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News

Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations.
14 July 2014
Zhao J, Kardashliev T, Joelle
Biotechnol Bioeng 2014
Diversity generation by random mutagenesis is often the first key step in directed evolution...
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Bacillus gibsonii alkaline protease
1 November 2012
Martinez R, Jakob F, Tu R, Sie
Biotechnol Bioeng. 2013 Mar
Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting...
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Review: To get what we aim for - progress in diversity generation methods
5 June 2013
Ruff AJ, Dennig A, Schwaneberg
FEBS J., 280 (2013), 2961-2978
Protein re-engineering by directed evolution has become a standard approach for tailoring enzymes...
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Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics
20 June 2013
Arango Gutierrez E, Mundhada H
Biosensors and Bioelectronics
Glucose oxidase is an oxidoreductase exhibiting a high β-d-glucose specificity and high stability...
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Bacillus gibsonii alkaline protease

Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution.
1 November 2012
Martinez R, Jakob F, Tu R, Siegert P, Maurer KH, Schwaneberg U
Biotechnol Bioeng. 2013 Mar

Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (K(cat)) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5-fold increased specific activity (15°C) and over 100 times prolonged half-life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity-altering amino acid substitutions were from non-charged to non-charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions.