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• W2548897357 abstract "Anchorage of recombinant proteins onto the outer membrane of gram-negative bacteria is an attractive solution for protein library screening and whole cell biocatalysis if a membrane environment is required or mass transfer into the cell is limiting. Autotransporters have been successfully applied for surface display of various heterologous proteins. Still, many underlying parameters for achieving active enzymes are not known. Here, we systematically tested different linkers between passenger and the membrane embedded β-barrel of the autotransporter. The linker can have influence on aspects such as steric orientation of the passenger, distance to the outer membrane and accessibility of active sites. Six linker variants for display of the cytochrome P450 reductase were tested. Cytochrome c reduction by the cytochrome P450 reductase varied fivefold and was highest by introduction of a flexible glycine-serine region. When these variants were co-expressed with surface displayed CYP1A2, product concentration for paracetamol differed between 0.22 μM and 2.5 μM and for resorufin between 0.23 μM to 1 μM. The best glycine/serine containing sequence, that turned out to be best for CPR display, was then introduced into the linker for displaying CYP1A2. In comparison, up to 7.9 μM paracetamol and up to 1.69 μM resorufin were obtained with this new variant. The differences were not caused by changes in the number of displayed enzymes. To our knowledge, this is the first systematic study on engineering the linker for surface display of recombinant enzymes." @default.
• W2548897357 created "2016-11-11" @default.
• W2548897357 creator A5008838350 @default.
• W2548897357 creator A5022601580 @default.
• W2548897357 creator A5055585179 @default.
• W2548897357 creator A5081645354 @default.
• W2548897357 date "2017-01-01" @default.
• W2548897357 modified "2023-09-26" @default.
• W2548897357 title "Improving the activity of surface displayed cytochrome P450 enzymes by optimizing the outer membrane linker" @default.
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• W2548897357 doi "https://doi.org/10.1016/j.bbamem.2016.10.022" @default.
• W2548897357 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/27814979" @default.
• W2548897357 hasPublicationYear "2017" @default.
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