Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions.
Rational shift of the optimum pH toward alkalinity and enhancement of thermostability were investigated by using a thermostable extremely alkaline protease (optimum pH, 12 to 13) from the alkaliphilic and thermophilic Bacillus sp. strain B18'. The protease gene (aprM) was cloned, and the sequence analysis revealed an open reading frame of 361 amino acids that was composed of a putative signal sequence (24 amino acids), a prosequence (69 amino acids), and a mature enzyme (268 amino acids) (molecular weight, 27,664). The amino acid sequence of this protease was compared with those of other serine proteases. A direct correlation of higher optimum pH with an increase in the number of arginine residues was observed. An even more thermostable mutant enzyme was created by introducing a point mutation. When the position of the beta-turn, Thr-203, was replaced by Pro, the residual activity of this mutant enzyme at 80 degrees C for 30 min was higher than that of the wild-type enzyme (50% versus 10%). The specific activity of this mutant enzyme at 70 degrees C was 105% of that of the wild-type enzyme under nondenaturation condition. These data suggest that the higher content of Arg residues favors the alkalinity of the serine protease and that introduction of a Pro residue into the beta-turn structure stabilizes the enzyme.
Masui
A
A
Osaka Prefectural Industrial Technology Research Institute, Japan.
Fujiwara
N
N
Imanaka
T
T
D000595:Amino Acid Sequence; D001120:Arginine / Q000032:analysis; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D001120:Arginine; C090777:AprM protein, Bacillus; D013381:Subtilisins