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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.


The flow chart of the ACS strategy to improve enzyme stability.
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f2: The flow chart of the ACS strategy to improve enzyme stability.

Mentions: Due to the complexity of enzyme structures and the key role of the active center where substrate recognition and catalysis takes place, we propose a general active center stabilization (ACS) strategy to improve enzymes thermostability (Fig. 2). In this strategy, we pay more attentions to the flexible fragments rather than the flexible residues. Therefore, we divided the flexible fragments into two parts, outer phase and active center. There are no obvious boundaries in small proteins, whereas the obvious boundaries in large enzymes. Here, we have used a relatively larger lipase, Candida rugosa lipase1 (LIP1) containing 534 residues (Fig. 1C), as a model system to investigate the effect of ACS strategy. Besides of a canonical core with the α/β hydrolase fold domain, LIP1 possesses 10 helixes and 5 strands around the core structure as well as a lid segment covering its catalytic triad. LIP1 is supposed to present in an equilibrium between its open and closed states in solution2122. The optimal temperature of LIP1 is 45 °C, and it loses nearly all activity after 60 °C incubation for 10 min2324. Those residues with high B-factor ranking located within 10 Å of the catalytic residue Ser209 were chosen as candidates for site-saturated mutagenesis and ordered recombination mutagenesis (ORM). To avoid enzymes inactivation caused by active center mutagenesis, a smart three-tier screening procedure was set up to obtain the thermostability improved variants with relatively high activity. The kinetic and thermodynamic stability of the mutants were systematically characterized. In this study, the ACS strategy as an efficient methodology have been used to enhance the thermostability of a large lipase and it showed that mutagenesis focusing on flexible residues around 10 Å of the catalytic residue might significantly increase the success rate.


A general and efficient strategy for generating the stable enzymes
The flow chart of the ACS strategy to improve enzyme stability.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: The flow chart of the ACS strategy to improve enzyme stability.
Mentions: Due to the complexity of enzyme structures and the key role of the active center where substrate recognition and catalysis takes place, we propose a general active center stabilization (ACS) strategy to improve enzymes thermostability (Fig. 2). In this strategy, we pay more attentions to the flexible fragments rather than the flexible residues. Therefore, we divided the flexible fragments into two parts, outer phase and active center. There are no obvious boundaries in small proteins, whereas the obvious boundaries in large enzymes. Here, we have used a relatively larger lipase, Candida rugosa lipase1 (LIP1) containing 534 residues (Fig. 1C), as a model system to investigate the effect of ACS strategy. Besides of a canonical core with the α/β hydrolase fold domain, LIP1 possesses 10 helixes and 5 strands around the core structure as well as a lid segment covering its catalytic triad. LIP1 is supposed to present in an equilibrium between its open and closed states in solution2122. The optimal temperature of LIP1 is 45 °C, and it loses nearly all activity after 60 °C incubation for 10 min2324. Those residues with high B-factor ranking located within 10 Å of the catalytic residue Ser209 were chosen as candidates for site-saturated mutagenesis and ordered recombination mutagenesis (ORM). To avoid enzymes inactivation caused by active center mutagenesis, a smart three-tier screening procedure was set up to obtain the thermostability improved variants with relatively high activity. The kinetic and thermodynamic stability of the mutants were systematically characterized. In this study, the ACS strategy as an efficient methodology have been used to enhance the thermostability of a large lipase and it showed that mutagenesis focusing on flexible residues around 10 Å of the catalytic residue might significantly increase the success rate.

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.