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A general and efficient strategy for generating the stable enzymes

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ABSTRACT

The local flexibility of an enzyme’s active center plays pivotal roles in catalysis, however, little is known about how the flexibility of these flexible residues affects stability. In this study, we proposed an active center stabilization (ACS) strategy to improve the kinetic thermostability of Candida rugosa lipase1. Based on the B-factor ranking at the region ~10 Å within the catalytic Ser209, 18 residues were selected for site-saturation mutagenesis. Based on three-tier high-throughput screening and ordered recombination mutagenesis, the mutant VarB3 (F344I/F434Y/F133Y/F121Y) was shown to be the most stable, with a 40-fold longer in half-life at 60 °C and a 12.7 °C higher Tm value than that of the wild type, without a decrease in catalytic activity. Further analysis of enzymes with different structural complexities revealed that focusing mutations on the flexible residues within around 10 Å of the catalytic residue might increase the success rate for enzyme stabilization. In summary, this study identifies a panel of flexible residues within the active center that affect enzyme stability. This finding not only provides clues regarding the molecular evolution of enzyme stability but also indicates that ACS is a general and efficient strategy for exploring the functional robustness of enzymes for industrial applications.

No MeSH data available.


Substrate spectra analysis of WT and its mutants towards a variety of acyl chain-length pNP-esters.The hydrolysis activities of enzymes toward pNP-ester were determined at 40 °C with a 50 mM Tris-HCl, pH 8.0 buffer containing 0.1% (w/v) gum arabic and 0.2% (w/v) sodium deoxycholate.
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f5: Substrate spectra analysis of WT and its mutants towards a variety of acyl chain-length pNP-esters.The hydrolysis activities of enzymes toward pNP-ester were determined at 40 °C with a 50 mM Tris-HCl, pH 8.0 buffer containing 0.1% (w/v) gum arabic and 0.2% (w/v) sodium deoxycholate.

Mentions: The substrate spectra for various acyl chain lengths were also measured for all of the mutants at 40 °C, and the optimum substrate (pNP-C8) was the same as the parent (Fig. 5), suggesting that mutations in the flexible residues do not lead to changes in the catalytic spectra. It is possible that the replacement of the phenylalanine by neutral or aromatic residues did not change the conformation of the binding pocket and thus retained the substrate preference.


A general and efficient strategy for generating the stable enzymes
Substrate spectra analysis of WT and its mutants towards a variety of acyl chain-length pNP-esters.The hydrolysis activities of enzymes toward pNP-ester were determined at 40 °C with a 50 mM Tris-HCl, pH 8.0 buffer containing 0.1% (w/v) gum arabic and 0.2% (w/v) sodium deoxycholate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036031&req=5

f5: Substrate spectra analysis of WT and its mutants towards a variety of acyl chain-length pNP-esters.The hydrolysis activities of enzymes toward pNP-ester were determined at 40 °C with a 50 mM Tris-HCl, pH 8.0 buffer containing 0.1% (w/v) gum arabic and 0.2% (w/v) sodium deoxycholate.
Mentions: The substrate spectra for various acyl chain lengths were also measured for all of the mutants at 40 °C, and the optimum substrate (pNP-C8) was the same as the parent (Fig. 5), suggesting that mutations in the flexible residues do not lead to changes in the catalytic spectra. It is possible that the replacement of the phenylalanine by neutral or aromatic residues did not change the conformation of the binding pocket and thus retained the substrate preference.

View Article: PubMed Central - PubMed

ABSTRACT

The local flexibility of an enzyme’s active center plays pivotal roles in catalysis, however, little is known about how the flexibility of these flexible residues affects stability. In this study, we proposed an active center stabilization (ACS) strategy to improve the kinetic thermostability of Candida rugosa lipase1. Based on the B-factor ranking at the region ~10 Å within the catalytic Ser209, 18 residues were selected for site-saturation mutagenesis. Based on three-tier high-throughput screening and ordered recombination mutagenesis, the mutant VarB3 (F344I/F434Y/F133Y/F121Y) was shown to be the most stable, with a 40-fold longer in half-life at 60 °C and a 12.7 °C higher Tm value than that of the wild type, without a decrease in catalytic activity. Further analysis of enzymes with different structural complexities revealed that focusing mutations on the flexible residues within around 10 Å of the catalytic residue might increase the success rate for enzyme stabilization. In summary, this study identifies a panel of flexible residues within the active center that affect enzyme stability. This finding not only provides clues regarding the molecular evolution of enzyme stability but also indicates that ACS is a general and efficient strategy for exploring the functional robustness of enzymes for industrial applications.

No MeSH data available.