Substantial Improvement of an Epimerase for the Synthesis of D-Allulose by Biosensor-Based High-Throughput Microdroplet Screening.

Biosynthesis of D-allulose has been achieved using ketose 3-epimerases (KEases), but its application is limited by poor catalytic performance. In this study, we redesigned a genetically encoded biosensor based on a D-allulose-responsive transcriptional regulator for real-time monitoring of D-allulose. An ultrahigh-throughput droplet-based microfluidic screening platform was further constructed by coupling with this D-allulose-detecting biosensor for the directed evolution of the KEases.

Directional immobilization of D-allulose 3-epimerase using SpyTag/SpyCatcher strategy as a robust biocatalyst for synthesizing D-allulose.

D-Allulose, as low-calorie rare sugar, possessed several notable biological activities and was biosynthesized by D-allulose 3-epimerase (DAEase). Here, CcDAE from Clostridium cellulolyticum was successfully immobilization via covalent attachment (RI-CcDAE), and Resin-SpyCatcher/SpyTag-CcDAE modular (DI-CcDAE). Both immobilized CcDAEs exhibited higher thermal and pH stabilities than the free form, and they maintained 80.

Customized exogenous ferredoxin functions as an efficient electron carrier.

Ferredoxin (Fdx) is regarded as the main electron carrier in biological electron transfer and acts as an electron donor in metabolic pathways of many organisms. Here, we screened a self-sufficient P450-derived reductase PRF with promising production yield of 9OHAD (9α-hydroxy4-androstene-3,17-dione) from AD, and further proved the importance of [2Fe-2S] clusters of ferredoxin-oxidoreductase in transferring electrons in steroidal conversion. The results of truncated Fdx domain in all oxidoreductases and mutagenesis data elucidated the indispensable role of [2Fe-2S] clusters in the electron transfer process.

Continuous Spectrophotometric Assay for High-Throughput Screening of Predominant d-Allulose 3-Epimerases.

d-Allulose is an attractive noncaloric sugar substitute with numerous health benefits, which can be biosynthesized by d-allulose 3-epimerases (DAEases). However, enzyme instability under harsh industrial reaction conditions hampered its practical applications. Here, we developed a continuous spectrophotometric assay (CSA) for the efficient analysis of d-allulose in a mixture.

Enhancing the sustainability of KsdD as a biocatalyst for steroid transformation by immobilization on epoxy support.

The Δ1-dehydrogenation of 3-ketosteroid substrates is a crucial reaction in the production of steroids. Although 3-ketosteroid Δ1-dehydrogenase (KsdD) catalyzes this reaction with high efficiency and selectivity, the low stability and high cost of the purified enzyme catalyst have limited its industrial application. In this study, an epoxy support was used to evaluate the covalent immobilization of KsdD from Pimelobacter simplex, and the best androsta-1,4-diene-317-dione (ADD) production was achieved after optimized immobilization of KsdD enzyme in 1.