- Email: [email protected]
Lessons on directed evolution of hydrolases and glucose oxidase
SYMPOSIUM 4: ROBUST BIOCATALYSTS FOR THE PRODUCTION OF NOVEL BIO-BASED PRODUCTS
immobilization methods to develop biofilm communities as well as their ability to perform biotransformations. References [1].Tsoligkas AN, Winn M, Bowen J, Overton TW, Simmons MJH, Goss RJM. Engineering biofilms for biocatalysis. ChemBioChem 2011;12:1391–5. [2].Tsoligkas AN, Bowen J, Winn M, Goss RJM, Overton TW, Simmons MJH. Characterisation of spin coated engineered Escherichia coli biofilms using atomic force microscopy. Colloids and Surfaces B: Biointerfaces 2012;89:152–60. [3].Perni S, Hackett L, Goss R, Simmons M, Overton T. Optimisation of engineered Escherichia coli biofilms for enzymatic biosynthesis of L-halotryptophans. AMB Express 2013;3:66.
http://dx.doi.org/10.1016/j.nbt.2014.05.1656
O4-4 Structural and biochemical characterization of two novel enzymes with promiscuous ene-reductase activity Tea Pavkov-Keller 1,∗ , Alexandra Binter 2 , Steinkellner Georg 1 , Christian C. Gruber 1 , Kerstin Steiner 2 , Christoph Winkler 3 , Helmut Schwab 4 , Kurt Faber 3 , Peter Macheroux 5 , Karl Gruber 6 1
ACIB GmbH, C/o ZMB, Austria 2 ACIB GmbH, Austria 3 Department of Chemistry, University of Graz, Austria 4 Institute of Molecular Biotechnology, Graz University of Technology, Austria 5 Institute of Biochemistry, Graz University of Technology, Austria 6 Institute of Molecular Biosciences, University of Graz, Austria
An approach using three-dimensional motifs reflecting specific active site arrangements (catalophore) was developed in our group. It does not depend on overall protein similarity and therefore enables the search across enzyme families and the detection of potential catalytic promiscuity. This catalophore approach led to the discovery of two novel enzymes with ene-reductase activity. Enzymes of this family have recently been shown to possess a great potential for (industrial) biotransformations. Neither the amino acid sequence of these two enzymes nor their overall structure is related to those of the well-known old yellow enzymes (OYE). These two flavoproteins contain FMN as a cofactor and exist as homodimers. We cloned, expressed and purified both enzymes and subjected them to crystallization trials. Obtained crystals were soaked with putative substrates/inhibitors. The enzymatic characterization was pursued by stopped-flow and difference titration experiments. Additionally, several typical OYE substrates (i.e. alkenes bearing an electron-withdrawing activating group) were tested to assess the biocatalytic performance. The analysis showed some salient features of typical OYEs as well as some striking differences, i.e. a stereocomplementary behaviour. In conclusion, the two novel enzymes can be described as NADPH-dependent quinone reductases with significant OYE-like side activities. http://dx.doi.org/10.1016/j.nbt.2014.05.1657
S20
www.elsevier.com/locate/nbt
New Biotechnology · Volume 31S · July 2014
O4-5 Lessons on directed evolution of hydrolases and glucose oxidase Ulrich Schwaneberg RWTH – Aachen University, Chair of Biotechnology, Germany
Protein engineering by directed evolution and semi-rational design has become a standard method to tailor enzyme properties to industrial demands. Improving thermal stability and activity simultaneously is often challenging since high activity often requires flexibility whereas thermal resistance relies and ‘strong’ interactions within a protein. On the example of proteases (BgAP [1,2], S41 [3]) and a phytase [4,5], lessons learned from improving both properties individually and simultaneously will be presented. Subsequently, lessons on improving detergent and salt (ionic liquid) resistance of a protease (subtilisin E [6,7]) and a cellulase (CelA2 [8,9]) will conclude the hydrolase reengineering examples. As a highlight, the generation of oxygen independent and highly active glucose oxidase variants (GOx from A. niger) [10,11] will conclude the presentation. References [1]. Martinez R, et al. Biotechnol Bioeng 2013;110:711–20. [2]. Jakob F, et al. Appl Microbiol Biotechnol 2013;52:2359–63. [3]. Martinez R, et al. Protein Eng Des Sel 2011;24:533–44. [4]. Shivange A, et al. J Biotechnol 2014;170:68–72. [5]. Shivange AV, et al. Appl Microbiol Biotechnol 2012;95:405–18. [6]. Li Z, et al. ChemBioChem 2012;13:691–9. [7]. Li Z, et al. J Biotechnol 2014;169:87–94. [8]. Lehmann C, et al. Green Chem 2012;14:2719–3272. [9]. Pottkämper J, et al. Green Chem 2009;11:691–7. [10].Arango Gutierreza E, et al. Biosens Bioelectron 2013;50:84–90. [11].Prodanovic R, et al. Anal Bional Chem 2012;404:1439–47.
