Purification, Characterization, and Substrate Specificity of a Novel Highly Glucose-Tolerant β-Glucosidase fromAspergillus oryzae

ABSTRACT Aspergillus oryzae was found to secrete two distinct β-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3′,4′,5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total β-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant β-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-β-d-glucoside as the substrate, we found that the enzyme was optimally active at 50°C and pH 5.0 and had a specific activity of 1,066 μmol min−1mg of protein−1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki , 1.36 M) or glucono-δ-lactone (Ki , 12.5 mM), another powerful β-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal β-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1→3)- and (1→6)-β-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-β-d-glucosides in a grape must (pH 2.9, 90 g of glucose liter−1). Other flavor precursors (benzyl- and 2-phenylethyl-β-d-glucosides) and prunin (4′,5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel β-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.

Download Full-text

Cadmium-binding proteins of rat testes. Characterization of a low-molecular-mass protein that lacks identity with metallothionein

Biochemical Journal ◽

10.1042/bj2200811 ◽

1984 ◽

Vol 220 (3) ◽

pp. 811-818 ◽

Cited By ~ 50

Author(s):

M P Waalkes ◽

S B Chernoff ◽

C D Klaassen

Keyword(s):

Metal Binding ◽

Gel Electrophoresis ◽

Binding Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Polyacrylamide Gel ◽

Or Groups

Cadmium-binding proteins in the cytosol of testes from untreated rats were separated by Sephadex G-75 gel filtration. Three major testicular metal-binding proteins (TMBP), or groups of proteins, with relative elution volumes of approx. 1.0 (TMBP-1), 1.7 (TMBP-2) and 2.4 (TMBP-3) were separated. Elution of Zn-binding proteins exhibited a similar pattern. TMBP-3 has previously been thought to be metallothionein (MT), and hence this protein was further characterized and compared with hepatic MT isolated from Cd-treated rats. Estimation of Mr by gel filtration indicated a slight difference between MT (Mr 10000) and TMBP-3 (Mr 8000). Two major forms of MT (MT-I and MT-II) and TMBP-3 (TMBP-3 form I and TMBP-3 form II) were obtained after DEAE-Sephadex A-25 anion-exchange chromatography, with the corresponding subfractions being eluted at similar conductances. Non-denaturing polyacrylamide-gel electrophoresis on 7% acrylamide gels indicated that the subfractions of TMBP-3 had similar mobilities to those of the corresponding subfractions of MT. However, SDS (sodium dodecyl sulphate)/12% (w/v)-polyacrylamide-gel electrophoresis resulted in marked differences in migration of the two corresponding forms of MT and TMBP-3. Co-electrophoresis of MT-II and TMBP-3 form II by SDS/polyacrylamide-gel electrophoresis revealed two distinct proteins. Amino acid analysis indicated much lower content of cysteine in the testicular than in the hepatic proteins. TMBP-3 also contained significant amounts of tyrosine, phenylalanine and histidine, whereas MT did not. U.v.-spectral analysis of TMBP-3 showed a much lower A250/A280 ratio than for MT. Thus this major metal-binding protein in testes, which has been assumed to be MT is, in fact, a quite different protein.

Download Full-text

Purification and some properties of the extracellular α-amylase from Aspergillus awamori

Canadian Journal of Microbiology ◽

10.1139/m85-029 ◽

1985 ◽

Vol 31 (2) ◽

pp. 149-153 ◽

Cited By ~ 28

Author(s):

Resham S. Bhella ◽

Illimar Altosaar

Keyword(s):

Enzyme Activity ◽

Gel Electrophoresis ◽

Culture Medium ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Aspergillus Awamori ◽

Original Activity

Alpha-amylase was purified from the extracellular culture medium of Aspergillus awamori by means of ethanol precipitation. Sephacryl-200 gel filtration and anion-exchange chromatography on Dowex (AG1-X4) resin. The enzyme preparation was found to be homogeneous by means of sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of 54 000 ± 2 500 and its isoelectric point was pH 4.2. The enzyme was found to be most active between pH 4.8 and 5.0 and was stable between pH 3.5 and 6.5. The optimal temperature for the enzyme activity was around 50 °C and the enzyme was stable for at least 1 h up to 45 °C retaining more than 80% of its original activity. The Km (37 °C, pH 5.3) for starch hydrolysis was 1.0 g∙L−1 and maltose inhibited the enzyme activity uncompetitively with a K1 value of 20.05 g∙L−1

