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Extracellular production of Pseudozyma (Candida) antarctica lipase B with genuine primary sequence in recombinant Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
20038 43154 2016 7 PDF Available
Title
Extracellular production of Pseudozyma (Candida) antarctica lipase B with genuine primary sequence in recombinant Escherichia coli
Abstract

•Pseudozyma (Candida) antarctica lipase B (CALB) was produced extracellularly by E. coli.•The target in the culture supernatant amounted to 550 mg/L.•5.67 mg of the purified CALB was obtained from 50 mL culture.•The structural and catalytic integrity of the recombinant CALB was confirmed.•This is the first example of E. coli-based extracellular production of CALB with the correct primary sequence.

An Escherichia coli expression system was established to produce recombinant extracellular Pseudozyma (Candida) antarctica lipase B (CALB). With the aim of producing the genuine CALB without additional amino acid residues, the mature portion of the CALB gene was fused seamlessly to a pelB signal sequence and expressed in E. coli BL21(DE3) using the pET system. Inducing gene expression at low temperature (20°C) was crucial for the production of active CALB; higher temperatures caused inclusion body formation. Prolonged induction for 48 h at 20°C allowed for the enzyme to be released into the culture medium, with more than half of the activity detected in the culture supernatant. A catalytically inactive CALB mutant (S105A) protein was similarly released, suggesting that the lipid-hydrolyzing activity of the enzyme was not the reason for the release. The CALB production level was further improved by optimizing the culture medium. Under the optimized conditions, the CALB in the culture supernatant amounted to 550 mg/L. The recombinant CALB was purified from the culture supernatant, yielding 5.67 mg of purified CALB from 50 mL of culture. N-terminal sequencing and ESI-MS analyses showed proper removal of the pelB signal sequence and the correct molecular weight of the protein, respectively, confirming the structural integrity of the recombinant CALB. The kinetic parameters towards p-nitrophenylbutyrate and the enantiomeric selectivity on rac-1-phenylethylacetate of the recombinant CALB were consistent with those of the authentic CALB. This is the first example of E. coli-based extracellular production of a CALB enzyme without extra amino acid residues.

Keywords
Recombinant Escherichia coli; Extracellular production; Pseudozyma (Candida) antarctica lipase B; pelB signal sequence; BL21(DE3)
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Extracellular production of Pseudozyma (Candida) antarctica lipase B with genuine primary sequence in recombinant Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 121, Issue 3, March 2016, Pages 303–309
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