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2020 ◽  
Vol 21 (1) ◽  
pp. 335 ◽  
Author(s):  
Benjamin Begander ◽  
Anna Huber ◽  
Manuel Döring ◽  
Josef Sperl ◽  
Volker Sieber

Successful directed evolution examples span a broad range of improved enzyme properties. Nevertheless, the most challenging step for each single directed evolution approach is an efficient identification of improved variants from a large genetic library. Thus, the development and choice of a proper high-throughput screening is a central key for the optimization of enzymes. The detection of low enzymatic activities is especially complicated when they lead to products that are present in the metabolism of the utilized genetic host. Coupled enzymatic assays based on colorimetric products have enabled the optimization of many of such enzymes, but are susceptible to problems when applied on cell extract samples. The purpose of this study was the development of a high-throughput screening for D-glycerate dehydratase activity in cell lysates. With the aid of an automated liquid handling system, we developed a high-throughput assay that relied on a pre-treatment step of cell extract prior to performing the enzymatic and assay reactions. We could successfully apply our method, which should also be transferable to other cell extract-based peroxidase assays, to identify an improved enzyme for the dehydration of D-glycerate.


Cell Systems ◽  
2018 ◽  
Vol 6 (2) ◽  
pp. 153-155
Author(s):  
Julien Lagarde ◽  
Rory Johnson
Keyword(s):  

Yeast ◽  
2011 ◽  
Vol 28 (3) ◽  
pp. 237-252 ◽  
Author(s):  
Juergen H. Nett ◽  
Terrance A. Stadheim ◽  
Huijuan Li ◽  
Piotr Bobrowicz ◽  
Stephen R. Hamilton ◽  
...  

2009 ◽  
Vol 18 (2) ◽  
pp. 200-203 ◽  
Author(s):  
Courtney Sebold ◽  
Christine Koil

2009 ◽  
Vol 18 (2) ◽  
pp. 384-398 ◽  
Author(s):  
Mariejoy Therese Jumawid ◽  
Tsuyoshi Takahashi ◽  
Toshimasa Yamazaki ◽  
Hiroshi Ashigai ◽  
Hisakazu Mihara

Genomics ◽  
2006 ◽  
Vol 88 (6) ◽  
pp. 841-845 ◽  
Author(s):  
Xingxing Zang ◽  
P'ng Loke ◽  
Jayon Kim ◽  
Kathleen Wojnoonski ◽  
Leonard Kusdra ◽  
...  

2005 ◽  
Vol 301 ◽  
pp. 9-22 ◽  
Author(s):  
RK Naviaux ◽  
B Good ◽  
JD McPherson ◽  
DL Steffen ◽  
D Markusic ◽  
...  
Keyword(s):  

2004 ◽  
Vol 13 (5) ◽  
pp. 447-452
Author(s):  
Clara Gaff
Keyword(s):  

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