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High level expression of a hydrophobic poly(ethylene terephthalate)-hydrolyzing carboxylesterase from Thermobifida fusca KW3 in Escherichia coli BL21(DE3)

Paper ID Volume ID Publish Year Pages File Format Full-Text
24453 43516 2010 5 PDF Available
Title
High level expression of a hydrophobic poly(ethylene terephthalate)-hydrolyzing carboxylesterase from Thermobifida fusca KW3 in Escherichia coli BL21(DE3)
Abstract

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 His6 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 41.6 mg/L was achieved.

Keywords
CAI, codon adaption index; IPTG, isopropyl-β-d-thiogalactopyranoside; LB, Luria–Bertani medium; PET, poly(ethylene terephthalate); p-NPB, p-nitrophenyl butyrate; TfCa, carboxylesterase from Thermobifida fusca KW3; Tris, tris(hydroxymethyl)aminomethane; w/
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High level expression of a hydrophobic poly(ethylene terephthalate)-hydrolyzing carboxylesterase from Thermobifida fusca KW3 in Escherichia coli BL21(DE3)
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 146, Issue 3, 1 April 2010, Pages 100–104
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us