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Autodisplay of functional CYP106A2 in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
23791 43473 2012 9 PDF Available
Title
Autodisplay of functional CYP106A2 in Escherichia coli
Abstract

Cytochrome P450 enzymes catalyse a wide variety of reactions, including the hydroxylation and epoxidation of CC bonds, and dealkylation reactions. There is high interest in these reactions for biotechnology and pharmaceutical processes. Many P450s require membrane surroundings and have substrates that do not cross biological membranes. To circumvent these obstacles, CYP106A2 from Bacillus megaterium was expressed on the outer membrane of Escherichia coli cells by Autodisplay. Exposure on the surface was confirmed by a protease accessibility test and flow cytometry after immunolabelling. HPLC assays showed that 0.5 ml of cells displaying the enzyme (OD578 = 6) converted 9.13 μmol of deoxycorticosterone to 15β-OH-deoxycorticosterone within 1 h. Imipramine and abietic acid were also accepted as substrates. The number of active enzyme molecules per cell was calculated to be 20,000. Surprisingly, surface-exposed CYP106A2 was active in E. coli BL21 without the external addition of the heme group. However, when CYP106A2 was expressed on the surface of an E. coli strain lacking the TolC channel protein (JW5503), enzymatic activity was almost completely abolished. The activity of CYP106A2 on the surface of E. coli JW5503 could be restored by the external addition of the heme group. This suggests, as has been reported before, that E. coli uses a TolC-dependent mechanism to export heme into the growth media, where it can be scavenged by a surface-displayed apoenzyme. Our results indicate that Autodisplay enables the functional surface display of P450 enzymes and provides a new platform to access their synthetic potential.

► The P450 enzyme CYP106A2 was displayed on the surface of E. coli. CYP106A2 was transported to the cell surface as apo-protein by Autodisplay and heme was incorporated after transport. ► The heme group was provided by the host cell and exported to the supernatant by TolC. ► The displayed CYP106A2 could be used for the conversion of deoxycorticosterone, abietic acid and imipramine.

Keywords
Autodisplay; Cytochrome; P450 106A2; Biocatalysis; Surface display; Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 161, Issue 2, 15 October 2012, Pages 104–112
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