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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Making glucose oxidase fit for biofuel cell applications by directed protein evolution
Ziwei Zhu1, Carmen Momeu, Maxim Zakhartsev
1International University Bremen (IUB), Campus Ring 1, 28759 Bremen, Germany.
Biosensors & Bioelectronics
|January 4, 2006
Summary
Researchers developed a new assay to improve glucose oxidase for biofuel cells. Directed evolution yielded a mutant with 1.5-fold higher activity, enhancing power for medical devices.
Area of Science:
- Biotechnology
- Bioengineering
- Enzyme Engineering
Background:
- Miniaturized chip design necessitates advanced implantable power sources for healthcare applications like continuous glucose monitoring.
- Enzymatic biofuel cells (mBCs) convert glucose to electrical energy, but their power output and lifetime are limited by enzyme performance, particularly glucose oxidase (GOx).
Purpose of the Study:
- To develop a screening system for improving glucose oxidase (GOx) properties through directed protein evolution.
- To enhance the bioelectrochemical performance of GOx for increased power output and lifetime in miniaturized biofuel cells (mBCs).
Main Methods:
- Developed a glucose oxidase detection assay (GODA) based on coupled enzymatic reactions measuring NADPH formation at 340 nm.
- Generated a mutagenic library of Aspergillus niger GOx and screened for improved activity in Saccharomyces cerevisiae using the GODA system.
- Validated the screening system by identifying a GOx mutant with enhanced kinetic properties.
Main Results:
- The GODA system effectively detects d-gluconolactone, enabling the improvement of GOx bioelectrochemical properties.
- Directed evolution identified a GOx mutant (I115V) with 1.4-1.5-fold increased activity for beta-d-glucose and improved oxygen consumption kinetics.
- The evolved GOx mutant exhibited Vmax values of 10.81 micromol min(-1) mg(-1) for beta-d-glucose and 8.34 micromol min(-1) mg(-1) for oxygen.
Conclusions:
- The developed mutagenic protocol and GODA assay provide a proof-of-principle for evolving GOx using directed evolution in Saccharomyces cerevisiae.
- This approach can significantly improve GOx performance, paving the way for more efficient and durable biofuel cells for medical applications.
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