http://dx.doi.org/10.1016/j.nbt.2014.05.1658
O4-6 Immobilization of carbonic anhydrase for biomimetic CO2 capture in slurry absorber Sara Peirce 1,∗ , Maria Elena Russo 2 , Viviana De Luca 3 , Clemente Capasso 3 , Mosè Rossi 3 , Giuseppe Olivieri 4 , Piero Salatino 4 , Antonio Marzocchella 4 1
Dipartimento di Ingegneria Chimica dei Materiali e della Produzione Industriale – Università degli Studi di Napoli Federico II, Italy 2 Consiglio Nazionale delle Ricerche – Istituto di Ricerche sulla Combustione, Italy 3 Consiglio Nazionale delle Ricerche – Istituto di Bioscienze e Biorisorse, Italy 4 Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale – Università degli Studi di Napoli Federico II, Italy
Novel post-combustion treatments include biomimetic Carbon Capture and Storage (CCS) processes based on CO2 absorption into aqueous solution assisted by enzyme catalysis. Carbonic anhydrase (EC 4.2.1.1) catalyzes CO2 hydration and it has been proposed as industrial biocatalyst for biomimetic CCS processes [Lacroix and Larachi, 2008, Recent Patent Chem Eng; Russo et al., 2013, Sep Pur Technol].
immobilization methods to develop biofilm communities as well as their ability to perform biotransformations. References [1].Tsoligkas AN, Winn M, Bowen J, Overton TW, Simmons MJH, Goss RJM. Engineering biofilms for biocatalysis. ChemBioChem 2011;12:1391–5. [2].Tsoligkas AN, Bowen J, Winn M, Goss RJM, Overton TW, Simmons MJH. Characterisation of spin coated engineered Escherichia coli biofilms using atomic force microscopy. Colloids and Surfaces B: Biointerfaces 2012;89:152–60. [3].Perni S, Hackett L, Goss R, Simmons M, Overton T. Optimisation of engineered Escherichia coli biofilms for enzymatic biosynthesis of L-halotryptophans. AMB Express 2013;3:66.
