Improving the activity and expression level of a phthalate-degrading enzyme by a combination of mutagenesis strategies and strong promoter replacement

Abstract

Phthalic acid esters (PAEs) are widely used plasticizers found in consumer products, which enter the environment and pose severe threats to human health. Here, a new PAE-degrading enzyme EstJ6 was modified by combining mutagenesis strategies and a strong promoter replacement to improve its catalytic activity and expression level. Four mutants with enhanced activity were obtained by random mutation, among which EstJ6M1.1 exhibited the highest catalytic activity with an increase in catalytic activity by 2.9-fold toward dibutyl phthalate (DBP) than that of the wild-type (WT) enzyme. With these mutants as a template, a variant EstJ6M2 with 3.1-fold higher catalytic activity and 4.61 times higher catalytic efficiency (Kcat/Km) was identified by staggered extension PCR. Targeting four mutation sites of EstJ6M2, a variant EstJ6M3.1 was gained by site-directed saturation mutagenesis and displayed 4.3-fold higher activity and 5.97 times higher Kcat/Km than WT. The expression level of three mutants EstJ6M1.1, EstJ6M2, and EstJ6M3.1, as well as the WT, increased nearly threefold after a strong promoter replacement. These results provide a proof-theoretical basis and practicable pipeline for applying PAE-degrading enzymes.

Keywords: Combinatorial mutation strategies; DBP and DEHP; Directed evolution; Docking study; PAE-degrading enzyme; Strong promoter replacement.