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Applied and Environmental Microbiology, March 2000, p. 890-894, Vol. 66, No. 3
Department of Biological Sciences, Korea
Advanced Institute of Science and Technology, 373-1, Kusong-dong, Yusong-gu, Taejon 305-701, Korea
Received 24 September 1999/Accepted 1 December 1999
The thermal stability and catalytic activity of phospholipase
A1 from Serratia sp. strain MK1 were improved
by evolutionary molecular engineering. Two thermostable mutants were
isolated after sequential rounds of error-prone PCR performed to
introduce random mutations and filter-based screening of the resultant
mutant library; we determined that these mutants had six (mutant TA3) and seven (mutant TA13) amino acid substitutions. Different types of
substitutions were found in the two mutants, and these substitutions resulted in an increase in nonploar residues (mutant TA3) or in differences between side chains for polar or charged residues (mutant
TA13). The wild-type and mutant enzymes were purified, and the effect
of temperature on the stability and catalytic activity of the enzymes
was investigated. The melting temperatures of the TA3 and TA13 enzymes
were increased by 7 and 11°C, respectively, compared with the melting
temperature of the wild-type enzyme. Thus, we found that evolutionary
molecular engineering was an effective and efficient approach for
increasing thermostability without compromising enzyme activity.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Simultaneous Enhancement of Thermostability and
Catalytic Activity of Phospholipase A1 by Evolutionary
Molecular Engineering
*
Corresponding author. Mailing address: Department of
Biological Sciences, Korea Advanced Institute of Science and
Technology, 373-1, Kusong-dong, Yusong-gu, Taejon 305-701, Korea.
Phone: 82-42-869-2613. Fax: 82-42-869-2610. E-mail:
jsrhee{at}sorak.kaist.ac.kr.
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