Heterologous Expression and Characterization of Aphid Myrosinase (BbMyr) in Escherichia coli: Enhanced Production of Sulforaphane

Myrosinase plays a pivotal role in the hydrolysis of glucosinolates within plants, leading to the formation of a diverse range of biologically active metabolites. This research presents the successful heterologous expression of the aphid myrosinase gene (BbMyr) in Escherichia coli, resulting in an engineered strain displaying robust hydrolytic activity towards glucoraphanin (GRA). Following purification, the engineered strain exhibited a specific enzyme activity of 15.45 U/mg. Under optimal reaction conditions of pH 4.5 and 45ﾰC, the engineered E. coli whole-cell effectively degraded glucoraphanin, yielding a remarkable concentration of 1.6 g/L sulforaphane (SFN). This represents the highest reported yield of SFN per gram of broccoli seeds, signifying a significant advancement in biocatalytic production.

Furthermore, the immobilized engineered E. coli demonstrated exceptional stability, retaining 64% activity after seven consecutive reaction cycles. In contrast, free cells exhibited a significantly lower activity retention of only 29%. These findings highlight the potential for utilizing immobilized E. coli cells as a robust and efficient biocatalyst for industrial-scale production of sulforaphane.

To gain deeper insights into the enzyme's active site, molecular dynamics simulations were employed to determine the protonation state of Glu167, a key residue within the BbMyr active center. These simulations provide a crucial foundation for future molecular modifications aimed at enhancing the catalytic efficiency and stability of BbMyr. This research paves the way for the development of sustainable and scalable biocatalytic processes for the production of valuable bioactive compounds like sulforaphane.