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Biotechnology and Applied Biochemistry (2001) 34, (81–84) (Printed in Great Britain)

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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

Jae Woo Jang*¹, Jung Ho Ko*¹, Eun Kyung Kim*, Won Hee Jang†, Joo Hyun Kang* and Ook Joon Yoo*²

*BioMedical Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea, and †The Paik-Inje Memorial Institute for Biomedical Science, Inje University, 633-165 Kaekum-dong, Pusanjin-gu, Pusan 614-735, Korea

Key words: autoproteolytic degradation, catalytic efficiency, thermostability.

¹These authors contributed equally to the study.

²To whom correspondence should be addressed (e-mail ojyoo@sorak.kaist.ac.kr).

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.

Received 10 May 2001; accepted 3 July 2001

Portland Press Ltd © 2001