A halophilic extracellular protease from a halophilic archaebacterium strain 172 P1

1990 ◽  
Vol 68 (1) ◽  
pp. 352-359 ◽  
Author(s):  
Masahiro Kamekura ◽  
Yukio Seno
Keyword(s):  
Serine Protease ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Amino Acid Residues ◽  
High Concentration ◽  
Extracellular Proteases ◽  
Complete Inactivation ◽  
Halophilic Archaebacteria ◽  
Terminal Amino

An unidentified halophilic archaebacterium strain 172 P1 produced three extracellular proteases in media containing 15–27% salts. One component, F-II, was purified to homogeneity. It is a serine protease that can be inhibited by phenylmethylsulfonyl fluoride and chymostatin. A high concentration of NaCl was required for its stability; in the presence of 25% NaCl, only 4% of the activity was lost by incubating at 60 °C for 30 min, while complete inactivation occurred in the presence of 5% NaCl. F-II is a thermophilic and halophilic protease. High activity was obtained at 75–80 °C when F-II was assayed in the presence of 25% NaCl. The optimal concentration of NaCl required was 10–14% when assayed at 70 °C with azocasein as substrate, though a halophilic characteristic was not distinct at lower temperatures. Hydrolyses of the synthetic substrates succinyl-alanyl-alanyl-prolyl-phenylalanyl-4-methylcoumaryl-7-amide or succinyl-alanyl-alanyl-alanyl-p-nitroanilide at 26 °C were maximal at 25 and 30% NaCl, respectively. F-II was most stable at pH 6–7, and its optimal pH was 10.7. Its molecular weight was estimated as 44 000 – 46 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and by gel filtration – high-pressure liquid chromatography. The sequence of the 35 N-terminal amino acid residues was determined and compared with that of other serine proteases.Key words: halophilic, archaebacteria, serine protease, thermophilic.

scholarly journals Thermophilic, Reversible γ-Resorcylate Decarboxylase from Rhizobium sp. Strain MTP-10005: Purification, Molecular Characterization, and Expression

2004 ◽  
Vol 186 (20) ◽  
pp. 6855-6863 ◽  
Author(s):  
Masahiro Yoshida ◽  
Nobuhiro Fukuhara ◽  
Tadao Oikawa
Keyword(s):  
Rattus Norvegicus ◽  
Homo Sapiens ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hypothetical Protein ◽  
Cell Extract ◽  
Amino Acid Residues ◽  
Sequence Identities ◽  
Rhizobium Sp

ABSTRACT We found the occurrence of thermophilic reversible γ-resorcylate decarboxylase (γ-RDC) in the cell extract of a bacterium isolated from natural water, Rhizobium sp. strain MTP-10005, and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be about 151 kDa by gel filtration, and that of the subunit was 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; in other words, the enzyme was a homotetramer. The enzyme was induced specifically by the addition of γ-resorcylate to the medium. The enzyme required no coenzyme and did not act on 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3,5-dihydroxybenzoate, 2-hydroxybenzoate, or 3-hydroxybenzoate. It was relatively thermostable to heat treatment, and its half-life at 50°C was estimated to be 122 min; furthermore, it catalyzed the reverse carboxylation of resorcinol. The values of k cat/Km (mΜ−1 · s−1) for γ-resorcylate and resorcinol at 30°C and pH 7 were 13.4 and 0.098, respectively. The enzyme contains 327 amino acid residues, and sequence identities were found with those of hypothetical protein AGR C 4595p from Agrobacterium tumefaciens strain C58 (96% identity), 5-carboxyvanillate decarboxylase from Sphingomonas paucimobilis (32%), and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylases from Bacillus cereus ATCC 10987 (26%), Rattus norvegicus (26%), and Homo sapiens (25%). The genes (graA [1,230 bp], graB [888 bp], and graC [1,056 bp]) that are homologous to those in the resorcinol pathway also exist upstream and downstream of the γ-RDC gene. Judging from these results, the resorcinol pathway also exists in Rhizobium sp. strain MTP-10005, and γ-RDC probably catalyzes a reaction just before the hydroxylase in it does.

