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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.


Correlations between the distances of the mutated residues to the catalytic residue serine, relative B-factors, and the thermostabilities  of the lipases.LipA (181 AA); CalB (317 AA); LIP1 (534 AA); the data analysis is based on their mutated results.
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f8: Correlations between the distances of the mutated residues to the catalytic residue serine, relative B-factors, and the thermostabilities of the lipases.LipA (181 AA); CalB (317 AA); LIP1 (534 AA); the data analysis is based on their mutated results.

Mentions: To propose a general and efficient rule as guidance for enzyme stabilization, three model lipases were performed analysis, namely Bacillus subtilis LipA, Candida antarctica CalB and Candida rugosa LIP1 (in ‘open’ and ‘closed’ states). These enzymes share the same canonical α/β fold core structure, but exhibit different structural complexities (Fig. 1). As a minimal α/β hydrolase, LipA is the first enzyme to improve the thermostability based on B-factor13 and contains only 181 amino acids. Due to that most of the residues in LipA are located within 10 Å of the catalytic residue Ser77 (Fig. 1A), its thermostability improvement engineering can be regarded as a special case in the application of ACS strategy. For simplicity and clarity, only the effects of all single mutations on enzyme thermostability were evaluated. The other selected sites were found to exert no change in stability (). As shown in Fig. 8, the correlations among the distances of the mutated residues to the catalytic serine, the relative B-factor (percentage with the highest B-factor within 10 Å of the catalytic residue) and the effect on the enzyme thermostability () were analyzed. The mutated flexible residues with a 60–100 relative B-factor and within 10 Å of the catalytic serine were most effective in improving kinetic stability () of enzymes. The mutations beyond ~14 Å and within ~6 Å did not improve stability.


A general and efficient strategy for generating the stable enzymes
Correlations between the distances of the mutated residues to the catalytic residue serine, relative B-factors, and the thermostabilities  of the lipases.LipA (181 AA); CalB (317 AA); LIP1 (534 AA); the data analysis is based on their mutated results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Correlations between the distances of the mutated residues to the catalytic residue serine, relative B-factors, and the thermostabilities of the lipases.LipA (181 AA); CalB (317 AA); LIP1 (534 AA); the data analysis is based on their mutated results.
Mentions: To propose a general and efficient rule as guidance for enzyme stabilization, three model lipases were performed analysis, namely Bacillus subtilis LipA, Candida antarctica CalB and Candida rugosa LIP1 (in ‘open’ and ‘closed’ states). These enzymes share the same canonical α/β fold core structure, but exhibit different structural complexities (Fig. 1). As a minimal α/β hydrolase, LipA is the first enzyme to improve the thermostability based on B-factor13 and contains only 181 amino acids. Due to that most of the residues in LipA are located within 10 Å of the catalytic residue Ser77 (Fig. 1A), its thermostability improvement engineering can be regarded as a special case in the application of ACS strategy. For simplicity and clarity, only the effects of all single mutations on enzyme thermostability were evaluated. The other selected sites were found to exert no change in stability (). As shown in Fig. 8, the correlations among the distances of the mutated residues to the catalytic serine, the relative B-factor (percentage with the highest B-factor within 10 Å of the catalytic residue) and the effect on the enzyme thermostability () were analyzed. The mutated flexible residues with a 60–100 relative B-factor and within 10 Å of the catalytic serine were most effective in improving kinetic stability () of enzymes. The mutations beyond ~14 Å and within ~6 Å did not improve stability.

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.