title: Recombinant thermoactive phosphoenolpyruvate carboxylase (PEPC) from Thermosynechococcus elongatus and its coupling with mesophilic/thermophilic bacterial carbonic anhydrases (CAs) for the conversion of CO2 to oxaloacetate.
abstract: With the continuous increase of atmospheric CO2 in the last decades, efficient methods for carbon capture, sequestration, and utilization are urgently required. The possibility of converting CO2 into useful chemicals could be a good strategy to both decreasing the CO2 concentration and for achieving an efficient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for the catalytic conversion of CO2 mainly to bicarbonate have been implemented. In order to design and construct a catalytic system for the conversion of CO2 to organic molecules, we implemented an in vitro multienzyme system using mesophilic and thermophilic enzymes. The system, in fact, was constituted by a recombinant phosphoenolpyruvate carboxylase (PEPC) from the thermophilic cyanobacterium Thermosynechococcus elongatus, in combination with mesophilic/thermophilic bacterial carbonic anhydrases (CAs), for converting CO2 into oxaloacetate, a compound of potential utility in industrial processes. The catalytic procedure is in two steps: the conversion of CO2 into bicarbonate by CA, followed by the carboxylation of phosphoenolpyruvate with bicarbonate, catalyzed by PEPC, with formation of oxaloacetate (OAA). All tested CAs, belonging to α-, β-, and γ-CA classes, were able to increase OAA production compared to procedures when only PEPC was used. Interestingly, the efficiency of the CAs tested in OAA production was in good agreement with the kinetic parameters for the CO2 hydration reaction of these enzymes. This PEPC also revealed to be thermoactive and thermostable, and when coupled with the extremely thermostable CA from Sulphurhydrogenibium azorense (SazCA) the production of OAA was achieved even if the two enzymes were exposed to temperatures up to 60 °C, suggesting a possible role of the two coupled enzymes in biotechnological processes.
authors.affiliation: Istituto di Bioscienze e Biorisorse-CNR, Via P. Castellino 111, 80131 Naples, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Del Prete
authors.affiliation: Istituto di Bioscienze e Biorisorse-CNR, Via P. Castellino 111, 80131 Naples, Italy.
authors.forename: Viviana
authors.initials: V
authors.lastname: De Luca
authors.affiliation: Istituto di Bioscienze e Biorisorse-CNR, Via P. Castellino 111, 80131 Naples, Italy.
authors.forename: Clemente
authors.initials: C
authors.lastname: Capasso
authors.affiliation: Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy; Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche, Polo Scientifico, Sesto Fiorentino, Florence, Italy.
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
authors.affiliation: Istituto di Bioscienze e Biorisorse-CNR, Via P. Castellino 111, 80131 Naples, Italy. Electronic address: vincenzo.carginale@cnr.it.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Carginale
mesh_terms: D002245:Carbon Dioxide / Q000737:chemistry / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D062907:Oxaloacetic Acid / Q000737:chemistry / Q000378:metabolism*; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D046940:Synechococcus / Q000201:enzymology*; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D002245:Carbon Dioxide; D062907:Oxaloacetic Acid; D010730:Phosphoenolpyruvate Carboxylase; D002256:Carbonic Anhydrases
keywords: CO(2) conversion; Carbonic anhydrase; Oxaloacetate; Phosphoenolpyruvate carboxylase; Thermostability