title: Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis.
abstract: The serine alkaline protease, SAPB, from Bacillus pumilus CBS is characterized by its high thermoactivity, pH stability and high catalytic efficiency (k(cat)/K(m)) as well as its excellent stability and compatibility with an alkaline environment under harsh washing conditions. Based on sequence alignments and homology-modeling studies, the present study identified five amino acids Leu31, Thr33, Asn99, Phe159 and Gly182 being putatively important for the enzymatic behaviour of SAPB. To corroborate the role of these residues, 12 mutants were constructed by site-directed mutagenesis and then purified and characterized. The findings demonstrate that the single mutants F159T, F159S and G182S and combined double substitutions were implicated in the decrease of the optimum pH and temperature to 8.0-9.0 and 50 degrees C, respectively, and that mutant F159T/S clearly affected substrate affinity and catalytic efficiency. With regards to the single L31I, T33S and N99Y and combined double and triple mutations, the N99Y mutation strongly improved the half-life times at 50 degrees C and 60 degrees C to 660 and 295 min from of 220 and 80 min for the wild-type enzyme, respectively. More interestingly, this mutation also shifted the optimum temperature from 65 degrees C to 75 degrees C and caused a prominent 31-fold increase in k(cat)/K(m) with N-succinyl-l-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF). The L31I and T33S mutants were observed to improve mainly the optimum pH from 11.0 to 11.5 and from 11.0 to 12.0, respectively. Kinetic studies of double and triple mutants showed that the cumulative effect of polar uncharged substitutions had a synergistic effect on the P1 position preference using synthetic peptide substrates, which confirms the implication of these amino acids in substrate recognition and catalytic efficiency.
authors.affiliation: Laboratoire d'Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax (CBS), Route de Sidi Mansour Km 6, Sfax, Tunisia.
authors.forename: Bassem
authors.initials: B
authors.lastname: Jaouadi
authors.forename: Nushin
authors.initials: N
authors.lastname: Aghajari
authors.forename: Richard
authors.initials: R
authors.lastname: Haser
authors.forename: Samir
authors.initials: S
authors.lastname: Bejar
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics*
chemical_list: D013381:Subtilisins