Enhanced Thermostability of Pseudomonas nitroreducens Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus Mutagenesis

Abstract

Vanillin has many applications in industries. Isoeugenol monooxygenase (IEM) can catalyze the oxidation of isoeugenol to vanillin in the presence of oxygen under mild conditions. However, the low thermal stability of IEM limits its practical application in the biosynthesis of natural vanillin. Herein, two rational strategies were combined to improve the thermostability of IEM from Pseudomonas nitroreducens Jin1. Two variants (K83R and K95R) with better thermostability and one mutant (G398A) with higher activity were identified from twenty candidates based on the Surface Residue Replacement method. According to the Consensus Mutagenesis method, one mutant (I352R) with better thermostability and another mutant (L273F) with higher activity were also identified from nine candidates. After combinatorial mutation, a triple mutant K83R/K95R/L273F with the best thermostability and catalytic efficiency was generated. Compared with the wild-type IEM, the thermal inactivation half-lives (t1/2) of K83R/K95R/L273F at 25 °C, 30 °C, and 35 °C increased 2.9-fold, 11.9-fold, and 24.7-fold, respectively. Simultaneously, it also exhibited a 4.8-fold increase in kcat, leading to a 1.2-fold increase in catalytic efficiency (kcat/Km). When the whole cell of K83R/K95R/L273F was applied to the biotransformation of isoeugenol on preparative scale, the vanillin concentration reached 240.1 mM with space-time yield of 109.6 g/L/d, and vanillin was achieved in 77.6% isolated yield and >99% purity.