Enhanced activity and stability in the presence of organic solvents by increased active site polarity and stabilization of a surface loop in a metalloprotease.
Salinivibrio zinc-metalloprotease (SVP) is an enzyme which was isolated from Salinivibrio proteolyticus, a moderately halophilic species from a hypersaline lake in Iran. A195E and G203D mutants were constructed to increase polarity near the active site in order to preserve the hydration layer against organic solvents [dimethylformamide (DMF), methanol, isopropanol and n-propanol]. A268P was constructed to stabilize a surface loop far from the active site and A195E/A268P was constructed to investigate the combined effects of these two mutations. Results showed that relative C(50) values of A195E increased to approximately 26 and 11% in DMF and methanol whereas an increase of approximately 32 and 41% was observed in the presence of isopropanol and n-propanol. The irreversible thermoinactivation rate (k(i)) for A195E was estimated to be 60 and 130 (x10(-3) min(-1)) in the presence of DMF and n-propanol, respectively, while k(i) for SVP was 90 and 190 (x10(-3) min(-1)). G203D exhibited similar k(i) as A195E in the presence of methanol and isopropanol, but the calculated k(i) in the presence of DMF and n-propanol was 70 and 160 (x10(-3) min(-1)), respectively. A268P and A268P/A195E variants marginally increased the thermoresistance of the enzyme in this condition.
Badoei-Dalfard
Arastoo
A
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Khajeh
Khosro
K
Asghari
S Mohsen
SM
Ranjbar
Bijan
B
Karbalaei-Heidari
Hamid Reza
HR
D020134:Catalytic Domain; D004795:Enzyme Stability / Q000187:drug effects; D006358:Hot Temperature; D045726:Metalloproteases / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012997:Solvents / Q000494:pharmacology; D014737:Vibrionaceae / Q000201:enzymology
D012997:Solvents; D045726:Metalloproteases