Proteases of enhanced stability: characterization of a thermostable variant of subtilisin.


Abstract

A 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 one-fourth the rate of the wild-type enzyme when incubated at elevated temperatures. Moreover, the mid-point in the thermally induced transition from the folded to unfolded state was found to be 2.4-3.9 degrees C higher for 7150 as determined by differential scanning calorimetry under a variety of conditions. The refined, 1.8-A 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. Study holds ProTherm entries: 2998, 2999, 3000 Extra Details: thermal stability; protein engineering; mutagenesis; plate assay;,thermophilic enzymes

Submission Details

ID: 73cAcisp3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:20 p.m.

Version: 1

Publication Details
Bryan PN;Rollence ML;Pantoliano MW;Wood J;Finzel BC;Gilliland GL;Howard AJ;Poulos TL,Proteins (1986) Proteases of enhanced stability: characterization of a thermostable variant of subtilisin. PMID:3329733
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1A2Q 1998-01-08T00:00:00+0000 1.8 SUBTILISIN BPN' MUTANT 7186
1AK9 1997-05-30T00:00:00+0000 1.8 SUBTILISIN MUTANT 8321
1AQN 1997-07-31T00:00:00+0000 1.8 SUBTILISIN MUTANT 8324
1AU9 1997-09-12T00:00:00+0000 1.8 SUBTILISIN BPN' MUTANT 8324 IN CITRATE
1DUI 2000-01-17T00:00:00+0000 2.0 Subtilisin BPN' from Bacillus amyloliquefaciens, crystal growth mutant
1GNS 2001-10-06T00:00:00+0000 1.8 SUBTILISIN BPN'
1GNV 2001-10-10T00:00:00+0000 1.9 CALCIUM INDEPENDENT SUBTILISIN BPN' MUTANT
1LW6 2002-05-30T00:00:00+0000 1.5 Crystal Structure of the Complex of Subtilisin BPN' with Chymotrypsin Inhibitor 2 at 1.5 Angstrom Resolution
1S01 1989-08-21T00:00:00+0000 1.7 LARGE INCREASES IN GENERAL STABILITY FOR SUBTILISIN BPN(PRIME) THROUGH INCREMENTAL CHANGES IN THE FREE ENERGY OF UNFOLDING
1S02 1991-02-20T00:00:00+0000 1.9 EFFECTS OF ENGINEERED SALT BRIDGES ON THE STABILITY OF SUBTILISIN BPN'

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Subtilisin BPN' P00782 SUBT_BACAM