scholarly journals Purification and Characterization of a Novel 5-Oxoprolinase (without ATP-Hydrolyzing Activity) fromAlcaligenes faecalis N-38A

1999 ◽  
Vol 65 (2) ◽  
pp. 712-717 ◽  
Author(s):  
Atsuhisa Nishimura ◽  
Yasunori Ozaki ◽  
Hiroshi Oyama ◽  
Takashi Shin ◽  
Sawao Murao
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Alcaligenes Faecalis ◽  
Cell Extract ◽  
Homogeneous State ◽  
Amino Terminal ◽  
A Cell ◽  
Terminal Amino

ABSTRACT A novel type of 5-oxoprolinase was found in a cell extract of strain N-38A, which was later identified as Alcaligenes faecalis. The enzyme in the cell extract was purified to a homogeneous state with a yield of 16.6%. The molecular weight of the purified enzyme was estimated to be 47,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, suggesting that the enzyme is a monomeric protein. The enzyme specifically catalyzed a decyclization of l-pyroglutamate without hydrolyzing ATP and also without any requirements for metal ions such as Mg2+ and K+. The optimal pH for the decyclization was 7.4. The reaction was reversible. The equilibrium constant of the reaction, K eq = [l-glutamate]/[l-pyroglutamate], was evaluated to be approximately 0.035, which indicates that the reaction tends to form l-pyroglutamate. The amino-terminal amino acid sequence of the enzyme was H-Glu-Pro-Arg-Leu-Asp-Thr-Ser-Gln-Leu-Tyr-Ala-Asp-Val-His-Phe-. No protein with a similar sequence was found in the DNASIS database. Based on these data, it was strongly suggested that the enzyme described here is a novel type of 5-oxoprolinase.

Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7

1992 ◽  
Vol 38 (9) ◽  
pp. 891-897 ◽  
Author(s):  
Hiroshi Tsujibo ◽  
Yukio Yoshida ◽  
Katsushiro Miyamoto ◽  
Chiaki Imada ◽  
Yoshiro Okami ◽  
...  
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Amino Terminal

Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 °C, respectively. Chi-A was stable in the range of pH 5–10 up to 40 °C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulons WL-12. Key words: marine bacterium, Alteromonas sp., chitinase.

scholarly journals Alternaria brassicae Produces a Host-Specific Protein Toxin from Germinating Spores on Host Leaves

2008 ◽  
Vol 98 (4) ◽  
pp. 458-463 ◽  
Author(s):  
R. Y. Parada ◽  
E. Sakuno ◽  
N. Mori ◽  
K. Oka ◽  
M. Egusa ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Protein ◽  
Gray Leaf Spot ◽  
Proteinase K ◽  
Alternaria Brassicae ◽  
Amino Acid Residues ◽  
New Host ◽  
Protein Toxin ◽  
Terminal Amino

Spore suspensions of Alternaria brassicae, the causal agent of gray leaf spot in Brassica plants, were incubated on the leaves of cabbage (B. oleracea) and spore germination fluid (SGF) was collected after 48 h. A high molecular weight (HMW) fraction (>10 kDa) was separated from the SGF by ultrafiltration. In a detached leaf assay, the HMW fraction induced visible symptoms only on host leaves and the toxicity was lost by treatment with proteinase K or heat at 60°C for 15 min, indicating the presence of host-specific protein toxin(s). A protein toxin in the HMW fraction was purified by several chromatography steps. The toxin induced water-soaked symptoms followed by chlorosis at concentrations of 0.5 to 1 μg/ml on host leaves, but not on nonhost leaves even at 50 μg/ml. The toxin also had infection-inducing activity when added to spore suspension of a nonpathogenic isolate of A. alternata, causing symptoms similar to the infection of A. brassicae only on host leaves. These results indicate that a new host-specific protein toxin named ABR-toxin is released from germinating spores of A. brassicae on host leaves. ABR-toxin migrated as a protein of 27.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of ABR-toxin was estimated to be approximately 7.0 and 21 N-terminal amino acid residues were sequenced.

