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.
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ABSTRACT: The ciliated protozoon Euplotes focardii, originally isolated from the coastal seawaters of Terra Nova Bay in Antarctica, shows a strictly psychrophilic phenotype, including optimal survival and multiplication rates at 4-5 °C. This characteristic makes E. focardii an ideal model species for identifying the molecular bases of cold adaptation in psychrophilic organisms, as well as a suitable source of novel cold-active enzymes for industrial applications. In the current study, we characterized the patatin-like phospholipase from E. focardii (EfPLP), and its enzymatic activity was compared to that of the homologous protein from the mesophilic congeneric species Euplotes crassus (EcPLP). Both EfPLP and EcPLP have consensus motifs conserved in other patatin-like phospholipases. By analyzing both esterase and phospholipase A2 activity, we determined the thermostability and the optimal pH, temperature dependence and substrates of these enzymes. We demonstrated that EfPLP shows the characteristics of a psychrophilic phospholipase. Furthermore, we analyzed the enzymatic activity of three engineered versions of the EfPLP, in which unique residues of EfPLP, Gly80, Ala201 and Val204, were substituted through site-directed mutagenesis with residues found in the E. crassus homolog (Glu, Pro and Ile, respectively). Additionally, three corresponding mutants of EcPLP were also generated and characterized. These analyses showed that the substitution of amino acids with rigid and bulky charged/hydrophobic side chain in the psychrophilic EfPLP confers enzymatic properties similar to those of the mesophilic patatin-like phospholipase, and vice versa. This is the first report on the isolation and characterization of a cold-adapted patatin-like phospholipase from eukaryotes. The results reported in this paper support the idea that enzyme thermal-adaptation is based mainly on some amino acid residues that influence the structural flexibility of polypeptides and that EfPLP is an attractive biocatalyst for industrial processes at low temperatures.Biochimie 06/2013; · 3.14 Impact Factor
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ABSTRACT: An extracellular alkaline elastase was produced from Pseudomonas aeruginosa CTM50182. It was chromatographically purified using HPLC and Mono Q Sepharose column. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme (called AMPP) was a monomer with a molecular mass of 33015.18Da. The N-terminal 29 amino acid sequence of AMPP showed high homology with those of Pseudomonas elastases. It showed optimal activity at pH 12 and 80°C and was stable at a pH range of 9 to 12 after 120h of incubation. Its thermoactivity and thermostability were upgraded in the presence of 5mM Co(2+). Its half-life times at 70 and 80°C were 16 and 10h, respectively. It was completely inhibited by ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 1,10-phenanthroline, suggesting that it belongs to the metalloprotease family. AMPP also exhibited high catalytic efficiency, organic solvent-tolerance, and hydrolysis. The lasB gene encoding AMPP was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rAMPP) were similar to those of native AMPP. This organic solvent-stable protease could be considered a potential candidate for application as a biocatalyst in the synthesis of enzymatic peptides.International journal of biological macromolecules 05/2013; · 2.37 Impact Factor
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ABSTRACT: Keratinases are proteolytic enzymes capable of degrading insoluble keratins. The importance of these enzymes is being increasingly recognized in fields as diverse as animal feed production, textile processing, detergent formulation, leather manufacture, and medicine. To enhance the thermostability of Bacillus licheniformis BBE11-1 keratinase, the PoPMuSiC algorithm was applied to predict the folding free energy change (ΔΔG) of amino acid substitutions. Use of the algorithm in combination with molecular modification of homologous subtilisin allowed the introduction of four amino acid substitutions (N122Y, N217S, A193P, N160C) into the enzyme by site-directed mutagenesis, and the mutant genes were expressed in Bacillus subtilis WB600. The quadruple mutant displayed synergistic or additive effects with an 8.6-fold increase in the t 1/2 value at 60 °C. The N122Y substitution also led to an approximately 5.6-fold increase in catalytic efficiency compared to that of the wild-type keratinase. These results provide further insight into the thermostability of keratinase and suggest further potential industrial applications.Journal of Industrial Microbiology 04/2013; · 1.80 Impact Factor