Functional expression of broccoli myrosinase in Bacillus subtilis: A GRAS microbial platform for enzyme production

Myrosinase (β-thioglucoside glucohydrolase, EC 3.2.1.147) catalyzes the conversion of glucosinolates into bioactive isothiocyanates such as sulforaphane, a compound of considerable interest for food and nutraceutical applications. However, the limited availability and variable stability of plant-derived myrosinase constrain its industrial use, underscoring the need for alternative production platforms. Here, we report the functional heterologous expression of broccoli myrosinase in Bacillus subtilis, a Generally Recognized As Safe (GRAS) microorganism widely employed for industrial enzyme production. A codon-optimized myrosinase cDNA was expressed under the constitutive P43 promoter using the pTTB2 system, generating constructs with and without an N-terminal His tag. Two competence-based transformation strategies were evaluated, showing that induction in SM1/SM2 minimal media significantly enhanced transformation efficiency compared with a conventional HS/LS protocol. Recombinant B. subtilis clones exhibited measurable myrosinase activity in crude protein extracts, whereas no activity was detected in the non-transformed control strain. Specific activities ranged from 0.316 to 2.614 U mg⁻¹ depending on the construct and clone, with noticeable variability between independent measurements. SDS-PAGE analysis revealed a protein band consistent with the expected molecular mass of myrosinase (∼64.5 kDa), and affinity-based enrichment of the His-tagged enzyme confirmed retention of catalytic activity. Although the enzyme was not purified to homogeneity, the observed activity demonstrates the feasibility of B. subtilis as a food-grade host for myrosinase production. This study establishes a proof-of-concept microbial platform for further optimization through strain engineering, secretion strategies, and downstream process development for food and biotechnological applications.