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Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis

Laboratoire d'Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax (CBS), Route de Sidi Mansour Km 6, Sfax, Tunisia.
Biochimie (Impact Factor: 3.12). 04/2010; 92(4):360-9. DOI: 10.1016/j.biochi.2010.01.008
Source: PubMed

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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    • ". 6 ) . The most stable variant ( MT2 ) was generated by the combination of all of the latter amino acid substitutions ( S39E , N74D , D87E , and N253D ) . Directed evolution to improve thermostability has been previously reported for several psychro - and mesophilic subtilisins ( Almog et al . , 2002 ; Erwin et al . , 1990 ; Jang et al . , 2001 ; Jaouadi et al . , 2010 ; Takagi et al . , 1990 ; Zhao and Arnold , 1999 ) . Most substitutions comprised again sub - stitutions to charged amino acids that are located on the surface of subtilisins ; only a few occurred in secondary structure motifs . Position 76 in subtilisin E , structural equivalent of position 74 in BgAP , is described to increase stabili"
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    ABSTRACT: Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (K(cat) ) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5 fold increased specific activity (15°C) and over 100 times prolonged half-life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity-altering amino acid substitutions were from non-charged to non-charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 03/2013; 110(3). DOI:10.1002/bit.24766 · 4.16 Impact Factor
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    • "For subtilisins, the pH activity dependence on the protein surface charge was demonstrated by amino acid substitutions already in 1987 (Russell and Fersht 1987). Improvements in thermostability (up to 3–3.7-fold improved half-life times; Jaouadi et al. 2010) were achieved by single amino acid substitutions and by optimization of charge–charge interactions on the surface of serine proteases (Jaouadi et al. 2010; Loladze et al. 1999). "
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    Applied Microbiology and Biotechnology 11/2012; 97(15). DOI:10.1007/s00253-012-4560-8 · 3.81 Impact Factor
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    Biochimie 04/2011; 93(4):783-91. DOI:10.1016/j.biochi.2011.01.011 · 3.12 Impact Factor
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Nushin Aghajari