Enhancing H2O2 resistance of an esterase from Pyrobaculum calidifontis by structure-guided engineering of the substrate binding site

Applied Microbiology and Biotechnology
Pengfei ZhouYonghua Wang

Abstract

Green technologies are attracting increasing attention in industrial chemistry where enzymatic reactions can replace dangerous and environmentally unfriendly chemical processes. In situ enzymatic synthesis of peroxycarboxylic acid is an attractive alternative for several industrial applications although concentrated H2O2 can denature the biocatalyst, limiting its usefulness. Herein, we report the structure-guided engineering of the Pyrobaculum calidifontis esterase (PestE) substrate binding site to increase its stability and perhydrolysis activity. The L89R/L40A PestE mutant showed better tolerance toward concentrated H2O2 compared with wild-type PestE, and retained over 72% of its initial activity after 24-h incubation with 2 M H2O2. Surprisingly, the half-life (t 1/2, 80 °C) of PestE increased from 28 to 54 h. The k cat/K m values of the mutant increased 21- and 3.4-fold toward pentanoic acid and H2O2, respectively. This work shows how protein engineering can be used to enhance the H2O2 resistance and catalytic efficiency of an enzyme.

References

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Datasets Mentioned

BETA
AB078331.1

Methods Mentioned

BETA
chemical modification

Software Mentioned

NOVA
AMBER14
Modeller

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