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Enhanced thermal stability of an alkaline protease, AprP, isolated from a Pseudomonas sp. by mutation at an autoproteolysis site, Ser-331

Authors

  • Jae Woo Jang,

    1. BioMedical Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea
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    • These authors contributed equally to the study.

  • Jung Ho Ko,

    1. BioMedical Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea
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    • These authors contributed equally to the study.

  • Eun Kyung Kim,

    1. BioMedical Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea
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  • Won Hee Jang,

    1. The Paik-Inje Memorial Institute for Biomedical Science, Inje University, 633-165 Kaekum-dong, Pusanjin-gu, Pusan 614-735, Korea
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  • Joo Hyun Kang,

    1. BioMedical Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea
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  • Ook Joon Yoo

    Corresponding author
    1. BioMedical Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea
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BioMedical Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea (e-mail ojyoo@sorak.kaist.ac.kr).

Abstract

The thermal stability of the alkaline protease extracellular subtilisin-type serine protease (AprP) from Pseudomonas sp. KFCC 10818 was improved by altering an amino acid residue at an autoproteolytic cleavage site. N-terminal sequence analysis of the autoproteolytic products of the protein revealed the presence of two cleavage sites, Ser-307 and Ser-331. To increase the thermal stability of the enzyme, serine residues of these sites were replaced with aspartate. The S331D mutant enzyme was successfully purified and characterized whereas the S307D mutant was not. The half-lives of the S331D mutant at 55 °C and 60 °C were 1.5 and 2.4 times longer than that of the wild-type enzyme, respectively. In addition, the catalytic efficiency was also enhanced.

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