Adipic acid is an important six-carbon dicarboxylic acid with numerous industrial applications in polymers (nylon) and the food industry. Traditional manufacturing of adipic acid relies on petroleum feedstocks and involves energy-intensive chemical processes with negative environmental impacts. Consequently, alternative synthesis methods are being developed, including the hydrogenation of biobased muconic acid to adipic acid via chemical catalysis or enzymatic reduction with 2-enoate reductases. This study revealed that purified full-length 2-enoate reductase ERBC from Heyndrickxia (Bacillus) coagulans can reduce the three muconic acid isomers (cis,cis, cis,trans, trans,trans) using NADH as a reductant. Titration of the purified ERBC with different chemical reductants showed that its redox cofactors (FMN, FAD, and [4Fe-4S]) can also be reduced by dithionite and Ti(III)-citrate. However, only dithionite and NADH supported the biohydrogenation of trans-cinnamic acid and cis,cis-muconic acid. The individually expressed and purified large domain of ERBC also catalyzed muconic acid reduction with these reductants, but exhibited lower activity and produced only 2-hexenedioic acid as the product. Efficient conversion of muconic acid to adipic acid was demonstrated using lyophilized E. coli cells expressing full-length ERBC as the catalyst, with dithionite acting as both a reductant and an oxygen scavenger. The use of lyophilized recombinant Escherichia coli cells with dithionite for ERBC-mediated biohydrogenation of muconic acid eliminates the need for protein purification and costly natural cofactors (NAD(P)H), as well as enhances ERBC tolerance to high substrate concentrations and creates anaerobic conditions for ERBC activity. This approach shows promise for biobased adipic acid production and other applications of 2-enoate reductases.