A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

Burlini, Nedda; Magnani, Paola; Villa, Andrea; Macchi, Fabio; Tortora, Paolo; Guerritore, A

doi:10.1016/0167-4838(92)90406-4

Cited by 36 publications

(21 citation statements)

References 38 publications

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“…In contrast, the activity of the same enzyme from A. radiobacter is completely eliminated after 10 min at 75°C (41). In addition to showing high thermostability, the purified arylesterase also displays high stability against several protein-denaturing compounds, like other enzymes from thermophilic archaea (12,13,16,21), although it is not feasible to compare it with other arylesterases from the same thermophilic archaea due to a lack of reports on the other arylesterases. At 70°C the enzyme can withstand, to a great extent, 8 M urea, 5% Tween 20, 5% Lubrol, 5% CHAPS, 1% SDS, 90% of different water-miscible organic solvents, and 2 M NaCl ( Table 3).…”

Section: Discussionmentioning

confidence: 98%

“…At 70°C the enzyme can withstand, to a great extent, 8 M urea, 5% Tween 20, 5% Lubrol, 5% CHAPS, 1% SDS, 90% of different water-miscible organic solvents, and 2 M NaCl ( Table 3). The high stability of the arylesterase against urea, some detergents, and organic solvents suggests that strong hydrophobic interactions make up the stable core of the enzyme and also that the enzyme may have a high surface hydrophobicity (12,13,16). It is also speculated that the proximity of hydrophobic substrates to this arylesterase may be facilitated by the addition of detergents.…”

Section: Discussionmentioning

confidence: 99%

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A Novel Thermostable Arylesterase from the ArchaeonSulfolobus solfataricusP1: Purification, Characterization, and Expression

2008

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A novel thermostable arylesterase, a 35-kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 94°C and 7.0, respectively. The enzyme displayed remarkable thermostability: it retained 52% of its activity after 50 h of incubation at 90°C. In addition, the purified enzyme showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity besides showing an arylesterase activity toward aromatic esters: it exhibits not only carboxylesterase activity toward tributyrin and p-nitrophenyl esters containing unsubstituted fatty acids from butyrate (C 4 ) to palmitate (C 16 ), but also paraoxonase activity toward organophosphates such as p-nitrophenylphosphate, paraoxon, and methylparaoxon. The k cat /K m ratios of the enzyme for phenyl acetate and paraoxon, the two most preferable substrates among all tested, were 30.6 and 119.4 s ؊1 ⅐ M ؊1 , respectively. The arylesterase gene consists of 918 bp corresponding to 306 amino acid residues. The deduced amino acid sequence shares 34% identity with that of arylesterase from Acinetobacter sp. strain ADP1. Furthermore, we successfully expressed active recombinant S. solfataricus arylesterase in Escherichia coli. Together, our results show that the enzyme is a serine esterase belonging to the A-esterases and contains a catalytic triad composed of Ser156, Asp251, and His281 in the active site.Sulfolobus solfataricus P1 (DSM1616), an extreme thermoacidophilic archaeon, inhabits sulfur-rich volcanic hot springs in a high-temperature and low-pH environment (10). Enzymes from thermoacidophilic archaea have attracted considerable attention among researchers because of the significance of their evolutionary phylogeny, the structural and functional stability of their enzymes despite their extreme environments, and the broad applications in biotechnology and industry available due to their special properties (23,44).Esterases (ester hydrolases) (EC 3.1

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

A Novel Thermostable Arylesterase from the ArchaeonSulfolobus solfataricusP1: Purification, Characterization, and Expression

2008

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“…To date, only a few other proteases from thermophilic (6,14,18,24) and hyperthermophilic (8,22,29,33) archaea have been purified and characterized, and the genes for a putative prolyl oligopeptidase (37) and a putative subtilisin-like protease (50) have been sequenced. To further examine the significance of the intracellular protease identified by Blumentals et al (4) in P. furiosus and compare it with other multisubunit intracellular proteases such as La (51), ClpP (27), the archaeal proteasome (55,56), and the eukaryotic proteasome (16,32), efforts to isolate and clone the gene encoding the enzyme were undertaken.…”

