Enhancing the activity and thermal stability of a phthalate-degrading hydrolase by random mutagenesis

• Published in: Ecotoxicology and environmental safety Vol. 209; p. 111795
• Main Authors: Qiu, Jiarong, Yang, Haiyan, Shao, Yuting, Li, Longxiang, Sun, Shengwei, Wang, Luyao, Tan, Yuzhi, Xin, Zhihong
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Inc 01.02.2021; Elsevier
• Subjects: Catalysis; Dibutyl Phthalate; Enzyme activity; Enzyme Stability; Gene Library; Hydrolases - metabolism; Hydrolysis; Kinetics; Methanol - metabolism; Molecular docking; Molecular Docking Simulation; Mutagenesis; Organic solvent tolerance; Phthalate-degrading hydrolase; Phthalic Acids - metabolism; Random mutagenesis; Solvents; Thermal stability; Phthalate-degrading hydrolase; Organic solvent tolerance; Enzyme activity; Thermal stability; Molecular docking; Random mutagenesis
• ISSN: 0147-6513; 1090-2414
• DOI: 10.1016/j.ecoenv.2020.111795

Our previous work has reported that EstJ6 was a phthalate-degrading hydrolase. In the study, a random mutant library was constructed by two rounds of error-prone PCR, three mutants (ET1.1, ET2.1, and ET2.2) with enhanced hydrolytic activity against dibutyl phthalate (DBP) were obtained. The best mutant ET2.2, accumulated three amino acid substitutions (Thr91Met, Ala67Val, and Val249Ile) and exhibited 2.8-fold increase enzyme activity and 2.3-fold higher expression level. Meanwhile, compared with EstJ6, ET2.2 showed over 50% improvement in thermostability (at 50 °C for 1 h) and 1.2-fold increase in 50% methanol tolerance. Kinetic parameters analysis revealed that the Km value for ET2.2 decreased by 60% and the kcat/Km value increased by 166%. The molecular docking indicated that the shortening of hydrogen bond between Ser146-OH and DBP-CO, which may led to an increase in enzyme activity and catalytic efficiency, the enhancement of hydrophobicity of hydrophobic pocket was related to the improvement of organic solvents tolerance, and three hydrophobic amino acid substitutions Thr91Met, Ala67Val, and Val249Ile facilitated to improve the thermal stability and organic solvents tolerance. These results confirmed that random mutagenesis was an effective tool for improving enzyme properties and lay a foundation for practical applications of phthalate-degrading hydrolase in biotechnology and industrial fields. [Display omitted] •A mutant library of a phthalate ester hydrolase was constructed by error-prone PCR.•The best mutant ET2.2 with enhanced activity and thermostability was identified.•The shortening of hydrogen bond between Ser146-OH and DBP-CO may increase activity.•The enhanced hydrophobicity of the pocket surface increased solvents tolerance.•Thr91Met, Ala67Val, and Val249Ile in ET2.2 facilitated to enhance stability.