Download Full-text

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

Acta Endocrinologica ◽

10.1530/acta.0.0770485 ◽

1974 ◽

Vol 77 (3) ◽

pp. 485-497 ◽

Cited By ~ 30

Author(s):

P. A. Torjesen ◽

T. Sand ◽

N. Norman ◽

O. Trygstad ◽

I. Foss

Keyword(s):

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Deae Cellulose ◽

Disc Electrophoresis ◽

Exchange Chromatography ◽

Polyacrylamide Gel ◽

Molecular Weights ◽

Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

Download Full-text

Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli

Biochemical Journal ◽

10.1042/bj2130187 ◽

1983 ◽

Vol 213 (1) ◽

pp. 187-191 ◽

Cited By ~ 37

Author(s):

A Lewendon ◽

J R Coggins

Keyword(s):

Escherichia Coli ◽

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Polyacrylamide Gel ◽

Purification Step ◽

Phosphate Synthase

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.

Download Full-text

Purified Factor XII Has a Higher Specific Activity than the Parent Molecule in Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647478 ◽

1991 ◽

Vol 65 (02) ◽

pp. 169-173 ◽

Cited By ~ 3

Author(s):

Walter A Wuillemin ◽

Miha Furlan ◽

Isabella Huber ◽

Bernhard Lämmle

Keyword(s):

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Factor Xii ◽

Parent Molecule ◽

Proteolytic Activation ◽

Filtration Step

SummaryThe specific clot promoting activity of factor XII (F XII) in plasma samples from 50 healthy adults was between 30 and 48 U/ mg, whereas the specific activity of purified F XII ranged from 55 to 66 U/mg. This difference was neither due to partial proteolytic activation during purification of F XII nor to the influence of plasma protease inhibitors. Purified F XII showed normal size and charge, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing, respectively. The increase of the specific F XII activity during the purification process mainly occurred after anion exchange chromatography on DEAE-Sephadex and after the final gel filtration step. Upon dextran sulfate activation, proteolytic cleavage of F XII and generation of kallikrein-like amidolytic activity was faster in F XII deficient plasma containing purified F XII than in F XII deficient plasma containing a corresponding amount of pooled normal plasma (NHP). The binding to kaolin was similar for both, purified F XII and plasma F XII.In conclusion, purification alters the properties of F XII in an unknown way, resulting in an increased specific clot promoting activity.

Download Full-text

Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis

Biochemical Journal ◽

10.1042/bj2610483 ◽

1989 ◽

Vol 261 (2) ◽

pp. 483-488 ◽

Cited By ~ 40

Author(s):

K Takazawa ◽

H Passareiro ◽

J E Dumont ◽

C Erneux

Keyword(s):

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Bovine Brain ◽

Polyacrylamide Gel ◽

Maximal Velocity ◽

Inositol 1,4,5 Trisphosphate ◽

Maximal Activation

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). A method is presented for the rapid purification of InsP3 3-kinase from bovine brain by calmodulin (CaM)-Sepharose affinity chromatography. Maximal activation of the purified InsP3 3-kinase by Ca2+/CaM was 6-7-fold as compared with the activity measured in the presence of EGTA (1 mM) and 10 microM-InsP3. At 10 microM-InsP3 and 0.1 mM free Ca2+, half-maximal activation required about 2 nM-CaM. The mechanism of activation by CaM appeared to be an increase in the maximal velocity of the enzyme without a substantial change in the Km for InsP3. Further purification was achieved by phosphocellulose chromatography eluted with ATP. Specific activity of the purified enzyme at 37 degrees C and 10 microM-InsP3 was 10-20 mumol/min per mg. The apparent Mr of the enzyme, determined by f.p.l.c.-gel filtration, was estimated as about 44,000. The purified InsP3 3-kinase was subjected to SDS/10%-polyacrylamide-gel electrophoresis. InsP3 3-kinase activity was associated with three silver-stained bands, which migrated with apparent Mr values of approx. 52,000, 38,000 and 35,000.

