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 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 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.

scholarly journals Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

1996 ◽  
Vol 316 (3) ◽  
pp. 841-846 ◽  
Author(s):  
Stuart M. PITSON ◽  
Robert J. SEVIOUR ◽  
Barbara M. McDOUGALL ◽  
Bruce A. STONE ◽  
Maruse SADEK
Keyword(s):  
Molecular Mass ◽  
Filamentous Fungus ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Eisenia Bicyclis ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Sds Page ◽  
Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

scholarly journals Purification and properties of a 1,3-1,4-β-d-glucanase (lichenase, 1,3-1,4-β-d-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli

1988 ◽  
Vol 255 (3) ◽  
pp. 833-841 ◽  
Author(s):  
J D Erfle ◽  
R M Teather ◽  
P J Wood ◽  
J E Irvin
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Temperature Optimum ◽  
Glucanase Activity ◽  
Ph Optimum ◽  
Beta Glucanase ◽  
Purification And Properties ◽  
Hydrolysis Of

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.

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.

Extracellular proteolytic enzymes of psychrophilic bacteria. I. Purification and some properties of enzymes of an obligately psychrophilic red-pigmented bacterium

1968 ◽  
Vol 14 (3) ◽  
pp. 215-224 ◽  
Author(s):  
Y. Nunokawa ◽  
I. J. McDonald
Keyword(s):  
Metal Ions ◽  
Ammonium Sulfate ◽  
Synthetic Peptides ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Psychrophilic Bacterium ◽  
Psychrophilic Bacteria ◽  
Fraction I ◽  
Hydrolysis Of

Proteinase in culture fluids of an obligately psychrophilic bacterium was precipitated by ammonium sulfate and fractionated by gel filtration and DEAE-cellulose chromatography. Three purified fractions (I-1, I-2, and III-1) with proteinase activity were obtained. On the basis of reactions and characteristics (i.e. effect of pH, heat, and metal ions on activity and stability, hydrolysis of synthetic peptides and of natural proteins) fractions I-1 and III-1 appeared to be very similar whereas fraction I-2 was different. When proteinase preparations were examined by electrophoresis, fractions I-1 and III-1 gave similar patterns; fraction I-2 gave a different one. From the results it is suggested that the organism produces two proteinases and that possibly fraction I-1 represents an aggregation of molecules of III-1 and that fraction I-2 is a different proteinase.

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.

Characterization and substrate specificity of fumarylacetoacetate fumarylhydrolase

1977 ◽  
Vol 55 (1) ◽  
pp. 1-8 ◽  
Author(s):  
D. J. Mahuran ◽  
Ronald H. Angus ◽  
Carl V. Braun ◽  
S. S. Sim ◽  
Donald E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Sulfhydryl Groups ◽  
Ph Optimum ◽  
Amino Terminal ◽  
Diketo Acids ◽  
Hydrolysis Of

The molecular weight of fumarylacetoacetate fumarylhydrolase (EC 3.7.1.2) is 86 000 ± 10 000, as determined by gel filtration. The enzyme appears to be a dimer with a monomer molecular weight of 38 000 – 43 000, as determined by gel electrophoresis, gel filtration in guanidine–hydrochloride, and ultracentrifugation. The subunits appear to be identical, as only one band is seen in gel electrophoresis, only one protein peak is detected in gel filtration in guanidine–hydrochloride, and only one amino-terminal amino acid (proline) is detected. Three free sulfhydryl groups per denatured monomer are detected by reaction with 5,5′-dithiobis(2-nitrobenzoic acid), while for the active enzyme only two sulfhydryl groups react with this reagent. The extinction coefficients at 260 and 280 nm, the amino acid composition, and the isoelectric point (6.7) of the enzyme are also reported.The enzyme catalyzes the hydrolysis of six 2,4-diketo acids and three 3,5-diketo acids tested. The Km of the substrates is similar but V varies by a factor of 120. The pH optimum is 7.3. The enzyme did not catalyze the hydrolysis of a number of esters tested.

A new protease in hog thyroid lysosomes

1981 ◽  
Vol 98 (3) ◽  
pp. 390-395 ◽  
Author(s):  
H. Nakagawa ◽  
Y. Endo ◽  
S. Ohtaki
Keyword(s):  
Gel Filtration ◽  
Physiological Role ◽  
Gel Chromatography ◽  
Ph Optimum ◽  
Chloromethyl Ketone ◽  
Protein Substrates ◽  
Liver Lysosomes ◽  
Purification Ratio ◽  
Hydrolysis Of

Abstract. A leupeptin-sensitive new protease was partially purified from hog thyroid lysosomes. The purification procedure included solubilization by hypotonic treatment of lysosomes, and Sephacryl S-300 and Sephadex G-100 gel chromatography, and the purification ratio was 10-fold from lysosomes. The pH optimum of the protease activity was around 5.5 and its molecular weight was estimated to be 22 000 by gel filtration. 2-Mercaptoethanol activated the hydrolysis of protein substrates and its effect was most pronounced in the case of thyroglobulin as substrate. Among the inhibitors used, leupeptin, antipain, toluenesulfonyl-lysine chloromethyl ketone and, to a lesser degree, chymostatin and toluenesulfonyl-phenylalanine chloromethyl ketone effectively inhibited the hydrolysis of casein by the enzyme at pH 5.5, whereas pepstatin did not inhibit the activity significantly. The enzyme activity was also inhibited by sulfhydryl inhibitors such as iodoacetamide, p-chloromercuribenzoate, and N-ethylmaleimide. The release of iodoamino acids from thyroglobulin by the enzyme was inhibited in the same manner by the inhibitors used in the hydrolysis of casein. The physiological role of the new protease is discussed in comparison with cathepsin B and L found in liver lysosomes.

α-Glutamic acid-β-naphthylamidase from Neisseria catarrhalis

1969 ◽  
Vol 15 (11) ◽  
pp. 1293-1300 ◽  
Author(s):  
Sidney T. Cox ◽  
Francis J. Behal
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Metal Ion ◽  
Gel Filtration ◽  
Aminopeptidase Activity ◽  
Amino Acid Residues ◽  
Ph Optimum ◽  
Hydrolysis Of ◽  
Derivatives Of

A second bacterial peptidase-like enzyme, arylamidase-II, has been isolated from cell free extracts of Neisseria catarrhalis. Arylamidase-II action is limited primarily to the hydrolysis of α-glutamic acid and α-aspartic acid derivatives of β-naphthylamine and short peptides of glutamic acid. The enzyme was purified 450-fold by gel filtration, ion exchange, and calcium phosphate chromatography. Its pH optimum and molecular weight were 7.7 and 170 000, respectively. Aside from its restricted substrate specificity, arylamidase-II has been found to be closely related in its mechanism of action, molecular weight, pH optimum, and metal ion dependence to arylamidase-I, which catalyzes the hydrolysis of neutral amino acid derivatives of β-naphthylamine. Arylamidase-II exhibits aminopeptidase activity, requiring the amino acid residues in the N-terminal and penultimate position to be of the L-configuration in order for hydrolysis to occur.

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