able to catalyze efficient degalactosylation of polymeric galactomannans leading to precipitation of the polymers. Stereochemical course of hydrolysis of two substrates, 4-nitrophenyl α-galactopyranoside and galactosyl 1mannotriose, followed by 1H NMR spectroscopy, pointed out the α-anomer of d-galactose was the primary product of hydrolysis from which the β-anomer was formed by mutarotation. Hence the enzyme is a retaining glycosyl hydrolase. In accord with its retaining character the enzyme catalyzed transgalactosylation from 4-nitrophenyl α-galactopyranoside as a glycosyl donor. Amino acid sequence alignment of N-terminal and two internal sequences suggested that the enzyme is a member of family 27 of glycosyl hydrolases.

High-yield production and characterization of α-galactosidase from Bifidobacterium breve grown on raffinose

Bifidobacteria assimilated raffinose about 4-fold more effectively than other intestinal bacteria, and α-galactosidase was active in all strains of Bifidobacteria tested. The enzyme activity of Bifidobacterium breve grown on raffinose was highly and specifically increased. Its activity was 30-fold higher than that of B. breve grown on glucose. Melibiose was also effective for production of the enzyme. The enzyme was purified to homogeneity from B. breve. It is a homodimer with Mr of about 160 kDa, and its optimum pH for activity of 5.5–6.5. The enzyme showed strict substrate specificity for α-galactoside although it had slight activity for α-glucoside. It hydrolysed stachyose, melibiose (Km = 2 mM) and raffinose (Km = 0.7 mM).

Hydrolysis of raffinose by immobilized cells of saccharomyces oleaceus

The production, extraction, purification and characterization of α-galactosidase (melibiase) from Saccharomyces cereuisiae var. oleaceus (N.R.R.L. Y 12056) are described. The yeast cells were immobilized by entrapment into cellulose diacetate beads. The removal of raffinose from molasses using this catalyst was studied under different reactor configurations.

Production, Purification, and Characterization of α-Galactosidase from Monascus pilosus

A Monascus pilosus strain was selected for production of intracellular alpha-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Galactose was one of the best enzyme inducers. The enzyme was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography and was demonstrated to be homogeneous by slab gel electrophoresis. The molecular weight of this enzyme, estimated by gel filtration, was about 150,000. The optimum conditions for the enzyme reaction was pH 4.5 to 5.0 at 55 degrees C. The purified enzyme was stable at 55 degrees C or below and in buffer at pH 3 to 8. The activity was inhibited by mercury, silver, and copper ions. The kinetics of this enzyme, with p-nitrophenyl-alpha-d-galactoside as substrate, was determined: K(m) was about 0.8 mM, and V(max) was 39 mumol/min per mg of protein. Enzymatic hydrolysis of melibiose, raffinose, and stachyose was analyzed by thin-layer chromatography.

Purification and characterization of alpha-galactosidase from watermelon.

An alpha-galactosidase [EC 3.2.1.22] was isolated from the fruit of the watermelon, Citrullus battich. The enzyme was purified by procedures including extraction, ammonium sulfate precipitation, and chromatographies on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. The final preparation was found to be fairly homogeneous on disc and SDS-polyacrylamide gel electrophoresis, and sufficiently free from other glycosidase activities. The molecular weight of the enzyme was estimated to be 45,000 by Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4.5 for natural substrates and at 5.9 for artificial substrates. The enzyme liberates the alpha-galactose units from oligosaccharides of the raffinose series and ceramide trihexoside, and the hemagglutination-inhibiting activities of human ovarian cyst B-glycoprotein and blood group B-type ghosts were abolished by the enzyme.

Purification and Characterization of α-Galactosidase from Waste Lager Yeast (Saccharomyces Carlsbergensis)

Purification and Characterization of α-Galactosidase from Waste Lager Yeast (Saccharomyces Carlsbergensis)

Characterization of α-Galactosidase from Cucumber Leaves

Two forms of alpha-galactosidase (alpha-d-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total alpha-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total alpha-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000- and 25,000-dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-alpha-d-galactoside sub-strate. K(m) values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, K(m) values were 10 and 30 millimolar for the 50,000- and 25,000- molecular weight forms, respectively.