2017
DOI: 10.1038/srep41212
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Two strategies to engineer flexible loops for improved enzyme thermostability

Abstract: Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. In this study, two parallel strategies were applied to identify mutation candidates within the flexible loops of Escherichia coli transketolase (TK). The first was a "back to consensus mutations" approach, and the second was computational design based on ΔΔG cal… Show more

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Cited by 133 publications
(108 citation statements)
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“…Transitioning from harsh to mild conditions between process steps typically requires further energy or material inputs and brings significant cost implications . Purified enzymes are often active in only mild conditions, but variants can be sourced or engineered that are stable to extremes . However, even robust enzyme variants have no capacity to regenerate themselves, as they inevitably accumulate damage and denaturation over time.…”
Section: Introductionmentioning
confidence: 99%
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“…Transitioning from harsh to mild conditions between process steps typically requires further energy or material inputs and brings significant cost implications . Purified enzymes are often active in only mild conditions, but variants can be sourced or engineered that are stable to extremes . However, even robust enzyme variants have no capacity to regenerate themselves, as they inevitably accumulate damage and denaturation over time.…”
Section: Introductionmentioning
confidence: 99%
“…5 Purified enzymes are often active in only mild conditions, but variants can be sourced or engineered that are stable to extremes. 6 However, even robust enzyme variants have no capacity to regenerate themselves, as they inevitably accumulate damage and denaturation over time. One option to make enzymes both selfrenewing and tolerant to non-mild conditions is to deploy them while they are still sequestered within a host cell, socalled whole cell biocatalysis.…”
Section: Introductionmentioning
confidence: 99%
“…Finally, this work has provided useful insights that will guide the future engineering of TK and other similarly complex enzymes. Improvements might be obtained by focussing enzyme engineering within the cofactor-binding loops and neighbouring regions close to the protein surface, as we have shown previously to be effective for the improvement of TK thermostability (Yu et al, 2017). Table 3.…”
Section: Discussionmentioning
confidence: 99%
“…The synthetic potential of TK has already been significantly improved by successive rounds of directed evolution with smart libraries (; Affaticati et al, 2016;Yu et al 2017), to enhance activity towards polar Subrizi et al, 2016), and increasingly aliphatic Cazares et al, 2010: Strafford et al, 2012, and heterocyclic substrates (Cazares et al 2010;Galman et al, 2010), to enhance and reverse enantioselectivity . De novo pathways containing improved TK variants have also been designed for the synthesis of optically pure high-value amino-diols (Rios-Solis et al, 2011.…”
Section: Introductionmentioning
confidence: 99%
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