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.
". 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"
"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). "
[Show abstract][Hide abstract] ABSTRACT: In proteins, a posttranslational deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartic (Asp) and glutamic acid (Glu), respectively. This process changes the protein net charge affecting enzyme activity, pH optimum, and stability. Understanding the principles which affect these enzyme properties would be valuable for protein engineering in general. In this work, three criteria for selecting amino acid substitutions of the deamidation type in the Bacillus gibsonii alkaline protease (BgAP) are proposed and systematically studied in their influence on pH-dependent activity and thermal resistance. Out of 113 possible surface amino acids, 18 (11 Asn and 7 Gln) residues of BgAP were selected and evaluated based on three proposed criteria: (1) The Asn or Gln residues should not be conserved, (2) should be surface exposed, and (3) neighbored by glycine. "Deamidation" in five (N97, N253, Q37, Q200, and Q256) out of eight (N97, N154, N250, N253, Q37, Q107, Q200, and Q256) amino acids meeting all criteria resulted in increased proteolytic activity. In addition, pH activity profiles of the variants N253D and Q256E and the combined variant N253DQ256E were dramatically shifted towards higher activity at lower pH (range of 8.5-10). Variant N253DQ256E showed twice the specific activity of wild-type BgAP and its thermal resistance increased by 2.4 °C at pH 8.5. These property changes suggest that mimicking surface deamidation by substituting Gln by Glu and/or Asn by Asp might be a simple and fast protein reengineering approach for modulating enzyme properties such as activity, pH optimum, and thermal resistance.
"Although the sequences and structures of several members of the subtilase superfamily, such as M-protease (PDB code 1MPT), psychrophilic proteinase Vibrio (PDB code 1SH7), thermitase (PDB code 1THM), subtilisin BPN 0 (PDB code 1UPS) and Carlsberg (PDB code 2SEC) have been determined and applied to analyze the mechanism of the adaptation of thermophilic and psychrophilic condition [10,11,14e16], structural analysis of high-alkaline proteases is still relatively limited. Determining the 3D structures is of particular importance to provide insights into the structural principles that control the variety of enzyme properties and further conduct the molecular direct evolution  . "
[Show abstract][Hide abstract] ABSTRACT: High-alkaline proteases are of great importance because of their proteolytic activity and stability under high-alkaline condition. We have previously isolated a new protease (AprB) which has potential industrial applications based on its high-alkaline adaptation. However, the molecular and structural basis for alkaline adaptation of this enzyme has not been fully elucidated. In the present study, AprB gene was cloned and expressed in the Bacillus subtilis WB600. This gene codes for a protein of 375 amino acids comprised with a 28-residual signal peptide, a 78-residual pro-peptide, and a 269-residual mature protein. The deduced amino acid sequence has the highest homology of 63.2% with that of the high-alkaline proteases. Recombinant AprB was purified and determined to be monomeric with molecular mass of 26.755kDa. The NH(2)-terminal sequence of the purified AprB was A-Q-S-I-P-W-G-I-E-R. This enzyme exhibited high catalytic efficiencies (K(cat)/K(m)) towards natural, modified, and synthesis substrates with optimal activity at 60°C and pH 10. AprB was stable over a wide range of pH 5 to 11 and various surfactants, and could be activated by Mg(2+), Ca(2+) and Ba(2+). The structural properties of AprB, like a higher ratio of R/(R+K), a larger area of hydrophobic surface, increased number of ion pairs formed by Arg residue, and the exposure of Asp active residue on the surface, might be responsible for its alkaline adaptation. In contrast with members of subtilisin family, such as M-protease and subtilisin BPN', AprB harbored a high content of Glu and Asp residues, and a low content of Arg and Lys residues on the surface. Interestingly, these structural characters were similar with that of psychrophilic proteases, which suggested that these molecular factors were not restricted in the psychrophilic proteases, and therefore were not solely responsible for their cold-adaptation. Our results reveal a novel structural feature of AprB unique to subtilisin family and provide clues for its alkaline adaptation.
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