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Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
3863 196 2010 6 PDF Available
Title
Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli
Abstract

The production of a novel thermostable amidase (EC 3.5.1.4) from Geobacillus pallidus RAPc8 using recombinant Escherichia coli BL21 (DE3) was investigated. Volumetric and specific enzyme activities were investigated in relation to inducer concentration in a batch process using a defined medium with glucose as the carbon source. While IPTG is routinely used to induce expression of genes under the control of lac promoter, the impact of high biomass concentration on IPTG induction has not been reported rigorously. In this study, biomass production was unaffected by IPTG concentration across the range 0–1000 μM. Induction of recombinant protein expression by 400 μM IPTG at late lag phase of growth (3rd hour) inhibited cell growth while induction at early exponential phase of growth (5th hour) gave a 3 fold increase in volumetric amidase activity compared to induction at mid exponential phase (8th hour). Protein production increased by a factor of two with IPTG addition, independent of IPTG concentration in the range of 40–1000 μM. Amidase activity, measured on a volumetric basis and relative to protein and biomass concentrations, increased with increasing IPTG concentration up to 400 μM. While inducer concentrations are typically reported on a volumetric basis, their mode of action is consistent with a biomass dependence. Analysis of the data across a range of biomass concentration confirmed that induction was a function of inducer concentration per unit biomass. The amidase enzyme was predominantly soluble and cytoplasmic with less than 3% retained within the cell debris.

Keywords
Thermostable amidase; Iso-propyl-β-d-1-thiogalactopyranoside (IPTG); Volumetric amidase activity; Heterologous protein expression
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Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 52, Issue 1, 15 October 2010, Pages 19–24
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