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Issue 31, 2012
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Redesign of enzyme for improving catalytic activity and enantioselectivity toward poor substrates: manipulation of the transition state

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Abstract

Secondary alcohols having bulky substituents on both sides of the hydroxy group are inherently poor substrates for most lipases. In view of this weakness, we redesigned a Burkholderia cepacia lipase to create a variant with improved enzymatic characteristics. The I287F/I290A double mutant showed a high conversion and a high E value (>200) for a poor substrate for which the wild-type enzyme showed a low conversion and a low E value (5). This enhancement of catalytic activity and enantioselectivity of the variant resulted from the cooperative action of two mutations: Phe287 contributed to both enhancement of the (R)-enantiomer reactivity and suppression of the (S)-enantiomer reactivity, while Ala290 created a space to facilitate the acylation of the (R)-enantiomer. The kinetic constants indicated that the mutations effectively altered the transition state. Substrate mapping analysis strongly suggested that the CH/π interaction partly enhanced the (R)-enantiomer reactivity, the estimated energy of the CH/π interaction being −0.4 kcal mol−1. The substrate scope of the I287F/I290A double mutant was broad. This biocatalyst was useful for the dynamic kinetic resolution of a variety of bulky secondary alcohols for which the wild-type enzyme shows little or no activity.

Graphical abstract: Redesign of enzyme for improving catalytic activity and enantioselectivity toward poor substrates: manipulation of the transition state

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Publication details

The article was received on 24 Mar 2012, accepted on 30 May 2012 and first published on 18 Jun 2012


Article type: Paper
DOI: 10.1039/C2OB25614B
Citation: Org. Biomol. Chem., 2012,10, 6299-6308
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    Redesign of enzyme for improving catalytic activity and enantioselectivity toward poor substrates: manipulation of the transition state

    T. Ema, Y. Nakano, D. Yoshida, S. Kamata and T. Sakai, Org. Biomol. Chem., 2012, 10, 6299
    DOI: 10.1039/C2OB25614B

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