Introduction of sulfhydryl groups and disulfide linkage to mungbean 8Sα globulin and effects on physicochemical and functional properties

Sulfhydryl groups and disulfide bond were introduced in mungbean's major storage protein, 8Sα globulin, by protein engineering to improve structural stability and functional properties. Five modified proteins or mutants (F59C, I99C, A213C, one free sulfhydryl group; I99C/A213, one disulfide bridge; F59C/I99C/A213C, one free sulfhydryl group and one disulfide linkage) were expressed in Escherichia coli at a yield similar to that of the unmodified protein or wild type (WT) in soluble form (38%). The number of introduced groups in the mutants was confirmed by Ellman analysis. Mutant and WT proteins exhibited similar elution patterns on gel filtration indicating their trimeric native conformation. Mutants had 2 to 3.8 °C higher T_m values than WT and were digested by chymotrypsin at 52–58% in 60 min but exhibited different digestion patterns. All mutants showed greater hardness of heat-induced gels than WT, especially I99C/A213C and F59C/I99C/A213C. Results indicate the improved structural stability of the modified 8Sα globulin.