Abstract:
Glutathione (GSH), a ubiquitous low-molecular-weight tripeptide thiol, exists in most organisms such as plants, mammals, fungi, and prokaryotes. Owing to its special physiological functions, it has been widely used in food, health products, cosmetic and medicine industry. Traditionally, glutathione is synthesized through a two-step ATP-dependent pathway consisting of
γ-glutamylcysteine synthase (
γ-GCS) and glutathione synthetase (GS) in organisms. Bifunctional glutathione synthase (GshF), possessing both enzymatic activity of
γ-GCS and GS in a single enzyme, can catalyze the two-step reaction sequentially, which contribute to the unique enzymatic properties for GSH biosynthesis. However, there is few research on enzymatic synthesis of glutathione through immobilization enzyme. Therefore, exploring the biosynthesis of GSH by immobilized GshF is promising and valuable for industrial application. In this study, we purified the GshF from
Streptococcus sanguinis (GshF) and studied the immobilized GshF by physical adsorption. Meanwhile, we firstly screened the enzyme immobilized carrier from six kinds of nano materials. Graphene oxide (GO) was found as the optimal carrier for the immobilization of GshF. Then the conditions of immobilization and the enzymatic properties of immobilized enzyme were studied. When the immobilization condition was at pH 8.0 and 4℃ with the time of absorption of 30 min, the maximum loading of enzymes reached 90% and the recovery rate of enzyme activity was 45%. The temperature and pH stability were further improved. In addition, the storage stability and recyclability of the immobilized enzyme were also enhanced. The free enzyme was almost completely inactivated on the third day under normal temperature conditions, whereas the enzyme activity of immobilized enzyme still retained about 63%. The immobilized GshF on GO could be reused 6 times to retain 80% relative activity. This study provides an efficient strategy for GSH synthesis through enzyme immobilization, providing a potential biotransformation platform for industrial production.