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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...
read more >>
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...
read more >>
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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Scientific publications

Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations.

Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations.
14 July 2014
Zhao J, Kardashliev T, Joelle Ruff A, Bocola M, Schwaneberg U
Biotechnol Bioeng 2014

Diversity generation by random mutagenesis is often the first key step in directed evolution experiments and screening of 1,000-2,000 clones is in most directed evolution campaigns sufficient to identify improved variants. For experimentalists important questions such as how many positions are mutated in the targeted gene and what amino acid substitutions can be expected after screening of 1,000-2,000 clones are surprisingly not answered by a statistical analysis of mutant libraries.

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.

Review: To get what we aim for - progress in diversity generation methods

Review: To get what we aim for - progress in diversity generation methods
5 June 2013
Ruff AJ, Dennig A, Schwaneberg U
FEBS J., 280 (2013), 2961-2978

Protein re-engineering by directed evolution has become a standard approach for tailoring enzymes in many fields of science and industry. Advances in screening formats and screening systems are fueling progress and enabling novel directed evolution strategies, despite the fact that the quality of mutant libraries can still be improved significantly.

Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics

Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics
20 June 2013
Arango Gutierrez E, Mundhada H, Meier T, Duefel H, Bocola M, Schwaneberg U
Biosensors and Bioelectronics 50 (2013) 84–90

Glucose oxidase is an oxidoreductase exhibiting a high β-d-glucose specificity and high stability which renders glucose oxidase well-suited for applications in diabetes care. Nevertheless, GOx activity is highly oxygen dependent which can lead to inaccuracies in amperometric β-d-glucose determinations. Therefore a directed evolution campaign with two rounds of random mutagenesis (SeSaM followed by epPCR), site saturation mutagenesis studies on individual positions, and one simultaneous site saturation library (OmniChange; 4 positions) was performed.

SeSaM-Tv-II generates a protein sequence space that is unobtainable by epPCR

SeSaM-Tv-II generates a protein sequence space that is unobtainable by epPCR
4 July 2011
Mundhada H, Marienhagen J, Scacioc A, Schenk A, Roccatano D, Schwaneberg U
Chembiochem. 2011 Jul 4;12 (10)

Generating high-quality mutant libraries in which each amino acid is equally targeted and substituted in a chemically diverse manner is crucial to obtain improved variants in small mutant libraries. The sequence saturation mutagenesis method (SeSaM-Tv(+)) offers the opportunity to generate such high-quality mutant libraries by introducing consecutive mutations and by enriching transversions.

dRTP and dPTP a complementary nucleotide couple for the Sequence Saturation Mutagenesis (SeSaM) method

dRTP and dPTP a complementary nucleotide couple for the Sequence Saturation Mutagenesis (SeSaM) method
26 April 2012
Ruff AJ, Marienhagen J, Verma R, Roccatano D, Genieser H-G, Niemann P, Shivange AV, Schwaneberg U
Journal of Molecular Catalysis B: Enzymatic 84 (2012) p. 40-47

Methods to generate random mutant libraries in directed evolution are limited in functional diversity generation. The Sequence Saturation Mutagenesis (SeSaM) method was reported as a four step random mutagenesis method overcoming the limitations of epPCR based mutagenesis methods. SeSaM targets in contrast to epPCR each nucleotide “equally” avoiding mutagenic hot spots, achieving subsequent mutations in a codon (up to 37.1%), and allowing to adjust mutational biases through employed universal bases.

Enzyme aus Metagenomen für die Biokatalyse in ionischen Flüssigkeiten

Enzyme aus Metagenomen für die Biokatalyse in ionischen Flüssigkeiten
10 January 2010 - 11 January 2010
N. Ilmberger, J. Pottkämper, W. R. Streit
Chemie Ingenieur Technik (2010) 82(1-2), 77-80

Ionische Flüssigkeiten (ILs) sind neue und chemisch inerte Lösungsmittel, von denen einige die Eigenschaft besitzen, Cellulose zu lösen. Allerdings sind keine in ILs stabile und aktive Cellulasen bekannt. In dieser Arbeit wurden metagenomische Banken durchmustert und die 24 resultierenden Klone auf ihre Aktivität und Stabilität in ILs getestet. Drei Enzyme zeigten moderate Aktivität in 30 % 1-Butyl-1-methyl-pyrrolidinium-trifluormethansulfonat.

Applying metagenomics for the identification of bacterial cellulases that are stable in ionic liquids

Applying metagenomics for the identification of bacterial cellulases that are stable in ionic liquids
10 December 2009 - 11 December 2009
Pottkämper J, Barthen P, Ilmberger N, Schwaneberg U, Schenk A, Schulte M, Ignatiev N, Streit WR
Green Chem. (2009) 11, 957 – 965

Ionic liquids (ILs) are novel and chemically inert solvents for a wide range of reactions in organic synthesis and biocatalysis, and at least one of them is known to dissolve cellulose. ILs would provide novel options for cellulose degradation in homogenous catalysis if cellulases were sufficiently stable and active. By screening metagenomic libraries 24 novel cellulase clones were identified and tested for their performance in the presence of ILs. Most enzyme clones showed only very poor or no activities. Three enzyme clones (i.e.

Advances in generating functional diversity for directed protein evolution

Advances in generating functional diversity for directed protein evolution
10 January 2009 - 11 January 2009
Shivange AV, Marienhagen J, Mundhada H, Schenk A, Schwaneberg U
Curr Opin Chem Biol. (2009) 13(1):19-25

Despite advances in screening technologies, only a very small fraction of theoretical protein sequence can be sampled in directed evolution experiments. At the current state of random mutagenesis technologies mutation frequencies have often been adjusted to values that cause a limited number of amino acid changes (often one to four amino acid changes per protein). For harvesting the power of directed evolution algorithms it is therefore important that generated mutant libraries are rich in diversity and enriched in active population.

Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+): a random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error-prone PCR

Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+): a random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error-prone PCR
10 January 2008 - 11 January 2008
Wong TS, Roccatano D, Loakes D, Tee KL, Schenk A, Hauer B, Schwaneberg U
Biotechnol J. (2008) 3(1):74-82

The sequence saturation mutagenesis (SeSaM) method has been advanced to a random mutagenesis method with adjustable mutational biases. SeSaM offers, for example, a bias that is complementary to error-prone (ep) PCR and is enriched in transversions (SeSaM-Tv(+)). dNTP alpha S and three degenerate bases (P, K and I) are used to control mutational bias flexibly.

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