scholarly journals Characterization of β-galactosidase and α-galactosidase activities from the halophilic bacterium Gracilibacillus dipsosauri

2021 ◽  
Vol 71 (1) ◽  
Author(s):  
Charles E. Deutch ◽  
Amy M. Farden ◽  
Emily S. DiCesare
Keyword(s):  
Gel Filtration ◽  
Halophilic Bacterium ◽  
Peptide Sequencing ◽  
Exchange Chromatography ◽  
Galactosidase Activity ◽  
Ph Optimum ◽  
Whole Cells ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Hydrolysis Of

Abstract Purpose Gracilibacillus dipsosauri strain DD1 is a salt-tolerant Gram-positive bacterium that can hydrolyze the synthetic substrates o-nitrophenyl-β-d-galactopyranoside (β-ONP-galactose) and p-nitrophenyl-α-d-galactopyranoside (α-PNP-galactose). The goals of this project were to characterize the enzymes responsible for these activities and to identify the genes encoding them. Methods G. dipsosauri strain DD1 was grown in tryptic soy broth containing various carbohydrates at 37 °C with aeration. Enzyme activities in cell extracts and whole cells were measured colorimetrically by hydrolysis of synthetic substrates containing nitrophenyl moieties. Two enzymes with β-galactosidase activity and one with α-galactosidase activity were partially purified by ammonium sulfate fractionation, ion-exchange chromatography, and gel-filtration chromatography from G. dipsosauri. Coomassie Blue-stained bands corresponding to each activity were excised from nondenaturing polyacrylamide gels and subjected to peptide sequencing after trypsin digestion and HPLC/MS analysis. Result Formation of β-galactosidase and α-galactosidase activities was repressed by d-glucose and not induced by lactose or d-melibiose. β-Galactosidase I had hydrolytic and transgalactosylation activity with lactose as the substrate but β-galactosidase II showed no activity towards lactose. The α-galactosidase had hydrolytic and transgalactosylation activity with d-melibiose but not with d-raffinose. β-Galactosidase I had a lower Km with β-ONP-galactose as the substrate (0.693 mmol l−1) than β-galactosidase II (1.662 mmol l−1), was active at more alkaline pH, and was inhibited by the product d-galactose. β-Galactosidase II was active at more acidic pH, was partially inhibited by ammonium salts, and showed higher activity with α-PNP-arabinose as a substrate. The α-galactosidase had a low Km with α-PNP-galactose as the substrate (0.338 mmol l−1), a pH optimum of about 7, and was inhibited by chloride-containing salts. β-Galactosidase I activity was found to be due to the protein A0A317L6F0 (encoded by gene DLJ74_04930), β-galactosidase II activity to the protein A0A317KZG3 (encoded by gene DLJ74_12640), and the α-galactosidase activity to the protein A0A317KU47 (encoded by gene DLJ74_17745). Conclusions G. dipsosauri forms three intracellular enzymes with different physiological properties which are responsible for the hydrolysis of β-ONP-galactose and α-PNP-galactose. BLAST analysis indicated that similar β-galactosidases may be formed by G. ureilyticus, G. orientalis, and G. kekensis and similar α-galactosidases by these bacteria and G. halophilus.

scholarly journals Enzymes of Trichomonas foetus. Separation and properties of two β-galactosidases

1970 ◽  
Vol 117 (4) ◽  
pp. 667-675 ◽  
Author(s):  
G. J. Harrap ◽  
Winifred M. Watkins
Keyword(s):  
Metal Ions ◽  
Gel Filtration ◽  
Galactosidase Activity ◽  
Ph Optimum ◽  
Crude Preparation ◽  
Hydrolysis Of

The β-galactosidase activity in extracts of Trichomonas foetus is separable into two fractions by gel filtration on Sephadex G-200. When o-nitrophenyl β-d-galactoside is used as substrate the first fraction to be eluted, β-galactosidase 1, has 50 times the activity (units per mg of protein) of the crude preparation. This fraction is activated by Mn2+ and Co2+ and inhibited by Hg2+ and EDTA. In the presence of Mn2+ the pH optimum for the hydrolysis of o-nitrophenyl β-d-galactoside or lactose is 5.8–6.0. β-Galactosidase 1 is an exoglycosidase that releases β-linked galactose joined to aliphatic and various carbohydrate aglycones. Hydrolysis is prevented, however, by a substituent on either the subterminal sugar or the terminal non-reducing β-galactosyl residue in an oligosaccharide. The second fraction, β-galactosidase 2, is not activated by metal ions or inhibited by EDTA and has a broad pH optimum from 4.5 to 6.0.

