Substrate specificity of thermitase, a thermostable serine proteinase fromThermoactinomyces vulgaris

Brὂmme, Dieter; Kleine, R.

doi:10.1007/bf01567710

Cited by 18 publications

(5 citation statements)

References 18 publications

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“…1 mM, 3 mM and 10 mM Ca2+ had equal effects on the stability of the enzyme to treatment at 80°C for 15 min and 30 min (data not shown). After treatment at 90°C and 95 "C for 30 min in the presence of Ca2+ (1 mM), the remain- (B), thermitase (1 : lo3, by mass; 2 h) [31] (C) and subtilisin BPN' (1 : 1170, by mass; 2 h) [32] (D) are also shown. Larger arrowheads indicate major cleavage sites, and smaller arrowheads minor ones.…”

Section: Effect Of Temperature On the Proteolytic Activity And Stabilmentioning

confidence: 99%

“…Fig. 7 also shows the cleavage sites for several microbial alkaline serine proteases, proteinase K [30], thermitase [31] and subtilisin BPN' [32]. The Gln4-His', Ser9-His" and Le~"-Tyr'~ bonds are commonly cleaved by all of the proteases mentioned, and there are no proteases which have the same cleavage sites as aqualysin I.…”

Section: Cleavage Specificity Of Aqualysin 1 Toward Oxidized Insulin mentioning

confidence: 99%

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Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT‐1

Matsuzawa

Tokugawa

Hamaoki

et al. 1988

European Journal of Biochemistry

106

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Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1. Aqualysin I was purified, and its apparent relative molecular mass was determined to be 28 500. The enzyme contained four Cys residues (probably as two cystines), and its amino acids composition was similar to those of cysteine-containing serine proteases (proteinase K, etc.) as well as those of subtilisins. The NH,-terminal sequence of aqualysin I showed homology with those of the microbial serine proteases. The optimum pH for the proteolytic activity of aqualysin 1 was around 10.0. Ca2+ stabilized the enzyme to heat treatment, and the maximum proteolytic activity was observed at 80°C. Aqualysin I was stable to denaturing reagents (7 M urea, 6 M guanidine . HCl and 1 YO SDS) at 23 "C for 24 h. The enzyme hydrolyzed the ester bond of an alanine ester and succinyl-Ala-Ala-Ala p-nitroanilide, a synthetic substrate for mammalian elastase. The cleavage sites for aqualysin I in oxidized insulin B chain were not specific when it was digested completely.The enzymes of thermophilic bacteria are generally heatstable [I], and the stability of these proteins is interesting for protein chemistry. Besides their scientific interest, these heatstable enzymes are valuable because of their bioengineering and biotechnological applications. We have studied the enzymes of extremely thermophilic bacteria belonging to the genus Thermus, mainly L-lactate dehydrogenases [2 -51 and proteases [6,7]. Heat-stable proteases are expected to be useful for application [8].At present three Thermus species strains are known which produce heat-stable extracellular proteases. A metal protease (called caldolysin) produced by Thermus T-351 has been purified and characterized [9]. We reported that Thermus caldophilus GK24 produces a neutral serine protease which forms a complex with several kinds of proteins and which appears homogeneous on chromatographic analysis [6]. These two proteases are extremely heat-stable and resistant to denaturing treatments [6, 91. We previously reported the production of two kinds of extracellular proteases, aqualysins I and I1 [7], by Thermus aquaticus YT-1 [lo]. Aqualysin I is an alkaline serine protease, and aqualysin I1 an chelator-sensitive, neutral serine protease, the optimum temperatures for the proteolytic activities of the two enzymes being around 75°C and 95°C respectively [7]. Correspoiidence to H. Matsuzawa, Department of AgriculturalChemistry, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 113Abbreviations. iPr,P-F, diisopropylfluorophosphate; Z-AlaGly-PheCH2C1, benzyloxycarbonyl-Ala-Gly-Phe-chloromethane; CIHgBzOH, p-chloromercuribenzoate; TosPheCH2C1, N-tosylphenylalanylchloromethane; TosLysCH2Cl, Nu-tosyl-L-lysylchloromethane; Suc-Ala-Ala-Ala-NH-Np, succinyl-Ala-Ala-Ala p-nitroanilide; Cya, cysteic acid. Except where otherwise specified, the constituent amino acids were all of the L configuration.Enzymes. Serine proteinase (EC 3.4.21 .-); microbial serine proteinase (EC 3.4.21.14); subtili...

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Section: Effect Of Temperature On the Proteolytic Activity And Stabilmentioning

confidence: 99%

Section: Cleavage Specificity Of Aqualysin 1 Toward Oxidized Insulin mentioning

confidence: 99%

Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT‐1

Matsuzawa

Tokugawa

Hamaoki

et al. 1988

European Journal of Biochemistry

106

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“…The activity under standard assay conditions is given relative to the maximum value. (b) Cleavage sites of STABLE and other subtilases in the insulin B-chain[43][44][45][46].…”

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confidence: 99%

A hyperthermostable protease of the subtilisin family bound to the surface layer of the Archaeon Staphylothermus marinus

et al. 1996

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“…This is consistent with the action of Ak.1 protease and thermitase, which cleave substrates containing Leu and Ala at the P " site. These proteases also have a high specificity for Phe at the P " site [24]. Subtilisins also cleaves substrates containing hydrophobic residues, such as Phe, at the P " site and small neutral residues, such as Val or Pro, at the P # site [25].…”

Section: Comparison Of Ak1 Protease With Other Subtilisinsmentioning

confidence: 99%

Purification and characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the substrate-binding cleft

Toogood

Smith

Baker

et al. 2000

Biochemical Journal

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Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517. It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P(1) site, such as valine, alanine or phenylalanine. The K(m) and k(cat) of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specificity changes with temperature. The protease is markedly stabilized by Ca(2+) ions. At 70 degrees C, a 10-fold increase in Ca(2+) concentration increases the half-life by three orders of magnitude. Ak.1 protease is stabilized by Ca(2+) to a greater extent than is thermitase. This may be due, in part, to the presence of an extra Ca(2+)-binding site in Ak.1 protease. Other metal ions, such as Sr(2+), increase the thermostability of the enzyme, but to a significantly lower degree than does Ca(2+). The structure of the protease showed the presence of a disulphide bond located within the active-site cleft. This bond influences both enzyme activity and thermostability. The disulphide bond appears to have a dual role: maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease. The protease was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures. However, it was found that this protease has a limited substrate specificity, so this application was not explored further.

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Substrate specificity of thermitase, a thermostable serine proteinase fromThermoactinomyces vulgaris

Cited by 18 publications

References 18 publications

Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT‐1

Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT‐1

A hyperthermostable protease of the subtilisin family bound to the surface layer of the Archaeon Staphylothermus marinus

Purification and characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the substrate-binding cleft

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