Limits...
A general and efficient strategy for generating the stable enzymes

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.


Thermostability of WT and all mutants.(A) Thermostability, expressed as  values (defined as the temperature at which the enzyme retains 50% of its activity after 15 min of incubation) of all mutants considered in this study: WT (black bar), F434Y, F133Y and F121Y (purple bar), F344M and F344M combination mutants (orange bar), and F344I and F444I combination mutants (blue bar). (B) Residual activity curves of WT and all mutants. (C) Thermostability at 60 °C. The activities of the enzymes without heat treatment were defined as 100%. (D) Thermal unfolding of WT and its mutants. The scans were performed at 60 °C/h over a temperature range of 30–90 °C using VP-cap DSC. The combinational mutants are VarA1 (F344M/F434Y), VarA2 (F344M/F434Y/F133Y), VarA3 (F344M/F434Y/F133Y/F121Y), VarB1 (F344I/F434Y), VarB2 (F344I/F434Y/F133Y), and VarB3 (F344I/F434Y/F133Y/F121Y).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036031&req=5

f4: Thermostability of WT and all mutants.(A) Thermostability, expressed as values (defined as the temperature at which the enzyme retains 50% of its activity after 15 min of incubation) of all mutants considered in this study: WT (black bar), F434Y, F133Y and F121Y (purple bar), F344M and F344M combination mutants (orange bar), and F344I and F444I combination mutants (blue bar). (B) Residual activity curves of WT and all mutants. (C) Thermostability at 60 °C. The activities of the enzymes without heat treatment were defined as 100%. (D) Thermal unfolding of WT and its mutants. The scans were performed at 60 °C/h over a temperature range of 30–90 °C using VP-cap DSC. The combinational mutants are VarA1 (F344M/F434Y), VarA2 (F344M/F434Y/F133Y), VarA3 (F344M/F434Y/F133Y/F121Y), VarB1 (F344I/F434Y), VarB2 (F344I/F434Y/F133Y), and VarB3 (F344I/F434Y/F133Y/F121Y).

Mentions: Finally, approximately 2.0 × 104 clones in all mutant libraries were screened, accounting for about 95.6% library coverage of the variant space293031. The thermostability of the mutant was estimated on fluorescent soft agar plates after exposure to 60 °C using the three-tier screening protocol. After screening, five mutants that showed improved thermostability were selected and sequenced to identify their mutations: F344M, F344I, F434Y, F133Y and F121Y (Supplementary Table S2, Fig. 4A). For all mutants, phenylalanine was substituted with other amino acids. Except for position 344, the other three positions have a tyrosine substitution (Fig. 4A). The , being the temperature at which 50% of the activity is lost after 15 min of incubation, for each of these mutants varied from 57.5 °C (F121Y, WT + 3.0 °C) to 62.4 °C (F344M, WT + 7.9 °C) (Fig. 4B). The half-lives of the mutants at 60 °C were enhanced 1.3- (F121Y, 7.8 min) to 5.1-fold (F344M, 30.9 min) compared to WT (6.0 min) (Fig. 4C). The catalytic efficiencies of the mutants were also higher than that of WT, except for F344M, which was similar to WT (Table 1).


A general and efficient strategy for generating the stable enzymes
Thermostability of WT and all mutants.(A) Thermostability, expressed as  values (defined as the temperature at which the enzyme retains 50% of its activity after 15 min of incubation) of all mutants considered in this study: WT (black bar), F434Y, F133Y and F121Y (purple bar), F344M and F344M combination mutants (orange bar), and F344I and F444I combination mutants (blue bar). (B) Residual activity curves of WT and all mutants. (C) Thermostability at 60 °C. The activities of the enzymes without heat treatment were defined as 100%. (D) Thermal unfolding of WT and its mutants. The scans were performed at 60 °C/h over a temperature range of 30–90 °C using VP-cap DSC. The combinational mutants are VarA1 (F344M/F434Y), VarA2 (F344M/F434Y/F133Y), VarA3 (F344M/F434Y/F133Y/F121Y), VarB1 (F344I/F434Y), VarB2 (F344I/F434Y/F133Y), and VarB3 (F344I/F434Y/F133Y/F121Y).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Thermostability of WT and all mutants.(A) Thermostability, expressed as values (defined as the temperature at which the enzyme retains 50% of its activity after 15 min of incubation) of all mutants considered in this study: WT (black bar), F434Y, F133Y and F121Y (purple bar), F344M and F344M combination mutants (orange bar), and F344I and F444I combination mutants (blue bar). (B) Residual activity curves of WT and all mutants. (C) Thermostability at 60 °C. The activities of the enzymes without heat treatment were defined as 100%. (D) Thermal unfolding of WT and its mutants. The scans were performed at 60 °C/h over a temperature range of 30–90 °C using VP-cap DSC. The combinational mutants are VarA1 (F344M/F434Y), VarA2 (F344M/F434Y/F133Y), VarA3 (F344M/F434Y/F133Y/F121Y), VarB1 (F344I/F434Y), VarB2 (F344I/F434Y/F133Y), and VarB3 (F344I/F434Y/F133Y/F121Y).
Mentions: Finally, approximately 2.0 × 104 clones in all mutant libraries were screened, accounting for about 95.6% library coverage of the variant space293031. The thermostability of the mutant was estimated on fluorescent soft agar plates after exposure to 60 °C using the three-tier screening protocol. After screening, five mutants that showed improved thermostability were selected and sequenced to identify their mutations: F344M, F344I, F434Y, F133Y and F121Y (Supplementary Table S2, Fig. 4A). For all mutants, phenylalanine was substituted with other amino acids. Except for position 344, the other three positions have a tyrosine substitution (Fig. 4A). The , being the temperature at which 50% of the activity is lost after 15 min of incubation, for each of these mutants varied from 57.5 °C (F121Y, WT + 3.0 °C) to 62.4 °C (F344M, WT + 7.9 °C) (Fig. 4B). The half-lives of the mutants at 60 °C were enhanced 1.3- (F121Y, 7.8 min) to 5.1-fold (F344M, 30.9 min) compared to WT (6.0 min) (Fig. 4C). The catalytic efficiencies of the mutants were also higher than that of WT, except for F344M, which was similar to WT (Table 1).

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.