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Ultrahigh-throughput screening system for directed glucose oxidase evolution in yeast cells.
MedLine Citation:
PMID:  20958255     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
A compartmentalized tyramid labeling system (CoaTi) employing flow cytometry for sorting of yeast cells was developed as ultrahigh-throughput screening for Glucose oxidase (GOx) from Aspergillus niger. CoaTi combines in vitro compartmentalization technology with the CARD reporter system which uses fluorescein tyramide labels for detection of peroxidase activity. Physical connection between cells and fluorescein tyramide radicals was achieved by compartmentalization of yeast cells inside microdroplets of single water-in-oil emulsions. After reaction cells were recovered from single emulsions and sorted by flow cytometry, an error prone PCR mutant library of Glucose oxidase (GOx) containing 10(7) cells and ~10(5) of different GOx variants was screened. Mutagenic conditions of GOx mutant library were selected to generate <1 % of active GOx population in order to explore influence of high mutation frequency on GOx activity. GOx variant Mut12 that contains 5 mutations (N2Y, K13E, T30V, I94V, K152R) showed a 1.2 times decreased K(m) (22.0 vs 18.1 mM) and a 2.7 fold increased k(cat) (150 s(-1) vs 54.8 s(-1)) compared to wt GOx. Compared to the employed parent B11 GOx (16 mM, 80 s(-1)) it has a slightly increased K(m) and 1.8 times increased k(cat).
Authors:
Radivoje Prodanovic; Raluca Ostafe; Andreea Scacioc; Ulrich Schwaneberg
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Combinatorial chemistry & high throughput screening     Volume:  14     ISSN:  1875-5402     ISO Abbreviation:  Comb. Chem. High Throughput Screen.     Publication Date:  2011 Jan 
Date Detail:
Created Date:  2011-01-17     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9810948     Medline TA:  Comb Chem High Throughput Screen     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  55-60     Citation Subset:  IM    
Affiliation:
RWTH Aachen University, Lehrstuhl für Biotechnologie, Worringer Weg 1, 52056 Aachen, Germany. u.schwaneberg@biotec.rwth-aachen.de.
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