Key Laboratory of Industrial Biocatalysis

Key Laboratory of Industrial Biocatalysis

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Huang Z.,Key Laboratory of Industrial Biocatalysis | Huang Z.,Tsinghua University | Li G.,Key Laboratory of Industrial Biocatalysis | Li G.,Tsinghua University | And 4 more authors.
Enzyme and Microbial Technology | Year: 2016

Fusion strategy has been widely used to construct artificial multifunction proteins. The flexibility or rigidity of linkers between two fused partners is an important parameter that affects the function of fusion proteins. By combining the flexible unit GGGGS (F) and rigid unit EAAAK (R), ten linkers consisting of five elementary units that cover the fully rigid RRRRR linker to the fully flexible FFFFF linker were used to construct acid phosphatase-green fluorescence protein fusion protein (PhoC-GFP). By varying the linker flexibility in PhoC-GFPs, the relative specific activity of phosphotransferase and phosphatase varied from ~19.0% to 100% and ~9.35% to 100%, respectively. There exists an optimal linker capable of achieving the highest phosphotransferase/phosphatase activity and GFP fluorescence intensity. We found that the highest activities were achieved neither with the rigid RRRRR linker nor with the flexible FFFFF linker, but with the FFFRR linker. Linker flexibility could adjust the activity ratio between phosphotransferase and phosphatase and varied between ~30% to 100%. PhoC-GFP with FRRRR linker achieved the highest relative specific phosphotransferase activity/relative specific phosphatase activity (T/P) value. Our results show that applying a linker library with controllable flexibility to the fusion proteins will be an efficient way to adjust the function of fusion enzymes. © 2015 Elsevier Inc.

Li G.,Tsinghua University | Li G.,Key Laboratory of Industrial Biocatalysis | Huang Z.,Tsinghua University | Huang Z.,Key Laboratory of Industrial Biocatalysis | And 12 more authors.
Applied Microbiology and Biotechnology | Year: 2016

Flexibility or rigidity of the linker between two fused proteins is an important parameter that affects the function of fusion proteins. In this study, we constructed a linker library with five elementary units based on the combination of the flexible (GGGGS) and the rigid (EAAAK) units. Molecular dynamics (MD) simulation showed that more rigid units in the linkers lead to more helical conformation and hydrogen bonds, and less distance fluctuation between the N- and C-termini of the linker. The diversity of linker flexibility of the linker library was then studied by fluorescence resonance energy transfer (FRET) of cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) fusion proteins, which showed that there is a wide range of distribution of the FRET efficiency. Dissipative particle dynamics (DPD) simulation of CFP-YFP with different linkers also gave identical results with that of FRET efficiency analysis, and we further found that the combination manner of the linker peptide had a remarkable effect on the orientation of CFP and YFP domains. Our studies demonstrated that the construction of the linker library with the widely controllable flexibility could provide appropriate linkers with the desirable characteristics to engineer the fusion proteins with the expected functions. © 2015, Springer-Verlag Berlin Heidelberg.

Zhang J.,Key Laboratory of Industrial Biocatalysis | Yu C.,Key Laboratory of Industrial Biocatalysis | Jiang G.,Key Laboratory of Industrial Biocatalysis
Journal of Biomaterials Science, Polymer Edition | Year: 2016

The specific interaction between bile acids and the bile acids transporters provides a promising way for hepatoma-targeted drug delivery. We synthesized an amphipathic polymer containing cholic acid (CA), the main bile acids in body, and prepared CA-functionalized nanoparticles to target hepatoma cells. Poly-[3-(4-vinylbenzonate)-7, 12-dihydroxy-5-cholan-24-oic acid] (PVBCA) was synthesized by introducing methyl cholate onto polyvinyl benzoate polymer backbone, and was characterized by 1H-NMR, FT-IR, and GFC. PVBCA can be incorporated onto PLGA nanoparticles surface via the emulsion-solvent evaporation procedure, resulting in the nanoparticles carrying CA moieties on their surface. The binding of CA moieties to the bile acids’ transporters on the cell membrane enhances the cellular uptake of the nanoparticles significantly. The SMMC-7721 cell uptake of PVBCA-decorated nanoparticles increases with amount of incorporated PVBCA and is 2- to 2.8-fold higher than that of the normal PLGA nanoparticles. By exclusion of specific endocytosis pathways using chemical inhibitors, we found that the uptake mechanism of PVBCA-decorated nanoparticles was mainly attributed to clathrin-and-caveolae-independent endocytosis, which was distinct from that of PLGA nanoparticles. The present study provides a simple and versatile method for hepatoma-targeted delivery of nanoparticles. © 2016 Informa UK Limited, trading as Taylor & Francis Group

Li G.,Key Laboratory of Industrial Biocatalysis | Li G.,Tsinghua University | Zhang C.,Key Laboratory of Industrial Biocatalysis | Zhang C.,Tsinghua University | And 2 more authors.
Biochemical Engineering Journal | Year: 2016

Direct channeling is a well-known process in which intermediates are funneled between enzyme active sites through a physical tunnel and can be a potential way to enhance the biocatalytic efficiency for cascading bioreactions. However, the exact mechanism of the substrate channeling remains unclear. In this work, we used mathematical models to describe the mass transfer in the physical tunnels and to gain further understanding of direct proximity channeling. Simulation with a diffusion-reaction model showed that the reduction of the diffusion distance of intermediates could not cause proximity channeling. A second kinetic model, which considered the physical tunnel as a small sphere capable of preventing diffusion of the intermediate into the bulk, was then constructed. It was used to show that the maximum channeling degree in branched pathways depends on the strength of the side reactions, suggesting that proximity channeling in a physical tunnel is more suitable for a pathway with strong side reactions. On the other hand, for a linear pathway, proximity channeling is more beneficial when the constituting enzymes have relatively low activities and expression levels. Our kinetic model provides a theoretical basis for engineering proximity channeling between sequentially acting enzymes in microbial cell factories and enzyme engineering. © 2015 Elsevier B.V.

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