DOI: 10.1002/prot.340010406. Proteases of enhanced stability: Characteization of a thermostable variant of subtilisin

Proteases of enhanced stability: Characteization of a thermostable variant of subtilisin

10.1002/prot.340010406

Crossref journal-article

Wiley

Proteins: Structure, Function, and Bioinformatics (311)

Abstract

AbstractA Procedure has been developed for the isolation and identification of mutants in the bacterial serine protease subtilisin that exhibit enhanced thermal stability. The cloned subtilisin BPN'gene from Bacillus amyloliquefaciens was treated with bisulfite, a chemical mutagen that deaminates cytosine to uracil in single‐stranded DNA. Strains containing the cloned, mutagenized subtilisin gene which produced subtilisin with enhanced thermal stability were selected by a simple plate assay procedure which screens for esterase activity on nitrocellulose filters after preincubation at elevated temperatures. One thermostable subtilisin variant, designated 7150, has been fully characterized and found to differ from wild‐type subtilisin by a single substitution of Ser for Asn at position 218. The 7150 enzyme was found to undergo thermal inactivation at onefourth the rate of the wild‐type enzyme when incubated at elevated temperatures. Moreover, the midpoint in the thermally induced transition from the folded to unfolded state was found to be 2.4–3.9°C higher for 7150 as determined by differential scanning calorimetry under a variety of conditions. The refined, 1.8‐Å crystal structures of the wild‐type and 7150 subtilisin have been compared in detail, leading to the conclusion that slight improvements in hydrogen bond parameters in the vicinity of position 218 result in the enhanced thermal stability of 7150.

Authors 8

Philip N. Brayan (first)
Michele L. Rollence (additional)
Michael W. Pantoliano (additional)
James Wood (additional)
Barry C. Finzel (additional)
Gary L. Gilliland (additional)
Andrew J. Howard (additional)
Thomas L. Poulos (additional)

References 45 Referenced 125

10.1126/science.6356360
Pantoliano M. W. Ladner R. C. Bryan P. N. Rollence M. L. Wood J. F. andPoulos T. L.Protein engineering of subtilisin BPN: Enhanced stabilization through the enhanced stabilization through the introduction of two cysteines to form a disulfide bond.Biochemistry in press.
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10.1016/S0021-9258(19)84599-4
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10.1126/science.694508
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10.1073/pnas.83.6.1588
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Howard A. J. Gilliland G. L. Finzel B. C. Poulos T. L. Ohlendrof D. H. Salemme F. R. J.Appl. Cryst. submitted.1986.
{'key': 'e_1_2_1_30_2', 'first-page': '13.01', 'volume-title': 'Computing in Crystallography', 'author': 'Hendrickson W. A.', 'year': '1980'} / Computing in Crystallography by Hendrickson W. A. (1980)
10.1073/pnas.75.6.2574
Finzel B. C. Gilliland G. L. Howard A. J. Ohlendorf D. H. T. L. Poulos. Unpublished data.
{'key': 'e_1_2_1_33_2', 'volume-title': 'Proteins: Structure and Molecular Properties', 'author': 'Creighton T. E.', 'year': '1984'} / Proteins: Structure and Molecular Properties by Creighton T. E. (1984)
10.1128/jb.159.3.811-819.1984 / J. Bacteriol. / Gene for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open‐reading frame between the regions coding for signal sequence and mature protein by Vasantha N. (1984)
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Rollence M. L. Pantoliano M. W. Filpula D. Bryan P. N.Increasing thermostability in subtilisin BPN'.CRC Crit. Rev. Biotechnol. in press.

Dates

Type	When
Created	20 years, 3 months ago (May 28, 2005, 8:56 p.m.)
Deposited	1 year, 10 months ago (Oct. 21, 2023, 2:07 a.m.)
Indexed	3 days, 19 hours ago (Aug. 30, 2025, 12:23 p.m.)
Issued	39 years, 5 months ago (April 1, 1986)
Published	39 years, 5 months ago (April 1, 1986)
Published Online	21 years, 7 months ago (Feb. 3, 2004)
Published Print	39 years, 5 months ago (April 1, 1986)

Funders 0

None

BibTeX

@article{Brayan_1986, title={Proteases of enhanced stability: Characteization of a thermostable variant of subtilisin}, volume={1}, ISSN={1097-0134}, url={http://dx.doi.org/10.1002/prot.340010406}, DOI={10.1002/prot.340010406}, number={4}, journal={Proteins: Structure, Function, and Bioinformatics}, publisher={Wiley}, author={Brayan, Philip N. and Rollence, Michele L. and Pantoliano, Michael W. and Wood, James and Finzel, Barry C. and Gilliland, Gary L. and Howard, Andrew J. and Poulos, Thomas L.}, year={1986}, month=apr, pages={326–334} }

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