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confidence: 99%

Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus

et al. 1996

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A previously identified intracellular proteolytic activity in the hyperthermophilic archaeon Pyrococcus furiosus (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1992Microbiol. 56: -1998Microbiol. 56: , 1990) was found to be a homomultimer consisting of 18.8-kDa subunits. Dissociation of this native P. furiosus protease I (PfpI) into a single subunit was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but only after trichloroacetic acid precipitation; heating to 95؇C in the presence of 2% SDS and 80 mM dithiothreitol did not dissociate the protein. The gene (pfpI) coding for this protease was located in genomic digests by Southern blotting with probes derived from the N-terminal amino acid sequence. pfpI was cloned, sequenced, and expressed in active form in Escherichia coli as a fusion protein with a histidine tag. The recombinant protease from E. coli showed maximum proteolytic activity at 95؇C, and its half-life was 19 min at this temperature. This level of stability was significantly below that previously reported for the enzyme purified by electroelution of a 66-kDa band from SDS-PAGE after extended incubation of cell extracts at 98؇C in 1% SDS (>30 h). The pfpI gene codes for a polypeptide of 166 amino acid residues lacking any conserved protease motifs; no protease activity was detected for the 18.8-kDa PfpI subunit (native or recombinant) by substrate gel assay. Although an immunological relationship of this protease to the eukaryotic proteasome has been seen previously, searches of the available databases identified only two similar amino acid sequences: an open reading frame of unknown function from Staphylococcus aureus NCTC 8325 (171 amino acid residues, 18.6 kDa, 41% identity) and an open reading frame also of unknown function in E. coli (172 amino acid residues, 18.8 kDa, 47% identity). Primer extension experiments with P. furiosus total RNA defined the 5 end of the transcript. There are only 10 nucleotides upstream of the start of translation; therefore, it is unlikely that there are any pre-or pro-regions associated with PfpI which could have been used for targeting or assembly of this protease. Although PfpI activity appears to be the dominant proteolytic activity in P. furiosus cell extracts, the physiological function of PfpI is unclear.Hyperthermophilic microorganisms (those that grow above 90ЊC with an optimum temperature of at least 80ЊC), which are mostly archaea (53), have been investigated for clues to evolutionary processes as well as to uncover biological strategies underlying life at elevated temperatures. Most of the focus has been on characterization of new isolates (3, 45) and on the mechanisms responsible for heightened levels of biomolecular thermostability (1). Although just as important, the physiology of this novel group of organisms has been less studied (20). This is not surprising since hyperthermophiles are generally difficult to culture, in addition to the fact that no genetic systems are available for directed ...

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“…Besides proteasomes [1], several non-methanogenic Archaea are also known to produce extracellular proteases belonging to di¡erent classes, including (i) serine proteases produced by a number of halobacteria [2] and Sulfolobus solfataricus [3]; (ii) subtilisin-like proteases such as the one bound to the surface layer of Staphylothermus marinus [4]; (iii) thiol proteases, e.g. from Pyrococcus kodakaraensis KOD1 [5] ; (iv) a maltose-regulated prolyl oligopeptidase from Pyrococcus furiosus [6]; (v) metalloproteases such as S. solfataricus zinc carboxypeptidase [7] and P. furiosus cobalt-activated carboxypeptidase [8].…”

Section: Introductionmentioning

confidence: 99%

Pseudomurein endoisopeptidases PeiW and PeiP, two moderately related members of a novel family of proteases produced in Methanothermobacter strains

Luo

2002

FEMS Microbiology Letters

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Sequence comparison of pseudomurein endoisopeptidases PeiW encoded by the defective prophage 8M100 of Methanothermobacter wolfeii, and PeiP encoded by phage 8M2 of Methanothermobacter marburgensis, revealed that the two enzymes share only limited similarity. Their amino acid sequences comprise an N-terminal domain characterized by the presence of direct repeats and a C-terminal domain with a catalytic triad C-H-D as in thiol proteases and animal transglutaminases. Both PeiW and PeiP catalyze the in vitro lysis of M. marburgensis cells under reducing conditions and exhibit characteristics of metal-activated peptidases. Optimal temperature and pH were determined to be 63³C and 6.4 for His-tagged PeiP and 71³C and 6.4 for His-tagged PeiW, respectively. Database search results suggest that PeiW and PeiP are the first two experimentally identified members of a novel family of proteases in a superfamily of archaeal, bacterial, and eukaryotic protein homologs of animal transglutaminases. ß

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A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

Cited by 36 publications

References 38 publications

A Novel Thermostable Arylesterase from the ArchaeonSulfolobus solfataricusP1: Purification, Characterization, and Expression

A Novel Thermostable Arylesterase from the ArchaeonSulfolobus solfataricusP1: Purification, Characterization, and Expression

Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus

Pseudomurein endoisopeptidases PeiW and PeiP, two moderately related members of a novel family of proteases produced in Methanothermobacter strains

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