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Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification

Paper ID Volume ID Publish Year Pages File Format Full-Text
23703 43465 2012 8 PDF Available
Title
Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification
Abstract

An integrated bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed in this study had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography column while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange media, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level at 871 U/g DCW, of which more than 90% was localized in the extracellular medium. In addition, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. The formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. Clarified culture medium was applied to a strong anion-exchange (Q) column and PAC was purified by non-retentive separation, where most contaminant proteins bound to the chromatographic media with PAC being collected as the major component in the flow-through fraction. After removing the contaminant oligopeptides using ultrafiltration, purified PAC with a specific activity of 16.3 U/mg was obtained and the overall purification factor for this one-step downstream purification process was up to 3 fold.

► A novel bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. ► A genetically engineered Escherichia coli strain was constructed for extracellular production of PAC. ► Cultivation medium was optimized to sustain cell growth and high-level production and effective secretion PAC. ► Cell-free medium was loaded directly for purification using an anion-exchange column with excellent purification performance.

Keywords
Anion-exchange chromatography; Escherichia coli; Extracellular secretion; Outer-membrane mutant; Penicillin G acylase
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Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 161, Issue 1, 15 September 2012, Pages 19–26
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