title: Subtilase from Beauveria sp.: conformational and functional investigation of unusual stability.
abstract: Retention of total activity of the subtilisin-like serine protease from Beauveria sp. MTCC 5184 (Bprot) in the vicinity of (1) 3 M GdnHCl for 12 h, (2) 50% methanol and dimethyl sulfoxide each for 24 h, and (3) proteolytic enzymes (trypsin, chymotrypsin, and proteinase K) for 48 h led to expect the enzyme to be a kinetically stable protein. Also, the structure of the protein was stable at pH 2.0. Biophysical characterization and conformational transitions were monitored using steady-state and time-resolved fluorescence, FTIR, and CD spectroscopy. Single tryptophan in the protein exists as two conformers, in hydrophobic and polar environment. The secondary structure of Bprot was stable in 3 M GdnHCl as seen in far-UV CD spectra. The active fraction of Bprot obtained from size-exclusion chromatography in the presence of GdnHCl (1.0-3.0 M) eluted at reduced retention time. The peak area of inactive or denatured protein with the same retention time as that of native protein increased with increasing concentration of denaturant (1.0-4.0 M GdnHCl). However, the kinetics of GdnHCl-induced unfolding as studied from intrinsic fluorescence revealed k unf of native protein to be 5.407 × 10(-5) s(-1) and a half-life of 3.56 h. The enzyme is thermodynamically stable in spite of being resistant to the denaturant, which could be due to the effect of GdnHCl imparting rigidity to the active fraction and simultaneously unfolding the partially unfolded protein that exists in equilibrium with the folded active protein. Thermal and pH denaturation of Bprot exhibited interesting structural transitions.
authors.affiliation: Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr Homi Bhabha Road, Pune, 411008, India.
authors.forename: Sayli A
authors.initials: SA
authors.lastname: Dalal
authors.forename: Snehal V
authors.initials: SV
authors.lastname: More
authors.forename: Shiv
authors.initials: S
authors.lastname: Shankar
authors.forename: R
authors.initials: R
authors.lastname: Seeta Laxman
authors.forename: Sushama M
authors.initials: SM
authors.lastname: Gaikwad
mesh_terms: D052982:Beauveria / Q000201:enzymology*; D004795:Enzyme Stability / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D059748:Proteolysis; D012997:Solvents / Q000494:pharmacology; D020860:Subtilisin / Q000737:chemistry* / Q000378:metabolism*; D013696:Temperature
chemical_list: D012997:Solvents; D020860:Subtilisin; D019791:Guanidine