scholarly journals The Modified β-Ketoadipate Pathway in Rhodococcus rhodochrous N75: Enzymology of 3-Methylmuconolactone Metabolism

1998 ◽  
Vol 180 (24) ◽  
pp. 6668-6673 ◽  
Author(s):  
Chang-Jun Cha ◽  
Ronald B. Cain ◽  
Neil C. Bruce
Keyword(s):  
Ammonium Sulfate ◽  
Gel Filtration ◽  
Sole Source ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Rhodococcus Rhodochrous ◽  
Natural Substrate ◽  
Ph Optimum ◽  
A Subunit ◽  
Hydrolysis Of

ABSTRACT Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified β-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of R. rhodochrous N75 on p-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native M r of 128,000, and a subunitM r of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.

The Purification and Properties of an Amidohydrolase from Soybean

1974 ◽  
Vol 52 (10) ◽  
pp. 903-910 ◽  
Author(s):  
Robert E. Hoagland ◽  
George Graf
Keyword(s):  
Arrhenius Plot ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Hydrolysis Rate ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Hydrolysis Rates ◽  
Hydrolysis Of

An amidohydrolase (EC 3.5.1.13) was isolated from the roots of soybean (Glycine max Merril, var. Hawkeye) seedlings and purified 130-fold over the crude extract with 30% recovery. The purification steps entailed ammonium sulfate precipitation, gel filtration, cellulose ion-exchange chromatography, and polyacrylamide gel electrophoresis. The specific activity of the purified enzyme for the hydrolysis of Nα-benzoyl-DL-arginine p-nitroanilide (BAPA) was 810 mU/mg. The Km of the enzyme for this substrate was 5.78 × 10−6 M. The enzyme possessed a broad substrate specificity and catalyzed the hydrolysis of BAPA, glycine p-nitroanilide, L-leucine p-nitroanilide, and L-lysine p-nitroanilide. Specificity studies with a series of aminoacyl β-naphthylamides revealed a high hydrolysis rate on Nα-benzoyl-L-arginine β-naphthylamide, and lower hydrolysis rates on several other aminoacyl-substituted β-naphthylamides. The enzyme also displayed dipeptide hydrolase activity on several dipeptide substrates. The enzyme had a pH optimum of 8.0 in 0.05 M phosphate buffer with Nα-benzoyl-DL-arginine p-nitroanilide as substrate. The temperature optimum was 50 °C. The apparent activation energy determined from an Arrhenius plot was 6.3 kcal/mol (26 400 J/mol). The molecular weight estimated by gel filtration was approximately 63 000. Mercury (II) ion, silver (I) ion, p-benzoquinone, p-chloromercuribenzoate, and N-ethylmaleimide were effective inhibitors of the enzyme.

Synthetic 1-thio-β-D-glucosiduronic acids as substrates for rat-liver β-glucuronidase

1970 ◽  
Vol 48 (7) ◽  
pp. 799-804 ◽  
Author(s):  
C. Hétu ◽  
R. Gianetto
Keyword(s):  
Rat Liver ◽  
Molecular Sieve ◽  
Enzyme Preparation ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Filtration Step ◽  
Hydrolysis Of ◽  
Menten Constant

The hydrolysis of 1-thio-β-D-glucosiduronic acids by rat liver was studied using synthetic phenyl 1-thio-β-D-glucosiduronic acid, sodium (2-benzothiazolyl 1-thio-β- D-glucosid)uronate, and sodium (p-nitrophenyl 1-thio-β-D-glucosid)uronate. It was found that rat liver preparations can hydrolyze the β-D-glucuronides of 2-benzothiazolethiol and p-nitrothiophenol but not the β-D-glucuronide of thiophenol.Partial purification of the enzyme from a lysosomal preparation using ammonium sulfate fractionation, gel filtration on a molecular sieve, and anion-exchange chromatography showed that β-glucuronidase (EC 3.2.1.31) is the enzyme responsible for the hydrolysis of these thioglucuronides. The pH optimum and Michaelis–Menten constant (Km) were determined for both substrates using an enzyme preparation obtained after the gel filtration step. The glucuronide of 2-benzothiazolethiol was found to be almost as good a substrate as that of phenolphthalein for rat-liver β-glucuronidase, while the glucuronide of p-nitrothiophenol is hydrolyzed at a much slower rate. Possible explanations of the fact that β-glucuronidase hydrolyzes only certain thioglucuronides are suggested.

Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts

1984 ◽  
Vol 4 (6) ◽  
pp. 1003-1012
Author(s):  
R L Nelson ◽  
P E Branton
Keyword(s):  
Chicken Embryo ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Soluble Form ◽  
Ph Optimum ◽  
Cell Extracts ◽  
Phosphoprotein Phosphatases ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

scholarly journals Purification and properties of three (1→3)-β-d-glucanase isoenzymes from young leaves of barley (Hordeum vulgare)

1993 ◽  
Vol 289 (2) ◽  
pp. 453-461 ◽  
Author(s):  
M Hrmova ◽  
G B Fincher
Keyword(s):  
Hordeum Vulgare ◽  
Elevated Temperatures ◽  
Gel Filtration ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Purification And Properties ◽  
Young Leaves ◽  
Monomeric Proteins ◽  
Ph Range ◽  
Hydrolysis Of

Three (1->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes GI, GII and GIII were purified from young leaves of barley (Hordeum vulgare) using (NH4)2SO4 fractional precipitation, ion-exchange chromatography, chromatofocusing and gel-filtration chromatography. The three (1->3)-beta-D-glucanases are monomeric proteins of apparent M(r)32,000 with pI values in the range 8.8-10.3. N-terminal amino-acid-sequence analyses confirmed that the three isoenzymes represent the products of separate genes. Isoenzymes GI and GII are less stable at elevated temperatures and are active over a narrower pH range than is isoenzyme GIII, which is a glycoprotein containing 20-30 mol of hexose equivalents/mol of enzyme. The preferred substrate for the enzymes is laminarin from the brown alga Laminaria digitata, an essentially linear (1->3)-beta-D-glucan with a low degree of glucosyl substitution at 0-6 and a degree of polymerization of approx. 25. The three enzymes are classified as endohydrolases, because they yield (1->3)-beta-D-oligoglucosides with degrees of polymerization of 3-8 in the initial stages of hydrolysis of laminarin. Kinetic analyses indicate apparent Km values in the range 172-208 microM, kcat. constants of 36-155 s-1 and pH optima of 4.8. Substrate specificity studies show that the three isoenzymes hydrolyse substituted (1->3)-beta-D-glucans with degrees of polymerization of 25-31 and various high-M(r), substituted and side-branched fungal (1->3;1->6)-beta-D-glucans. However, the isoenzymes differ in their rates of hydrolysis of a (1->3;1->6)-beta-D-glucan from baker's yeast and their specific activities against laminarin vary significantly. The enzymes do not hydrolyse (1->3;1->4)-beta-D-glucans, (1->6)-beta-D-glucan, CM-cellulose, insoluble (1->3)-beta-D-glucans or aryl beta-D-glycosides.

Physicochemical properties of a novel α-L-arabinofuranosidase fromRhizomucor pusillusHHT-1

2001 ◽  
Vol 47 (8) ◽  
pp. 767-772 ◽  
Author(s):  
A KM Shofiqur Rahman ◽  
Shinya Kawamura ◽  
Masahiro Hatsu ◽  
M M Hoq ◽  
Kazuhiro Takamizawa
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Rhizomucor Pusillus ◽  
Terminal Amino ◽  
Metal Cofactors

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular α-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0–10.0, respectively. The temperature optimum was 65°C, and it was stable up to 70°C. The Kmand Vmaxfor p-nitrophenyl α-L-arabinofuranoside were 0.59 mM and 387 µmol·min–1·mg–1protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with p-nitrophenyl α-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat–spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.Key words: α-L-arabinofuranosidase, enzyme purification, amino acid sequence, Rhizomucor pusillus.

scholarly journals A high-molecular-mass neutral endopeptidase-24.5 from human lung

1987 ◽  
Vol 241 (1) ◽  
pp. 129-135 ◽  
Author(s):  
R Zolfaghari ◽  
C R Baker ◽  
P C Canizaro ◽  
A Amirgholami ◽  
F J Bĕhal
Keyword(s):  
Metal Ions ◽  
Human Lung ◽  
Proteinase Inhibitors ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Neutral Endopeptidase ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Apparent Homogeneity

