New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 4: ROBUST BIOCATALYSTS FOR THE PRODUCTIONOF NOVEL BIO-BASED PRODUCTS

Symposium 4: Robust biocatalysts for the pro-duction of novel bio-based products

O4-1

Molecular design of transglucosidases for polysaccharideand oligosaccharide synthesis

P. Monsana,b,c,d,e,f

a Université de Toulouse; INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077Toulouse, Franceb CNRS, UMR 5504, F-31400 Toulouse, Francec INRA, UMR 792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400Toulouse, Franced Toulouse White Biotechnology, 3 rue des Satellites, F-31400 Toulouse, Francee INRA, UMS 1337TWB, F31400 Toulouse, Francef CNRS 3582, F-31400 Toulouse, France

Among the diversity of transglucosidase enzymes, a specificinterest is given to the glucansucrases of the GH70 family, whichare produced extracellularly by different lactic acid bacteria: Leu-conostoc sp., Lactococcus sp., Streptococcus sp., Weissella sp. Theseenzymes are able to use the energy of the osidic linkage of thesucrose molecule (27.6 kJ/mol) to catalyse the efficient transfer ofits �-D-glucopyranosyl unit. This results in the synthesis of a widevariety of products, polysaccharides, oligosaccharides and gluco-conjugates, which contain a diversity of linkages: �-1,2; �-1,3;�-1,4; �-1,6.

It is possible to combine the biochemical and structural char-acterization of these transglucosidases with gene sequence dataand alignement analysis, to develop structure-function relation-ship studies and molecular engineering based on both rational insilico and combinatorial in vitro approaches. The resulting variantspresent new and improved catalytic properties, resulting in thesynthesis of new products.

In addition, original transglucosidases have been obtained fromsequencing the genome of several lactic acid bacteria and isola-tion of the corresponding genes. They are able to decorate dextranpolysaccharides (�-1,6 main backbone linkage) with �-1,2 and �-1,3 osidic branching and generate a new series of polysaccharidesand oligosaccharides.

http://dx.doi.org/10.1016/j.nbt.2014.05.1654

O4-2

Structural studies on transaminase enzymes and appli-cations in biocatalysis

Jenny Littlechild1,∗ , C. Sayer1, M. Isupov1, J. Ward2, J. Littlechild1

1 University of Exeter, United Kingdom2 University College London, United Kingdom

The transaminases (TAs) catalyse the transfer of an amino groupfrom an amino acid to a keto acid using the cofactor pyridoxal 5’-phosphate (PLP) and are important for the production of opticallypure amines and amino alcohols used in the synthesis of manyimportant drugs.

We have solved the structures of TAs of the Pfam classes, III,IV and V in order to further understand their mechanism andsubstrate specificities.

The class III �-amino acid TAs catalyse transamination of �-amino acids such as �-alanine or �-aminobutyric acid where thetransferred amino group is not adjacent to the carboxyl group.The crystal structures and inhibitor complexes of two �-TAs fromPseudomonas aeruginosa and Chromobacterium violaceumhave beendetermined to understand differences in their substrate specificity.

The class V TA enzymes include the serine:pyruvate transami-nases having broad substrate specificity including a reaction witha �-hydroxyl substrate. The structure of the thermophilic archaealSulfolobus solfataricus serine TA has been determined to 1.8 A res-olution and in complex with the inhibitor, gabaculine. Thesestructures have shown the conformational changes in the enzymeactive site during the course of the catalytic reaction.

The structure of the class IV Nectria haematococca transam-inase enzyme has been determined in the holo and inhibitorbound form which offers a detailed insight into the structuralbasis for substrate specificity and enantioselectivity of (R)-selectiveamine:pyruvate transaminases [1–4].

References

[1].Sayer S, et al. Acta Cryst 2007;F63:117–9.[2].Sayer S, et al. Acta Cryst 2012;D68:763–72.[3].Sayer S, et al. Acta Cryst 2013;D69:564–76.[4].Sayer S, et al. FEBS J 2014 [in press].

http://dx.doi.org/10.1016/j.nbt.2014.05.1655

O4-3

Current developments on the engineering of Escherichiacoli biofilms for enzymatic biosynthesis of halotrypto-phans

Isaac Vizcaino-Caston1,∗ , James Thomas Leech1, Tania Triscari

Barberi 2, Rebeca J.M. Goss2, Mark J.H. Simmons1, TimW. Overton1

1 The University of Birmingham, United Kingdom2 University of St. Andrews, United Kingdom

Halogenated molecules are being utilized on a daily basis bythe fine chemistry and pharmaceutical industries in diverse reac-tions for the production of enantiomerically pure compounds.The chemical synthesis of such molecules requires harsh solventsand conditions which result in expensive and environmen-tally unfriendly processes. We propose an alternative to suchmethods utilizing recombinant bacterial biofilms to synthesise5-halotryptophans. The plasmid pSTB7 expressing recombinanttryptophan synthase TrpBA was transformed into diverse strainsof E. coli and used to generate engineered biofilms [1,2]. Thesebiofilms were used as biocatalysts for the bioconversion of 5-haloindoles into 5-halotryptophans. Biofilm cultures showedhigher yield than planktonic bacteria, bacterial lysates or immo-bilised TrpBA enzyme [3]. Biofilm topology and viability wereassessed using confocal microscopy before and after reactions. Inorder to enable scale-up, a novel method to adhere bacteria tosupports was devised. We will discuss the ability of different cell

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SYMPOSIUM 4: ROBUST BIOCATALYSTS FOR THE PRODUCTIONOF NOVEL BIO-BASED PRODUCTS New Biotechnology · Volume 31S · July 2014

immobilization methods to develop biofilm communities as wellas their ability to perform biotransformations.