scholarly journals Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

2001 ◽  
Vol 67 (6) ◽  
pp. 2445-2452 ◽  
Author(s):  
Yuji Kannan ◽  
Yuichi Koga ◽  
Yohei Inoue ◽  
Mitsuru Haruki ◽  
Masahiro Takagi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Heat Inactivation ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Hyperthermophilic Archaeon

ABSTRACT The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensissubtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding,T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly−82 to Gly316), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests thatT. kodakaraensis subtilisin exists in a monomeric form.T. kodakaraensis subtilisin hydrolyzed the synthetic substrateN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca2+ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.

scholarly journals Purification of a neutral proteinase, associated with the actomyosin complex, from uterine myometrium

1984 ◽  
Vol 222 (3) ◽  
pp. 613-620 ◽  
Author(s):  
R Barth ◽  
M Hoechst ◽  
E G Afting
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Thiol Group ◽  
Hydrolytic Activity ◽  
Proteinase Activity ◽  
Molar Ratio ◽  
High Concentration ◽  
Neutral Proteinase ◽  
Actomyosin Complex

We have purified and characterized a neutral proteinase activity from pig uterine myometrium. The proteinase co-purified with the actomyosin complex and could only be separated from it by a high concentration of a chaotropic ion, 3M-NaBr. The proteinase was further purified by gel filtration and affinity chromatography. The purified protein showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to an Mr of 28 000. Gel filtration on Sephadex G-100 in a buffer containing 3M-NaBr gave an Mr of 27 500. Without the addition of the chaotropic Br- ion, the proteinase aggregates to high-Mr forms of more than 10(6)Da. The proteinase has optimum hydrolytic activity with casein as substrate at pH 7.5-8.0. The thiol-group-blocking reagents p-chloromercuribenzoate, p-chloromercuribenzenesulphonate and Hg2+, as well as soya-bean trypsin inhibitor and 4-aminobenzamidine, inhibited the proteinase. Other bivalent cations, chelating agents and the serine-specific reagents 7-amino-1-chloro-3-tosylamido-L-heptan-2-one and phenylmethanesulphonyl fluoride were without any effect on proteinase activity. The proteinase degraded myosin very rapidly at a molar ratio of proteinase to myosin of 1:50, concomitant with the rate of loss of the ATPase activity. Compared with myosin, actin was only a poor substrate and was degraded at a much lower rate, even at a high molar ratio of the proteinase to actin.

scholarly journals Purification and Properties of an Esterase from the YeastSaccharomyces cerevisiae and Identification of the Encoding Gene

1999 ◽  
Vol 65 (8) ◽  
pp. 3470-3472 ◽  
Author(s):  
Giuliano Degrassi ◽  
Lasse Uotila ◽  
Raffaella Klima ◽  
Vittorio Venturi
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Terminal Amino Acid ◽  
Purification And Properties ◽  
Terminal Amino ◽  
Encoding Gene ◽  
Esterase Activities ◽  
Null Mutants

ABSTRACT We purified an intracellular esterase that can function as anS-formylglutathione hydrolase from the yeastSaccharomyces cerevisiae. Its molecular mass was 40 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was 5.0 by isoelectric focusing. The enzyme activity was optimal at 50°C and pH 7.0. The corresponding gene, YJLO68C, was identified by its N-terminal amino acid sequence and is not essential for cell viability. Null mutants have reduced esterase activities and grow slowly in the presence of formaldehyde. This enzyme may be involved in the detoxification of formaldehyde, which can be metabolized toS-formylglutathione by S. cerevisiae.

scholarly journals Enzyme instability and proteolysis during the purification of an alcohol dehydrogenase from Drosophila melanogaster

1977 ◽  
Vol 163 (2) ◽  
pp. 317-323 ◽  
Author(s):  
D R Thatcher
Keyword(s):  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Alcohol Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Evolutionary Significance ◽  
Nitrobenzoic Acid ◽  
A Subunit ◽  
Terminal Amino

The alcohol dehydrogenase of the Drosophila melanogaster adhUF allele (alloenzyme with ultra-fast electrophoretic mobility) was unstable in crude or partially purified preparations. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that inactivation was porbably due to proteolytic degradation, and new method of purification of the enzyme was developed. After three steps, namely salmine sulphate precipitation, hydroxyapatite chromatography and Sephadex G-100 gel filtration, a 10-fold purified preparation was obtained. The enzyme produced was relatively stable compared with alcohol dehydrogenase purified by other methods, and was shown to be proteinase-free. The enzyme had a subunit mol.wt. of 24000 and had a single thiol residue per subunit available for titration with 5,5′-dithiobis-(2-nitrobenzoic acid). The amino acid composition and C-terminal amino acid sequence of the enzyme were determined. The substrate specificity of this alcohol dehydrogenase was also characterized. These results are discussed in relation to experiments on the evolutionary significance of thermostability at the adh locus.

Characterization and purification of extracellular proteases of Trichomonas vaginalis

1989 ◽  
Vol 35 (10) ◽  
pp. 903-909 ◽  
Author(s):  
Gary E. Garber ◽  
Laurel T. Lemchuk-Favel
Keyword(s):  
Gel Electrophoresis ◽  
Trichomonas Vaginalis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Extracellular Proteases ◽  
Chloromethyl Ketone ◽  
Acid Ph ◽  
Molecular Masses

Extracellular protease activity was detected in serum-free culture filtrates of Trichomonas vaginalis. The activity was demonstrated by hydrolysis of hide powder azure and possessed the characteristics of cysteine type proteases: inhibition by N-ethyl maleimide, Cu2+, antipain, N-tosyl-L-phenylalanine chloromethyl ketone, N-tosyl-L-lysine chloromethyl ketone, leupeptin, chymostatin, and iodoacetamide, and enhancement by cysteine, EDTA, and dithiothreitol. The activity was optimal at acid pH and the protease was also active on peptide nitroanilides with arginine derivatives. Purification of this activity by ethanol precipitation, ammonium sulfate fractionation, ion exchange chromatography, and gel filtration resulted in the isolation of two proteases estimated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis to have molecular masses of 60 and 30 kilodaltons (kDa), respectively. The larger molecular mass protease broke down during purifications to two subunits of approximately 23 and 43 kDa, as determined by gel electrophoresis. Rabbit sera derived by immunization with the 23-kDa subunit cross-reacted by immunoblot with the 60- and 43-kDa subunits, but not with the 30-kDa protease. These soluble products of T. vaginalis growth could be important pathogenically in establishing T. vaginalis infection in the normally acid (pH ≤ 4.5) environment of the vagina.Key words: Trichomonas vaginalis, trichomoniasis, vaginitis, protease, pathogenesis.

scholarly journals Restoration of β-galactosidase to Escherichia coli M15. Complementation studies

1977 ◽  
Vol 165 (3) ◽  
pp. 417-423 ◽  
Author(s):  
Dobrivoje V. Marinkovic ◽  
Jelka N. Marinkovic
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Galactosidase Activity ◽  
Terminal Amino Acid ◽  
E Coli ◽  
Molecular Weights ◽  
Terminal Amino

Carboxymethylated β-galactosidase from Escherichia coli was dissociated at 100°C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore β-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ‘complementable fractions’. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the β-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented β-galactosidase can exist.

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