Enhancement of thermostability and catalytic efficiency of AprP, an alkaline protease from Pseudomonas sp., by the introduction of a disulfide bond.
A site-directed mutagenesis in AprP, an alkaline protease isolated from Pseudomonas sp. KFCC 10818 was carried out in order to obtain increased thermostability. Sites for cysteine substitutions to form disulfide bond within AprP were chosen by comparing the sequences with aqualysin I, an alkaline thermostable serine protease whose disulfide bonds seems to be important for its thermostability. Gly199 and Phe236 residues were each replaced with cysteine by site-directed mutagenesis. The G199C/F236C mutant enzyme appeared to form a disulfide bond spontaneously during its expression. It also showed improved kinetic parameters for the hydrolysis of a synthetic peptide substrate at pH 8.5 and 10.5 compared to those of the wild-type enzyme. The half-life of the G199C/F236C mutant was found to be 2 to 4.8 times longer than that of wild-type under various experimental conditions, except when tested under reducing condition, where no significant differences in the half-life of the two types were observed. Therefore, it is concluded that the introduction of the disulfide bond enhanced the thermostability and the catalytic efficiency of the enzyme AprP.
Ko
J H
JH
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusong-gu, Taejon, Korea.
Jang
W H
WH
Kim
E K
EK
Lee
H B
HB
Park
K D
KD
Chung
J H
JH
Yoo
O J
OJ
D000469:Alkaline Phosphatase; D001426:Bacterial Proteins; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D006207:Half-Life; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D004220:Disulfides; D000469:Alkaline Phosphatase; C099349:AprP protease, Pseudomonas; D012697:Serine Endopeptidases; D002118:Calcium