Dual promoter strategy enhances co-expression of α-L-rhamnosidase and enhanced fluorescent protein for whole-cell catalysis and bioresource valorization

Abstract

Developing circular economy is the only way to improve the efficiency of resource utilization. Whole-cell catalysis is an effective method to recycle enzymes, improve catalytic efficiency, and reduce production costs. The enzyme, α-L-rhamnosidase has considerable application prospects in the field of biocatalysis as it can hydrolyze a variety of α-L rhamnoses. In the present study, the genes for α-L-rhamnosidase (rhaB1) and enhanced fluorescent protein (EGFP) were co-expressed using a bi-promoter expression vector pRSFDuet1 and their enzymatic properties were evaluated. To our knowledge, this study has established an effective rhamnosidase-fluorescent indicator and whole-cell catalytic system for the first time. Moreover, we analyzed the change in the activity of the crude rhaB1-EGFP as well as its whole-cell during the biocatalysis process using fluorescence intensity. Recombinant rhaB1-EGFP as a product which contains rhaB1 and EGFP showed higher thermal stability, pH stability, and conversion efficiency than rhaB1, and its optimum temperature for rutin catalysis was ideal for industrial applications. Moreover, under the optimal conditions of a rutin concentration of 0.05 g/L, pH of 6.0, temperature of 40 °C, a yield of 92.5% was obtained. Furthermore, we demonstrated the relationship between the fluorescence intensity and enzyme activity. This study established a highly efficient whole-cell catalytic system whose activity can be evaluated by fluorescence intensity, providing a reference for enzyme recycling.

Keywords: Biocatalysis; Co-expression; Dual promoter; Enhanced fluorescent protein; Whole-cell catalysis; α-L-rhamnosidase.