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Variations in the stability of NCR ene reductase by rational enzyme loop modulation
Affiliation:1. Max Planck Institute for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany;2. Milwaukee School of Engineering, Physics and Chemistry Department, 1025 N Broadway, Milwaukee, WI 53202, USA;1. Department of Life Science and Systems Biology, University of Turin, Viale P.A. Mattioli 25, 10125 Turin, Italy;2. Department of Chemistry, Materials and Chemical Engineering “G. Natta”, Politecnico di Milano, Via L. Mancinelli 7, 20131 Milan, Italy;2. Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco 9, 20131 Milano, Italy;1. Instituto de Química de São Carlos, Universidade de São Paulo, Av. João Dagnone, 1100, Ed. Química Ambiental, J. Santa Angelina, 13563-120, São Carlos, SP, Brazil;2. Universidade Federal do Amapá, Rod. Juscelino Kubitscheck, KM 02, S/N – J. Marco Zero, 68903-419, Macapá, AP, Brazil;3. Bioengineering Department, Instituto Superior Técnico, University of Lisbon, Institute for Bioengineering and Biosciences, Av. Rovisco Pais, 1049-001, Lisbon, Portugal;1. INTEQUI-CONICET, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, CP 5700 San Luis, Argentina;2. IMIBIO-CONICET, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, Argentina;3. Departamento de Biología Molecular, FCEFQN, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, CP 5800 Río Cuarto, Córdoba, Argentina
Abstract:The engineering of protein stability is of major importance for the application of enzymes in a wide range of industrial applications. Here we study the determinants of the thermo- and solvent stability of the Zymomonas mobilis ene reductase NCR using a rational protein engineering approach based on analyses of structural and sequence data. We designed and created two loop mutants with the aim to increase their overall stability. They all retained catalytic activity but exhibited altered thermostability relative to the wild-type enzyme. The modulation of one specific loop segment near the active site of NCR showed an increased tolerance to organic solvents along with an enhanced thermostability.
Keywords:Thermostability  Enzyme engineering  Old Yellow Enzyme  Solvent stability  Rational loop design  ThermoFAD
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