The Optimization of Assay Conditions and Characterization of the Succinic Semialdehyde Dehydrogenase Enzyme of Germinated Tartary Buckwheat

Abstract

In this study, the conditions for optimizing the determination of succinic semialdehyde dehydrogenase (SSADH, EC 1.2.1.79) activity in germinated Tartary buckwheat were investigated. Based on a single-factor test, the effects of temperature, pH, and succinic semialdehyde (SSA) concentration on the enzyme activity of germinated buckwheat SSADH were investigated by using the response surface method, and optimal conditions were used to study the enzymatic properties of germinated buckwheat SSADH. The results revealed that the optimum conditions for determining SSADH enzyme activity are as follows: temperature—30.8 °C, pH—8.7, and SSA concentration—0.3 mmol/L. Under these conditions, SSADH enzyme activity was measured as 346 ± 9.61 nmol/min. Furthermore, the thermal stability of SSADH was found to be superior at 25 °C, and its pH stability remained comparable at pH levels of 7.6, 8.1, and 8.6 in germinated Tartary buckwheat samples; however, a decline in stability was observed at pH 9.1. Cu2+, Co2+, and Ni2+ exhibited an activating effect on SSADH activity in germinating Tartary buckwheat, with Cu2+ having the greatest influence (p < 0.05), which was 1.21 times higher than that of the control group. Zn2+, Mn2+, and Na+ inhibited SSADH activity in germinating Tartary buckwheat, with Zn2+ showing the strongest inhibitory effect (p < 0.05). On the other hand, the Km and Vmax of SSADH for SSA in germinated Tartary buckwheat were 0.24 mmol/L and 583.24 nmol/min. The Km and Vmax of SSADH for NAD+ in germinated Tartary buckwheat were 0.64 mmol/L and 454.55 nmol/min.