High level expression of a hydrophobic poly(ethylene terephthalate)-hydrolyzing carboxylesterase from Thermobifida fusca KW3 in Escherichia coli BL21(DE3).
The gram-positive thermophilic actinomycete Thermobifida fusca KW3 secretes a highly hydrophobic carboxylesterase (TfCa) that is able to hydrolyze poly(ethylene terephthalate). TfCa was produced in the Escherichia coli strain BL21(DE3) as a fusion protein consisting of a pelB leader sequence to ensure periplasmic localization of the protein and a His(6) tag for use in its purification. To enhance the recombinant enzyme yield, the tfca gene from T. fusca KW3 was successfully optimized for codon usage in E. coli. In addition, the gene expression induction conditions were optimized and the temperature for cell cultivation was lowered to reduce inclusion body formation. The optimized codons and expression conditions yielded 4500-fold higher TfCa activity than the wild-type strain. Using a pH-controlled bioreactor for cultivation, a TfCa protein concentration of 41.6mg/L was achieved.
Department of Microbiology and Bioprocess Technology, University of Leipzig, Johannisallee 21-23, D-4103 Leipzig, Germany.
Thorsten
T
Oeser
Ren
R
Wei
Thomas
T
Baumgarten
Susan
S
Billig
Christina
C
Föllner
Wolfgang
W
Zimmermann
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D043182:Carboxylesterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods*; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D011092:Polyethylene Glycols / Q000737:chemistry*; D011093:Polyethylene Terephthalates; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*
D011093:Polyethylene Terephthalates; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D043182:Carboxylesterase