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Content archived on 2024-04-30

Towards designer peroxidases: application of protein engineering techniques to the design of novel peroxidase biocatalysts


Peroxidases are of strategic importance to industry because of their numerous applications, including: biosensor construction, diagnostics, food processing, remediation of waste water, decolourization of pulp paper and textiles so avoiding the use of chlorine, and in dye transfer inhibition (bio bleaching).
This project's objective is to design 'improved' peroxidase enzymes by a combination of protein engineering and chemical modification techniques with: (1) increased stability towards heat, organic solvents and denaturants. (2) greater resistance to catalytic inactivation and damage by radical products. (3) enhanced activity during steady-state catalytic and chemiluminescent turnover.
We will study four peroxidases with important commercial applications or potential: The most used cationic horseradish peroxidase (HRP C), Coprinus cinereus peroxidase (CIP), soybean peroxidase (SBP) and anionic horseradish peroxidase (HRP A2).
Scientifically the project will: (1) study the factors at the protein level which control the stability and unfolding of these enzymes, in particular the role of Ca in stabilising enzyme structures.
(2) Determine new peroxidase crystal structures. (3) Determine the structure of their high oxidation state intermediates using fast x-ray /
Microspectrophotometer methodology. resonance raman and NMR spectroscopy will support these investigations (1)-(3).
The industrial participants, Ortho-Clinical Diagnostics Ltd and Novo Nordisk A/S, desire improved peroxidases for diagnostic and industrial enzyme applications. For new peroxidases which are found to offer a significant performance advantage, a business evaluation will be completed to assess the overall benefit of replacing current reagents with those based on the new `engineered' peroxidases.
The project will have the following objectives:
. More stable variant forms of HRP C and CIP obtained by a combination of protein engineering and chemical modification.
. Peroxidase variants with increased specific activities and enhanced chemiluminescent turnover.
. Crystal and solution structures of SBP and HRP A2.
. Structure and decay of peroxidase intermediates.
. Development of advanced time-resolved crystallographic methods. . Insight into enzyme stabilization by calcium ion.

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Ëlborg Universitet
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SohngÄrdsholmsvej 57
9000 Ëlborg

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Participants (7)