caa a22 4500 605504113 CHVBK 20210128100614.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s00253-014-6172-y doi (NATIONALLICENCE)springer-10.1007/s00253-014-6172-y Spatially programmed assembling of oxidoreductases with single-stranded DNA for cofactor-required reactions [Elektronische Daten] [Tianwen Wang, Fei Ma, Xingyuan Ma, Ping Wang] Cofactor is especially important for biotransformation catalyzed by oxidoreductases. Many attempts in enhancing performance of the reactions by improving cofactor utilization have been reported. In this study, efficiency of cofactor-requiring biocatalysis was enhanced by improving cofactor recycling via spatially programmed assembling glycerol dehydrogenase (GlyDH, Escherichia coli MG1655) and glutamate dehydrogenase (GluDH, Bacillus subtilis str168), with the aid of single-stranded DNA (ssDNA). The two enzymes were first independently expressed as molecules fused with a phage protein A* that could covalently link ssDNA with certain features. After an enzymatic cross-linking reaction taking place under mild conditions, the conjugate of fused enzyme and ssDNA was assembled into desired structures through base pairing enabled by the ssDNA. Results showed that, to some extent, the fusion with protein A* could improve the specific activity of the enzymes (GlyDH-A*/GlyDH = 116.0%; GluDH-A*/GluDH = 105.2%). Additionally, in the coupled reaction system with glycerol and α-ketoglutaric acid as substrates, regarding the production of glutamic acid based on HPLC analysis, the efficiency of cofactor utilization was significantly enhanced (by 23.8- to 41.9-folds), indicating the existence of a substrate-channeling mechanism for cofactor utilization in the assembled reaction system due to the proximity effects. The degree of substrate channeling was calculated as from 1.65 to 1.73. Furthermore, the efficiency of cofactor utilization was influenced in an architecture-dependent manner when complexes with different stoichiometry of GlyDH and GluDH were utilized in biotransformation. This study demonstrated a novel strategy of cofactor recycling for enhanced performance of coupled oxidoreductive reactions. Springer-Verlag Berlin Heidelberg, 2014 Coupled oxidoreductive reaction nationallicence Cofactor recycling nationallicence Spatially assembled enzymes nationallicence Single-stranded DNA nationallicence Substrate channeling nationallicence Proximity effect nationallicence Wang Tianwen School of Biotechnology, State Key Laboratory of Bioreactor Engineering, NanoBioEngineering Laboratory, BioMedicine Nanotechnology Center, East China University of Science and Technology, 200237, Shanghai, China aut Ma Fei School of Biotechnology, State Key Laboratory of Bioreactor Engineering, NanoBioEngineering Laboratory, BioMedicine Nanotechnology Center, East China University of Science and Technology, 200237, Shanghai, China aut Ma Xingyuan School of Biotechnology, State Key Laboratory of Bioreactor Engineering, NanoBioEngineering Laboratory, BioMedicine Nanotechnology Center, East China University of Science and Technology, 200237, Shanghai, China aut Wang Ping School of Biotechnology, State Key Laboratory of Bioreactor Engineering, NanoBioEngineering Laboratory, BioMedicine Nanotechnology Center, East China University of Science and Technology, 200237, Shanghai, China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/8(2015-04-01), 3469-3477 0175-7598 99:8<3469 2015 99 253 https://doi.org/10.1007/s00253-014-6172-y text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6172-y text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Wang Tianwen School of Biotechnology, State Key Laboratory of Bioreactor Engineering, NanoBioEngineering Laboratory, BioMedicine Nanotechnology Center, East China University of Science and Technology, 200237, Shanghai, China aut NATIONALLICENCE 700 1- Ma Fei School of Biotechnology, State Key Laboratory of Bioreactor Engineering, NanoBioEngineering Laboratory, BioMedicine Nanotechnology Center, East China University of Science and Technology, 200237, Shanghai, China aut NATIONALLICENCE 700 1- Ma Xingyuan School of Biotechnology, State Key Laboratory of Bioreactor Engineering, NanoBioEngineering Laboratory, BioMedicine Nanotechnology Center, East China University of Science and Technology, 200237, Shanghai, China aut NATIONALLICENCE 700 1- Wang Ping School of Biotechnology, State Key Laboratory of Bioreactor Engineering, NanoBioEngineering Laboratory, BioMedicine Nanotechnology Center, East China University of Science and Technology, 200237, Shanghai, China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/8(2015-04-01), 3469-3477 0175-7598 99:8<3469 2015 99 253