In vivo enzyme immobilization by inclusion body display.
A novel strategy for in vivo immobilization of enzymes on the surfaces of inclusion bodies has been established. It relies on expression in Escherichia coli of the polyhydroxybutyrate synthase PhaC from Cupriavidus necator, which carries at its amino terminus an engineered negatively charged alpha-helical coil (Ecoil) and forms inclusion bodies upon high-level expression. Coexpression in the same cell of galactose oxidase (GOase) from Fusarium spp. carrying a carboxy-terminal positively charged coil (lysine-rich coil [Kcoil]) sequence results in heterodimeric coiled-coil formation in vivo and in the capture of the enzyme in active form on the surface of the inclusion body particle. These round-shaped enzyme-decorated microparticles, with sizes of approximately 0.7 mum, can be isolated from lysed cells simply by centrifugation. The cost-effective one-step generation and isolation of enzymes immobilized on inclusion body particles may become useful for various applications in bioprocessing and biotransformation.
Steinmann
Björn
B
Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
Christmann
Andreas
A
Heiseler
Tim
T
Fritz
Janine
J
Kolmar
Harald
H
D000217:Acyltransferases / Q000096:biosynthesis* / Q000235:genetics; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D004800:Enzymes, Immobilized / Q000096:biosynthesis / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D005692:Galactose Oxidase / Q000096:biosynthesis* / Q000235:genetics; D002479:Inclusion Bodies / Q000201:enzymology*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D005692:Galactose Oxidase; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase