Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis
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.
[Show abstract] [Hide abstract] ABSTRACT: The current paper reports on the purification of an extracellular thermostable keratinase (KERCA) produced from C. algeriensis strain TH7C1T, a thermophilic, anaerobic bacterium isolated from a hydrothermal hot spring in Algeria. The maximum keratinase activity recorded after 24-h of incubation at 50 °C was 21000 U/ml. The enzyme was purified by ammonium sulfate precipitation-dialysis and heat treatment (2 h at 50 °C) followed by UNO Q-6 FPLC anion exchange chromatography, and submitted to biochemical characterization assays. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 33246.10 Da. The sequence of the 23 N-terminal residues of KERCA showed high homology with those of bacterial keratinases. Optimal activity was achieved at pH 7 and 50 °C. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine keratinase family. KERCA displayed higher levels of hydrolysis and catalytic efficiency than keratinase KERQ7 from Bacillus tequilensis strain Q7. These properties make KERCA a potential promising and eco-friendly alternative to the conventional chemicals used for the dehairing of goat, sheep, and bovine hides in the leather processing industry.0Comments 0Citations
- "One keratin azure unit was defined as the amount of enzyme causing an increase of 0.01 in absorbance at 595 nm in one min under the experimental conditions described. Keratinolytic activity was also measured using the Folin-Ciocalteu method and as previously described elsewhere  with keratin as a substrate. One keratin unit was defined as the amount of enzyme that hydrolyzed the substrate and that produced 1 µg of amino acid equivalent to tyrosine per min at 50 °C and pH 7 in buffer A. "
[Show abstract] [Hide abstract] ABSTRACT: Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang, a Korean fermented soyfood. A gene, aprE176, encoding the major fibrinolytic enzyme was cloned from B. subtilis HK176 and overexpressed in E. coli BL21(DE3) using plasmid pET26b(+). The specific activity of purified AprE176 was 216.8 +/- 5.4 plasmin unit/mg protein and the optimum pH and temperature were pH 8.0 and 40 degrees C, respectively. Error-prone PCR was performed for aprE176, and the PCR products were introduced into E. coli BL21(DE3) after ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity of M179 was 2.2-fold higher than that of the wild-type enzyme, but the catalytic efficiency (k(cat)/K-m) of M179 was not different from the wild-type enzyme owing to reduced substrate affinity. Interestingly, M179 showed increased thermostability. M179 retained 36% of activity after 5 h at 45 degrees C, whereas AprE176 retained only 11%. Molecular modeling analysis suggested that the 176th residue of M179, threonine, was located near the cation-binding site compared with the wild type. This probably caused tight binding of M179 with Ca2+, which increased the thermostability of M179.0Comments 0Citations
- "Some intramolecular interactions among the aromatic 122 th residue, the active site (Asp 32 , His 63 , Ser 220 ), and substrate influenced to increase the catalytic efficiency , whereas other substitutions (N217S, A193P, and N160C), which were located far from the active site, did not significantly change the K m or k cat . For a serine alkaline protease from B. pumilus CBS, the catalytic efficiency was increased 42-fold by mutations (L31I/T33S/N99Y) . The substitutions occurred at residues surrounding the catalytic residue, Asp 32 , in serine alkaline proteases. "
- "However, structural analysis of high-alkaline proteases is still scantly reported. Determination of enzyme 3D structures is also of great importance to get the insights of biophysical and structural forces, which affect the enzyme properties and its function under changing environments [152,153]. "