Biotransformation of isoeugenol to vanillin is an attractive approach in industry for vanillin synthesis. However, the biotransformation efficiency is hampered by the low activity of isoeugenol monooxygenase (IEM) and the product inhibition towards IEM. Therefore, it is worth discovering a new IEM and improving its enzymatic properties by protein engineering. Herein, a new IEM720 gene was firstly cloned from microbial metagenome and successfully expressed in E. coli. Secondly, five potentially beneficial amino acid positions were selected by a "three-criteria" in silica system and visual inspection was performed for grouping them into two sets (regions A and B). The variant IEM720-F281Q displayed highest isoeugenol conversion and k(cat) value after screening the site-saturation libraries of these five positions. Furthermore, the structure-functional relationships of the beneficial substitution on IEM activity were investigated. The final vanillin concentration reached similar to 4.5 g/L similar to 75% conversion) by combining sol-gel chitosan membrane (for relieve of product inhibition) and E. colt cells harboring IEM720-F281Q.