In this study, a γPFD-SpyCatcher hydrogel scaffold with the capacity for spontaneous assembly was established. With a maximum loading capacity of a 1:1 molar ratio with SpyTag-enzymes, the immobilized proteins can not only rapidly provide pure enzymes but also exhibit improved thermal and pH stability. The results of the transmission electron microscopic analysis and the traits they present indicated that SpyCatcher promotes the aggregation of γPFD and the formation of hydrogels. In the cell-free pyruvate synthesis system, the γPFD-SpyCatcher coimmobilized SpyTag-hexokinase (HK), SpyTag-phosphofructokinase (PFK) and SpyTag-pyruvate kinase (PK) were employed, and the production of pyruvate increased by 43, 78 and 47% respectively. In in vitro experiments, the oxidative deamination activity of glutamate dehydrogenase (GDH) coimmobilized with γPFD-SpyCatcher was 38% higher than that of purified enzymes. These findings indicate that the γPFD-SpyCatcher-based hydrogels play an important role in breaking the barrier of regulatory enzymes and will provide more strategies for the development of synthetic biology.
Keywords: SpyTag/SpyCatcher; cell-free biosynthesis; coimmobilization; hydrogel.