Dear Priya,

The following publications cover the answer to your question:

1-Journal of Environmental Biology

Optimization of the whole-cell catalytic activity of

recombinant Escherichia coli cells with surface-immobilized

organophosphorus hydrolase

Hongxing Zhang, Qianqian Li, Ting Ye, Zhen Zhang and Lin Li*

State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology,

Huazhong Agricultural University, Wuhan, 430070, China

*Corresponding Author email : [email protected]

© Triveni Enterprises, Lucknow (India ) Journal of Environmental Biology, Vol. 34, 315-319, April 2013

ISSN: 0254-8704

CODEN: JEBIDP J

Abstract

Previous studies have verified the feasibility of using Escherichia coli systems that display organophosphorous hydrolase (OPH) on the cell surface as whole­cell catalysts. However, the inefficient display of the enzyme on cell surfaces remains unaddressed. In the present study, multiple optimization experiments on full­length and truncated ice nucleation protein anchors, E. coli host cells, culture media, and culture conditions were performed to optimize whole­cell OPH enzymatic activity. The results show that apart from the dramatic effect of isopropyl­­D­thiogalactoside

concentration and culture temperature, the coordination between the anchor protein, culture media, and host cells is essential for highly efficient OPH display. Under optimal conditions, namely, culturing in M9 medium, 20 °C induction temperature, 0.1 mmol l­1 IPTG, and 100 μmol l­1 Co2+, the engineered E. coli strain MB109­406 that expresses the fusion enzyme InaK­N­OPH exhibited a wholecell

OPH activity of 0.62 U mg­1 × cell d.wt. This result is much higher than that of several currently available OPH­displaying systems, which shows the potential of the current system for further largescale industrial or environmental applications.

Key words Cell surface display, Organophosphorus hydrolase, Optimization, Ice nucleation protein, Escherichia col

2-Jundishapur J Microbiol. 2015 April; 8(4): e16236. DOI: 10.5812/jjm.8(4)2015.16236

Published online 2015 April 18. Research Article

Optimization of Fermentation Conditions for Recombinant Human

Interferon Beta Production by Escherichia coli Using the Response

Surface Methodology

Mohammad Hossein Morowvat 1,2; Valiollah Babaeipour 3,4,*; Hamid Rajabi Memari 5 ;

Hossein Vahidi 1

Background: The periplasmic overexpression of recombinant human interferon beta (rhIFN-β)-1b using a synthetic gene in Escherichia coli BL21 (DE3) was optimized in shake flasks using Response Surface Methodology (RSM) based on the Box-Behnken Design (BBD).

Objectives: This study aimed to predict and develop the optimal fermentation conditions for periplasmic expression of rhIFN-β-1b in shake flasks whilst keeping the acetate excretion as the lowest amount and exploit the best results condition for rhIFN-β in a bench top bioreactor.

Materials and Methods: The process variables studied were the concentration of glucose as carbon source, cell density prior the induction (OD 600 nm) and induction temperature. Ultimately, a three-factor three-level BBD was employed during the optimization process. The rhIFN-β production and the acetate excretion served as the evaluated responses. Results: The proposed optimum fermentation condition consisted of 7.81 g L-1 glucose, OD 600 nm prior induction 1.66 and induction temperature of 30.27°C. The model prediction of 0.267 g L-1 of rhIFN-β and 0.961 g L-1 of acetate at the optimum conditions was verified experimentally as 0.255 g L-1 and 0.981 g L-1 of acetate. This agreement between the predicted and observed values confirmed the precision of the applied method to predict the optimum conditions.

Conclusions: It can be concluded that the RSM is an effective method for the optimization of recombinant protein expression using synthetic genes in E. coli.\

Keywords: Periplasmic; Acetic acid; Escherichia coli; Interferon-beta

Hoping this will be helpful,

Rafik

There are several phenomena to consider when comparing enzyme in whole cell extracts to purified enzyme:

1. Differences in the composition of the buffer (e.g. salt type and concentration, pH) can affect the temperature optimum.

2. If the enzyme has any binding partners in the whole cell extract that are not present in the purified enzyme, there could be an effect on the temperature optimum.

3. If the whole cell extract contains enzymes other than the one that is being studied but that have measurable activity in the enzyme assay (i.e. background), but with a different temperature optimum, the results could be affected.

Thankyou all for your suggestions ....I will take them into account for planning my experiments..