Dataset related to the immobilization of three microbial variants of carbonic anhydrase (CA) on the Ni2+-ReliZyme carrier, their contribution to CO2 reduction to formate by FDH, rational design and optimization of the trifunctional biocatalyst from cell lysates by evaluating different BhCA loadings, different BhCA concentrations in CO2 reduction, different GC-GlyDH loadings in the final biocatalyst, evaluation of the effect of BhCA in the multi-enzymatic system, operational stability, reusability of the biocatalyst over five reaction cycles, and performance metrics.

METHODOLOGICAL INFORMATION

1. Description of the methods used to collect and generate the data:

The production of FDH and GlyDH enzymes was carried out in a bioreactor using a defined medium with controlled temperature, pH, oxygen, and feeding strategy, followed by IPTG induction, cell recovery, mechanical cell disruption, and resuspension in phosphate buffer for subsequent purification by IMAC affinity chromatography and ion-exchange chromatography, respectively. CAs were produced in LB medium and auto-induction medium and purified by TALON IMAC chromatography in Tris-HCl buffer. The enzymatic activity of FDH and GlyDH was measured spectrophotometrically at 340 nm using a phosphate buffer, NAD+ at 1.67 and 5 mM, 100 mM sodium formate, 100 mM glycerol, and a variable amount of soluble enzyme. Carbonic anhydrase activity was also measured spectrophotometrically at 550 nm by monitoring CO2 release from a bicarbonate solution using a colorimetric pH indicator (phenolphthalein). The protein concentration was quantified spectrophotometrically at 595 nm using the Bradford method. Formate, DHA and glycerol quantification were performed in the liquid chromatograph using an ion exchange method with the IC-Sep COREGEL 87H3 column and sulfuric acid (H2SO4) 0.5 mM: Acetonitrile (65:35) as mobile phase. A flow rate of 0.6 mL·min-1, injection volume of 20 µL, column temperature of 30 °C, UV/Visible detector at 210 nm and RID detector at 30 °C. The immobilizations and co-immobilizations experiments were carried out in 100 mM phosphate buffer + 100 mM NaCl at pH 7.5, at a temperature of 4 °C, with a glutaraldehyde coating for GlyDH at 0.05% v/v.
The multi-enzymatic CO₂ Reduction reaction was carried out in 100 mM phosphate buffer at pH 7.5 as reaction media, 100 mM glycerol, 1 mM NADH, with CO₂ continuously bubbled at 0.1 VVM, and 20 grams of biocatalyst in a final volume of 200 mL. The reaction was stirred at 300 rpm, maintained at 30 °C, and run for 54 and 82 hours.

1. Data processing methods:

2. All data was processed using Excel.

3. Software or instruments needed to interpret the data:

4. Excel

5. Information about instruments, calibration and standards:

SpectraMax® M2 plate reader (Molecular Devices, San Jose, CA, USA) Liquid chromatograph Agilent 1260 Infinity (Agilent Technologies, Waldbronn, Alemania) Varian Cary 50 Bio UV-visible Spectrophotometer (Agilent) SPECTROstar Nano (BMG LABTECH) Spectrophotometer

1. Environmental or experimental conditions: All experiments were carried out in a fume hood at 30°C and within a pH range of 7.0 to 8.5.