Download Full-text

Purification and characterization of a chymosinlike protease from the gastric mucosa of harp seal (Pagophilus groenlandicus)

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o84-091 ◽

1984 ◽

Vol 62 (8) ◽

pp. 699-708 ◽

Cited By ~ 27

Author(s):

K. Shamsuzzaman ◽

N. F. Haard

Keyword(s):

Gastric Mucosa ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Exchange Chromatography ◽

Polyacrylamide Gel ◽

Ph Optimum ◽

Porcine Pepsin ◽

Milk Clotting

Four zymogens of acidic proteases A, B, C, and D were isolated from the gastric mucosa of harp seals by ion-exchange chromatography on a diethylaminoethyl-Sephadex A-50 column. The major zymogens were A and C, and the ratio of zymogen A to zymogen C was greater in extracts from 1-week-old animals than in extracts from adult animals. Zymogens A and C were further purified by affinity chromatography using carbobenzoxy-D-phenylalaninetriethylene tetramine Sepharose and gel filtration on a Sephadex G-100 column. Certain physical and catalytic properties of proteases A and C were compared with those of calf chymosin (EC 3.4.23.4) and porcine pepsin (EC 3.4.23.1). Zymogen C and the corresponding enzyme were homogeneous on analytical polyacrylamide gel electrophoresis. Zymogen A was homogeneous as judged by sodium dodecyl sulphate (SDS) – polyacrylamide gel electrophoresis and high performance liquid chromatography, but was heterogenous by polyacrylamide gel electrophoresis at pH 8.3. Zymogens A and C had molecular weights of 33 800 and 44 000, respectively, as estimated by SDS–polyacrylamide gel electrophoresis. Protease A had an isoelectric point of 4.90. Protease A was similar to calf chymosin with respect to several criteria. It had a higher ratio of milk-clotting to proteolytic activity than those of seal protease C and porcine pepsin and had a pH optimum of 2.2–3.5 for hemoglobin hydrolysis. It did not inactivate ribonuclease, had very low activity on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine and lost activity in 6 M urea. These results indicate protease A is chymosinlike.

Download Full-text

Purification and properties of a specific collagenase from rabbit synovial fibroblasts

Biochemical Journal ◽

10.1042/bj1510645 ◽

1975 ◽

Vol 151 (3) ◽

pp. 645-653 ◽

Cited By ~ 26

Author(s):

Z Werb ◽

J J Reynolds

Keyword(s):

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Synovial Fibroblasts ◽

Exchange Chromatography ◽

Polyacrylamide Gel ◽

Purification And Properties ◽

Fibroblast Collagenase ◽

Specific Rabbit

1. A specific collagenase from the culture medium of rabbit synovial fibroblasts was purified by gel filtration and ion-exchange chromatography. 2. The enzyme was homogenous on polyacrylamide-gel electrophoresis and showed only traces of contaminants when tested in gels with a non-specific antiserum. 3. The rabbit fibroblast collagenase could hydrolyse collagen both in solution and in fibrillar form. Viscometry showed that at 35°C the purified enzyme could hydrolyse >50 nmol of collagen/min per mg of enzyme. 4. The purified collagenase cleaved collagen in solution at either 24°or 35°C into the characteristic 1/4 and 3/4-length fragments. However, as compared with the impure enzyme, the purified enzyme at 35°C had a much decreased capacity to further degrade the initial specific cleavage products. 5. The specific rabbit collagenase had a mol. wt. of approx. 32000 as estimated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, and 35000 by gel filtration.

Download Full-text

Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

Biochemical Journal ◽

10.1042/bj2130225 ◽

1983 ◽

Vol 213 (1) ◽

pp. 225-234 ◽

Cited By ~ 107

Author(s):

N Lambert ◽

R B Freedman

Keyword(s):

Amino Acid ◽

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Sedimentation Coefficient ◽

Bovine Liver ◽

Polyacrylamide Gel ◽

Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

Download Full-text

Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

Bayero Journal of Pure and Applied Sciences ◽

10.4314/bajopas.v13i2.14 ◽

2021 ◽

Vol 13 (2) ◽

pp. 107-112

Author(s):

C.F. Okechukwu ◽

P.L. Shamsudeen ◽

R.K. Bala ◽

B.G. Kurfi ◽

A.M. Abdulazeez

Keyword(s):

Ion Exchange ◽

Phospholipase A2 ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Partial Purification ◽

Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km of 1.47mg/ml and Vmax of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

Download Full-text