http://dx.doi.org/10.1016/j.nbt.2014.05.1656
O4-4 Structural and biochemical characterization of two novel enzymes with promiscuous ene-reductase activity Tea Pavkov-Keller 1,∗ , Alexandra Binter 2 , Steinkellner Georg 1 , Christian C. Gruber 1 , Kerstin Steiner 2 , Christoph Winkler 3 , Helmut Schwab 4 , Kurt Faber 3 , Peter Macheroux 5 , Karl Gruber 6 1
ACIB GmbH, C/o ZMB, Austria 2 ACIB GmbH, Austria 3 Department of Chemistry, University of Graz, Austria 4 Institute of Molecular Biotechnology, Graz University of Technology, Austria 5 Institute of Biochemistry, Graz University of Technology, Austria 6 Institute of Molecular Biosciences, University of Graz, Austria
An approach using three-dimensional motifs reflecting specific active site arrangements (catalophore) was developed in our group. It does not depend on overall protein similarity and therefore enables the search across enzyme families and the detection of potential catalytic promiscuity. This catalophore approach led to the discovery of two novel enzymes with ene-reductase activity. Enzymes of this family have recently been shown to possess a great potential for (industrial) biotransformations. Neither the amino acid sequence of these two enzymes nor their overall structure is related to those of the well-known old yellow enzymes (OYE). These two flavoproteins contain FMN as a cofactor and exist as homodimers. We cloned, expressed and purified both enzymes and subjected them to crystallization trials. Obtained crystals were soaked with putative substrates/inhibitors. The enzymatic characterization was pursued by stopped-flow and difference titration experiments. Additionally, several typical OYE substrates (i.e. alkenes bearing an electron-withdrawing activating group) were tested to assess the biocatalytic performance. The analysis showed some salient features of typical OYEs as well as some striking differences, i.e. a stereocomplementary behaviour. In conclusion, the two novel enzymes can be described as NADPH-dependent quinone reductases with significant OYE-like side activities. http://dx.doi.org/10.1016/j.nbt.2014.05.1657
S20
www.elsevier.com/locate/nbt
New Biotechnology · Volume 31S · July 2014
O4-5 Lessons on directed evolution of hydrolases and glucose oxidase Ulrich Schwaneberg RWTH – Aachen University, Chair of Biotechnology, Germany
Protein engineering by directed evolution and semi-rational design has become a standard method to tailor enzyme properties to industrial demands. Improving thermal stability and activity simultaneously is often challenging since high activity often requires flexibility whereas thermal resistance relies and ‘strong’ interactions within a protein. On the example of proteases (BgAP [1,2], S41 [3]) and a phytase [4,5], lessons learned from improving both properties individually and simultaneously will be presented. Subsequently, lessons on improving detergent and salt (ionic liquid) resistance of a protease (subtilisin E [6,7]) and a cellulase (CelA2 [8,9]) will conclude the hydrolase reengineering examples. As a highlight, the generation of oxygen independent and highly active glucose oxidase variants (GOx from A. niger) [10,11] will conclude the presentation. References [1]. Martinez R, et al. Biotechnol Bioeng 2013;110:711–20. [2]. Jakob F, et al. Appl Microbiol Biotechnol 2013;52:2359–63. [3]. Martinez R, et al. Protein Eng Des Sel 2011;24:533–44. [4]. Shivange A, et al. J Biotechnol 2014;170:68–72. [5]. Shivange AV, et al. Appl Microbiol Biotechnol 2012;95:405–18. [6]. Li Z, et al. ChemBioChem 2012;13:691–9. [7]. Li Z, et al. J Biotechnol 2014;169:87–94. [8]. Lehmann C, et al. Green Chem 2012;14:2719–3272. [9]. Pottkämper J, et al. Green Chem 2009;11:691–7. [10].Arango Gutierreza E, et al. Biosens Bioelectron 2013;50:84–90. [11].Prodanovic R, et al. Anal Bional Chem 2012;404:1439–47.
http://dx.doi.org/10.1016/j.nbt.2014.05.1658
O4-6 Immobilization of carbonic anhydrase for biomimetic CO2 capture in slurry absorber Sara Peirce 1,∗ , Maria Elena Russo 2 , Viviana De Luca 3 , Clemente Capasso 3 , Mosè Rossi 3 , Giuseppe Olivieri 4 , Piero Salatino 4 , Antonio Marzocchella 4 1
Dipartimento di Ingegneria Chimica dei Materiali e della Produzione Industriale – Università degli Studi di Napoli Federico II, Italy 2 Consiglio Nazionale delle Ricerche – Istituto di Ricerche sulla Combustione, Italy 3 Consiglio Nazionale delle Ricerche – Istituto di Bioscienze e Biorisorse, Italy 4 Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale – Università degli Studi di Napoli Federico II, Italy
Novel post-combustion treatments include biomimetic Carbon Capture and Storage (CCS) processes based on CO2 absorption into aqueous solution assisted by enzyme catalysis. Carbonic anhydrase (EC 4.2.1.1) catalyzes CO2 hydration and it has been proposed as industrial biocatalyst for biomimetic CCS processes [Lacroix and Larachi, 2008, Recent Patent Chem Eng; Russo et al., 2013, Sep Pur Technol].