A high-Mr neutral endopeptidase-24.5 (NE) that cleaved bradykinin at the Phe5-Ser6 bond was purified to apparent homogeneity from human lung by (NH4)2SO4 fractionation, ion-exchange chromatography and gel filtration. The final enzyme preparation produced a single enzymically active protein band after electrophoresis on a 5% polyacrylamide gel. Human lung NE had an Mr of 650,000 under non-denaturing conditions, but after denaturation and electrophoresis on an SDS/polyacrylamide gel NE dissociated into several lower-Mr components (Mr 21,000-32,000) and into two minor components (Mr approx. 66,000). The enzyme activity was routinely assayed with the artificial substrate Z-Gly-Gly-Leu-Nan (where Z- and -Nan represent benzyloxycarbonyl- and p-nitroanilide respectively). NE activity was enhanced slightly by reducing agents, greatly diminished by thiol-group inhibitors and unchanged by serine-proteinase inhibitors. Human lung NE was inhibited by the univalent cations Na+ and K+. No metal ions were essential for activity, but the heavy-metal ions Cu2+, Hg2+ and Zn2+ were potent inhibitors. With the substrate Z-Gly-Gly-Leu-Nan a broad pH optimum from pH 7.0 to pH 7.6 was observed, and a Michaelis constant value of 1.0 mM was obtained. When Z-Gly-Gly-Leu-Nap (where -Nap represents 2-naphthylamide) was substituted for the above substrate, no NE-catalysed hydrolysis occurred, but Z-Leu-Leu-Glu-Nap was readily hydrolysed by NE. In addition, NE hydrolysed Z-Gly-Gly-Arg-Nap rapidly, but at pH 9.8 rather than in the neutral range. Although human lung NE was stimulated by SDS, the extent of stimulation was not appreciable as compared with the extent of SDS stimulation of NE from other sources.

Characterization of Muscle Sarcoplasmic and Myofibrillar Protein Hydrolysis Caused by Lactobacillus plantarum

1999 ◽  
Vol 65 (8) ◽  
pp. 3540-3546 ◽  
Author(s):  
Silvina Fadda ◽  
Yolanda Sanz ◽  
Graciela Vignolo ◽  
M.-Concepción Aristoy ◽  
Guillermo Oliver ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Myofibrillar Protein ◽  
Myofibrillar Proteins ◽  
Muscle Proteins ◽  
Whole Cells ◽  
Sarcoplasmic Proteins ◽  
Cell Extracts ◽  
Hydrophobic Nature ◽  
Hydrolysis Of

ABSTRACT Strains of Lactobacillus plantarum originally isolated from sausages were screened for proteinase and aminopeptidase activities toward synthetic substrates; on the basis of that screening,L. plantarum CRL 681 was selected for further assays on muscle proteins. The activities of whole cells, cell extracts (CE), and a combination of both on sarcoplasmic and myofibrillar protein extracts were determined by protein, peptide, and free-amino-acid analyses. Proteinase from whole cells initiated the hydrolysis of sarcoplasmic proteins. The addition of CE intensified the proteolysis. Whole cells generated hydrophilic peptides from both sarcoplasmic and myofibrillar proteins. Other peptides of a hydrophobic nature resulted from the combination of whole cells and CE. The action of both enzymatic sources on myofibrillar proteins caused maximal increases in lysine, arginine, and leucine, while the action of those on sarcoplasmic proteins mainly released alanine. In general, pronounced hydrolysis of muscle proteins required enzyme activities from whole cells in addition to those supplied by CE.

scholarly journals Characterization of Reutericyclin Produced by Lactobacillus reuteri LTH2584

2000 ◽  
Vol 66 (10) ◽  
pp. 4325-4333 ◽  
Author(s):  
Michael G. Gänzle ◽  
Alexandra Höltzel ◽  
Jens Walter ◽  
Günther Jung ◽  
Walter P. Hammes
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Culture Supernatant ◽  
Lactobacillus Reuteri ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Growth Media ◽  
Dependent Manner ◽  
Cell Extracts

ABSTRACT Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains ofEscherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C8 chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. Höltzel, M. G. Gänzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766–2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.

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