Engineered DyP Peroxidase for Bacterial Oxidation Yield
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Solution Overview
Problem
The challenge lies in efficiently expressing heavily-glycosylated horse radish peroxidase (HRP) in bacterial systems for industrial applications, as it is difficult to produce high-yielding oxidase reactions using conventional methods.
Innovation Solution
Engineering DyP-type peroxidase enzymes with specific polypeptide sequences derived from Anabaena (Nostoc) sp. (strain PCC 7120) that can oxidize 10-acetyl-3,7-dihydroxyphenoxazine using hydrogen peroxide as an electron donor, and providing polynucleotides, vectors, and host cells to facilitate production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If horse radish peroxidase (HRP) is used for oxidation reactions, then high catalytic activity is achieved, but difficult expression in bacterial systems occurs due to heavy glycosylation
Solution Approach 1:
The patent creates a bacterial expression system that copies the essential catalytic function of HRP using a simplified protein structure (DyP peroxidase from Anabaena) that does not require heavy glycosylation, thereby achieving HRP-like activity while being easily expressible in bacterial systems
Solution Approach 2:
The patent changes the protein structure parameters by replacing the heavily-glycosylated HRP structure with a non-glycosylated DyP peroxidase structure, maintaining catalytic functionality while eliminating the expression barrier in bacterial systems
2Quantity of substance
If conventional HRP expression methods are used, then peroxidase enzyme is produced, but low reaction yield occurs due to expression difficulties
Solution Approach 1:
The patent employs a self-service approach by using the bacterial cell's own protein synthesis machinery to produce the DyP peroxidase enzyme, eliminating the need for complex eukaryotic expression systems and achieving high-yield enzyme production directly in bacterial systems
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The engineered DyP peroxidases exhibit improved activity on substrates like 10-acetyl-3,7-dihydroxyphenoxazine, offering a viable alternative to HRP with enhanced expression and reaction yield.
Implementation Method 1
engineered peroxidases comprising polypeptide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4 and/or 6... capable of oxidation of 10-acetyl-3,7-dihydroxyphenoxazine using hydrogen peroxide as the electron donor
Implementation Method 2
The enzyme is a haem containing protein that catalyzes the oxidation of a number of substrates using hydrogen peroxide as the electron donor
Data Source
AI summary
The present invention provides engineered peroxidase enzymes, polypeptides having peroxidase activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing peroxidase enzymes are also provided. The present invention further provides compositions comprising the peroxidase enzymes and methods of using the engineered peroxidase enzymes. The present invention finds particular use in the production of pharmaceutical and other compounds.