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Directed Mutagenesis of the Conserved Asparagine Residues of Bacillus Stearothermophilus Leucine Aminopeptidase II

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Summary

To understand the structure–function relationships of Bacillus stearothermophilus leucine aminopeptidase II (LAPII), each of the four conserved asparagine residues was replaced with leucine, aspartate, and lysine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes with an apparent molecular mass of approximately 44.5 kDa were purified to homogeneity by nickel-chelate chromatography. Substitution of Asn-245, Asn-335, and Asn-341 with Lys generated variants with a dramatic loss of LAP activity. Kinetic analysis of Asn-373 variants with p-leucine-nitroanilide as the substrate revealed an increase in k cat with no significant change in K m, leading to a more than 2-fold increase in the catalytic efficiency. Thermostability assays showed that replacement of Asn-335, Asn-341, and Asn-373 by aspartic acid markedly increased the half-life of the enzyme at 70 °C, indicating that the deamination of these residues may have a deleterious effect on LAPII.

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Authors and Affiliations

  1. Graduate Institute of Biotechnology, National Chiayi University, 60083, Chiayi, Taiwan

    Ruey-Shyang Chen & Shin-Ling Yang

  2. Department of Applied Chemistry, National Chiayi University, 300 University Road, 60083, Chiayi, Taiwan

    Yu-Wen Hua, Meng-Chun Chi & Long-Liu Lin

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  1. Ruey-Shyang Chen
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  2. Shin-Ling Yang
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  3. Yu-Wen Hua
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Correspondence to Long-Liu Lin.

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Chen, RS., Yang, SL., Hua, YW. et al. Directed Mutagenesis of the Conserved Asparagine Residues of Bacillus Stearothermophilus Leucine Aminopeptidase II. World J Microbiol Biotechnol 21, 1477–1482 (2005). https://doi.org/10.1007/s11274-005-7023-x

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