In vivo enzyme immobilization by use of engineered polyhydroxyalkanoate synthase

Applied and Environmental Microbiology
Verena Peters, B H Rehm

Abstract

This study demonstrated that engineered polyhydroxyalkanoate (PHA) synthases can be employed as molecular tools to covalently immobilize enzymes at the PHA granule surface. The beta-galactosidase was fused to the N terminus of the class II PHA synthase from Pseudomonas aeruginosa. The open reading frame was confirmed to encode the complete fusion protein by T7 promoter-dependent overexpression. Restoration of PHA biosynthesis in the PHA-negative mutant of P. aeruginosa PAO1 showed a PHA synthase function of the fusion protein. PHA granules were isolated and showed beta-galactosidase activity. PHA granule attached proteins were analyzed and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Surprisingly, the beta-galactosidase-PHA synthase fusion protein was detectable at a high copy number at the PHA granule, compared with PHA synthase alone, which was barely detectable at PHA granules. Localization of the beta-galactosidase at the PHA granule surface was confirmed by enzyme-linked immunosorbent assay using anti-beta-galactosidase antibodies. Treatment of these beta-galactosidase-PHA granules with urea suggested a covalent bind...Continue Reading

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Citations

Sep 19, 2008·Biotechnology Letters·Jane A Atwood, Bernd H A Rehm
Nov 28, 2009·Biotechnology Letters·Ulrich RoesslBernd Nidetzky
Jan 30, 2013·Molecular Biotechnology·Stefan R Schmidt
May 26, 2009·Applied and Environmental Microbiology·Jun MifuneBernd H A Rehm
Jun 29, 2010·Applied and Environmental Microbiology·Björn SteinmannHarald Kolmar
Mar 5, 2013·Applied and Environmental Microbiology·David O HooksBernd H A Rehm
Aug 2, 2011·Applied and Environmental Microbiology·Katrin GrageBernd H A Rehm
Oct 11, 2011·Applied and Environmental Microbiology·Natalie A ParlaneBernd H A Rehm
Jan 29, 2008·Journal of Bacteriology·Liv NeumannAlexander Steinbüchel
Apr 21, 2012·International Journal of Microbiology·Sujatha KabilanGunasekaran Paramasamy
Jun 26, 2014·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·David O HooksBernd H A Rehm
Aug 29, 2013·BioMed Research International·Anupama ShrivastavYoung-Rok Kim
Feb 28, 2015·Nanomedicine : Nanotechnology, Biology, and Medicine·Nina Dinjaski, M Auxiliadora Prieto
Jun 19, 2012·Bioengineered·Jenny L Draper, Bernd H Rehm
Jan 6, 2015·Molecular Pharmaceutics·Gemma NavarroVladimir P Torchilin
Oct 19, 2016·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Fabian B H RehmBernd H A Rehm
Nov 24, 2017·Microbial Cell Factories·Katrin GrageBernd H A Rehm
Mar 13, 2014·Applied and Environmental Microbiology·Mark Venning-SlaterBernd H A Rehm
Jul 23, 2009·Chemical Society Reviews·Guo-Qiang Chen
Sep 9, 2017·Applied Microbiology and Biotechnology·Huibin ZouMo Xian
Mar 23, 2021·Enzyme and Microbial Technology·Sun Ah JangDong-Myung Kim
Sep 10, 2008·Bioconjugate Chemistry·Anika C JahnsBernd H A Rehm
Jun 30, 2021·ACS Applied Materials & Interfaces·Narshone SodaBernd H A Rehm

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