NAD(P)H-dependent oxidoreductases are crucial biocatalysts for synthesizing chiral compounds. Yet, the industrial implementation of enzymatic redox reactions is often hampered by an insufficient supply of expensive nicotinamide cofactors. Here, a cofactor self-sufficient whole-cell biocatalyst was developed for the enzymatic asymmetric reduction of 2-oxo-4-[(hydroxy)(-methyl)phosphinyl] butyric acid (PPO) to L-phosphinothricin (L-PPT). The endogenous NADP + pool was significantly enhanced by regulating Preiss-Handler pathway toward NAD(H) synthesis and, in the meantime, introducing NAD kinase to phosphorylate NAD(H) toward NADP + . The intracellular NADP(H) concentration displayed a 2.97-fold increase with the strategy compared with the wild-type strain. Furthermore, a recombinant multi-enzyme cascade biocatalytic system was constructed based on the Escherichia coli chassis. In order to balance multi-enzyme co-expression levels, the strategy of modulating rate-limiting enzyme Pm GluDH by RBS strengths regulation successfully increased the catalytic efficiency of PPO conversion. Finally, the cofactor self-sufficient whole-cell biocatalyst effectively converted 300 mM PPO to L-PPT in 2 h without the need to add exogenous cofactors, resulting in a 2.3-fold increase in PPO conversion (%) from 43% to 100%, with a high space-time yield of 706.2 g L −1 d −1 and 99.9% ee . Overall, this work demonstrates a technological example for constructing a cofactor self-sufficient system for NADPH-dependent redox biocatalysis.
How to balance the sensitivity and signal-to-noise ratio of immunosensor remains many challenges during various diseases diagnosis. Here we develop a new microfluidic immunosensor based on surface-modified mesoporous nanofibers , and simultaneously realize an ultra-sensitivity and high signal-to-noise ratio for the detection of multiple biomarkers. In the current study, we fabricated titanium dioxide (TiO 2 )-based mesoporous electrospinning nanofibers, and modified nanofiber surface with both octadecylphosphonic acid (OPA) and poly(ethylene oxide)-poly(propylene oxide) triblock copolymer (PEO-PPO-PEO). Such nanofibers as solid substrate are covered on microfluidic channels. The porosity of our nanofibers dramatically increased the adsorption capability of antibodies, realizing an ultra sensitivity of biomarker detection. PEO-PPO-PEO modification can significantly block non-specific absorptions, obtaining a satisfied signal-to-noise ratio. For the detection of HIV p24 and interleukin 5 (IL-5), our immunosensor increased 6.41 and 6.93 fold in sensitivity and improved 504.66% and 512.80% in signal-to-noise ratio, in compared with gold standard immunoassay (ELISA) used in the clinic. Our immunosensor also broaden the linear range for the detection of HIV p24 (0.86–800 pg/ml) and IL-5 (0.70–800 pg/ml), in compared with ELISA which is 5.54–500 pg/ml for HIV p24 and 4.84–500 pg/ml for IL-5. Our work provided a guideline for the construction of advanced point-of-care immunosensor with an ultra-sensitivity and high signal-to-noise ratio for disease diagnosis.
危险品化学品经营许可证（带存储）