谷胱甘肽双功能合成酶固定化研究

Immobilization of Bifunctional Glutathione Synthase

对谷胱甘肽双功能合成酶(GshF)固定化载体进行了筛选,最终选择氧化石墨烯(GO)作为GshF的固定化载体,并对其固定化条件和固定化酶学性质进行了探讨。结果表明,固定化体系最优pH为8.0,温度为4℃,固定化时间为30 min,此时最大加载率达到90%,酶活回收率为45%。固定化体系中温度和pH的适用范围都得到了进一步提高。另外,对固定化酶的贮存稳定性和循环利用性进行了研究,结果表明在常温条件下贮存时,游离酶在第3天就几乎完全失活,而固定化酶仍保留约63%的酶活力。在重复循环利用6次后,固定化酶的酶活力仍保持在80%左右。

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.