TY  - THES
U1  - Dissertation oder Habilitation
A1  - Zhu, Ziwei
T1  - Making glucose oxidase fir for biofuel cell applications by directed protein evolution
N2  - Inspired by nature Darwinian evolution, directed evolution involves mutation,recombination and screening or selection to accumulate the mutants required to achieve significant change in protein function. The gene encoding gox harboring á-pheromone signal sequence from Aspergillus niger was expressed as secretary product in yeast Saccharomyces cerevisiae. Error prone PCR has been used to generate mutant libraries of GOx by adjusting the concentration of MnCl2. 4000 mutants have been screened for catalytic activity and thermostability. One mutant (G4) showed two-fold higher activity than WT and an enhanced thermostability. For the screening of mutants, two novel and reliable assays were developed for medium-throughput screening. Glucose oxidase detection assay (GODA) and ferroecenemethanol based assay were developed for improving GOx properties by directed protein evolution. GODA is a reaction product detection assay based on coupled enzymatic reactions leading to NADPH formation which is recorded at 340 nm. The main advantage of the assay is that it detects the production of D-gluconolactone instead of the side-product hydrogen peroxide and enables to improve bioelectrochemical properties of GOx. Ferrocenemethanol based assay can directly screen electron transfer between oxidized form of ferrocenemethanol and reduced form of GOx by monitoring colorimetric development. It is a simple, less expensive and rapid assay, more importantly; it is insensitive to nature electron acceptor dioxygen in pH 8.
AB  - Inspired by nature Darwinian evolution, directed evolution involves mutation,recombination and screening or selection to accumulate the mutants required to achieve significant change in protein function. The gene encoding gox harboring á-pheromone signal sequence from Aspergillus niger was expressed as secretary product in yeast Saccharomyces cerevisiae. Error prone PCR has been used to generate mutant libraries of GOx by adjusting the concentration of MnCl2. 4000 mutants have been screened for catalytic activity and thermostability. One mutant (G4) showed two-fold higher activity than WT and an enhanced thermostability. For the screening of mutants, two novel and reliable assays were developed for medium-throughput screening. Glucose oxidase detection assay (GODA) and ferroecenemethanol based assay were developed for improving GOx properties by directed protein evolution. GODA is a reaction product detection assay based on coupled enzymatic reactions leading to NADPH formation which is recorded at 340 nm. The main advantage of the assay is that it detects the production of D-gluconolactone instead of the side-product hydrogen peroxide and enables to improve bioelectrochemical properties of GOx. Ferrocenemethanol based assay can directly screen electron transfer between oxidized form of ferrocenemethanol and reduced form of GOx by monitoring colorimetric development. It is a simple, less expensive and rapid assay, more importantly; it is insensitive to nature electron acceptor dioxygen in pH 8.
KW  - glucose oxidase
KW  - directed evolution
Y1  - 2006
U6  - https://nbn-resolving.org/urn:nbn:de:101:1-201305225938
UN  - https://nbn-resolving.org/urn:nbn:de:101:1-201305225938
ER  -