References

[1].Tsoligkas AN, Winn M, Bowen J, Overton TW, Simmons MJH,Goss RJM. Engineering biofilms for biocatalysis. ChemBioChem2011;12:1391–5.

[2].Tsoligkas AN, Bowen J, Winn M, Goss RJM, Overton TW, SimmonsMJH. Characterisation of spin coated engineered Escherichia colibiofilms using atomic force microscopy. Colloids and Surfaces B: Bioin-terfaces 2012;89:152–60.

[3].Perni S, Hackett L, Goss R, Simmons M, Overton T. Optimisationof engineered Escherichia coli biofilms for enzymatic biosynthesis ofL-halotryptophans. AMB Express 2013;3:66.

http://dx.doi.org/10.1016/j.nbt.2014.05.1656

O4-4

Structural and biochemical characterization of twonovel enzymes with promiscuous ene-reductase activity

Tea Pavkov-Keller1,∗ , Alexandra Binter2, Steinkellner Georg1,Christian C. Gruber1, Kerstin Steiner2, Christoph Winkler3, HelmutSchwab4, Kurt Faber3, Peter Macheroux5, Karl Gruber6

1 ACIB GmbH, C/o ZMB, Austria2 ACIB GmbH, Austria3 Department of Chemistry, University of Graz, Austria4 Institute of Molecular Biotechnology, Graz University of Technology, Austria5 Institute of Biochemistry, Graz University of Technology, Austria6 Institute of Molecular Biosciences, University of Graz, Austria

An approach using three-dimensional motifs reflecting specificactive site arrangements (catalophore) was developed in our group.It does not depend on overall protein similarity and thereforeenables the search across enzyme families and the detection ofpotential catalytic promiscuity. This catalophore approach led tothe discovery of two novel enzymes with ene-reductase activity.Enzymes of this family have recently been shown to possess a greatpotential for (industrial) biotransformations. Neither the aminoacid sequence of these two enzymes nor their overall structureis related to those of the well-known old yellow enzymes (OYE).These two flavoproteins contain FMN as a cofactor and exist ashomodimers. We cloned, expressed and purified both enzymesand subjected them to crystallization trials. Obtained crystalswere soaked with putative substrates/inhibitors. The enzymaticcharacterization was pursued by stopped-flow and difference titra-tion experiments. Additionally, several typical OYE substrates (i.e.alkenes bearing an electron-withdrawing activating group) weretested to assess the biocatalytic performance. The analysis showedsome salient features of typical OYEs as well as some strikingdifferences, i.e. a stereocomplementary behaviour. In conclusion,the two novel enzymes can be described as NADPH-dependentquinone reductases with significant OYE-like side activities.

http://dx.doi.org/10.1016/j.nbt.2014.05.1657

O4-5

Lessons on directed evolution of hydrolases and glucoseoxidase

Ulrich Schwaneberg

RWTH – Aachen University, Chair of Biotechnology, Germany

Protein engineering by directed evolution and semi-rationaldesign has become a standard method to tailor enzyme propertiesto industrial demands. Improving thermal stability and activ-ity simultaneously is often challenging since high activity oftenrequires 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 improvingboth properties individually and simultaneously will be presented.Subsequently, lessons on improving detergent and salt (ionic liq-uid) 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 andhighly 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 biomimeticCO2 capture in slurry absorber

Sara Peirce1,∗ , Maria Elena Russo2, Viviana De Luca3, ClementeCapasso3, Mosè Rossi 3, Giuseppe Olivieri 4, Piero Salatino4, Anto-nio Marzocchella4

1 Dipartimento di Ingegneria Chimica dei Materiali e della Produzione Industri-ale – Università degli Studi di Napoli Federico II, Italy2 Consiglio Nazionale delle Ricerche – Istituto di Ricerche sulla Combustione,Italy3 Consiglio Nazionale delle Ricerche – Istituto di Bioscienze e Biorisorse, Italy4 Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Indus-triale – Università degli Studi di Napoli Federico II, Italy

Novel post-combustion treatments include biomimetic CarbonCapture and Storage (CCS) processes based on CO2 absorption intoaqueous solution assisted by enzyme catalysis. Carbonic anhydrase(EC 4.2.1.1) catalyzes CO2 hydration and it has been proposed asindustrial biocatalyst for biomimetic CCS processes [Lacroix andLarachi, 2008, Recent Patent Chem Eng; Russo et al., 2013, Sep PurTechnol].

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