Myrosinase is responsible for the hydrolysis of glucosinolates in plants to yield a variety of biological activity metabolites Here the gene encoding aphid myrosinase BbMyr was successfully heterologo

Myrosinase plays a crucial role in the hydrolysis of glucosinolates in plants, yielding a diverse array of biologically active metabolites. In this study, the gene encoding aphid myrosinase (BbMyr) was successfully heterologously expressed in Escherichia coli. The engineered strain exhibited hydrolytic activity towards glucoraphanin (GRA), with a specific enzyme activity of 15.45 U/mg after purification. Under reaction conditions of pH 4.5 and 45°C, the engineered E. coli whole-cell effectively degraded glucoraphanin to produce sulforaphane (SFN) at a concentration of 1.6 g/L with a remarkable yield of 20.8 mg/g broccoli seeds, which represents the highest yield reported per gram of broccoli seeds.

Additionally, the immobilized engineered E. coli maintained 64% activity after seven batches of repeated reactions, in contrast to only 29% activity for the free cells. Furthermore, molecular dynamics simulations were utilized to determine the protonation state of Glu167, the active center of BbMyr. This finding provides a foundation for further molecular modification of BbMyr