Isolation, purification, characterization and mass spectroscopic analysis of carbonic anhydrase from Serratia sp. for sequestration of carbon dioxide and formation of calcite

Bacterial community isolated from marble rock was enriched in the chemostat by continuous culture in minimal salt mediumwith different concentrations of sodium bicarbonate. One of the isolate survived at 100 mM sodium bicarbonate exhibited capability to sequester carbon dioxide (CO2) was identified as Serratia sp. by 16S rRNA sequencing. CO2 sequestering capacity was detected by carbonic anhydrase enzyme activity and formation of calcite. The carbonic anhydrase enzyme from Serratia sp. was detected after isolation, partial purification by ammonium sulfate precipitation, gel filtration chromatography, affinity chromatography, sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF/MS. The purified enzyme molecular weight was 29 kDa on SDS-PAGE and gel filtration chromatography. The optimum pH and temperature of the enzyme were found to be 7.5 and 40 degrees C respectively. The Km of CA was 2.25 mM and the Vmax 940 mmol/min/mg. The Kcat and Kcat/Km value of purified carbonic anhydrase was 2.8 Chi 10(5) s(1) and 1.2 Chi 10(8) M-1 s(1) respectively. MALDI-TOF mass spectrometry data showing highest matched sequence in database was similar to carbonic anhydrase. Scanning electronmicroscopy ( SEM) analysis revealed the formation of calciumcarbonate. Major calcite phase diffraction peaks occurred at 2u = 29.48, 36.18, 39.58 and 43.58 determined by X-ray diffraction (XRD) analysis. Energy-dispersive X- ray (EDX) analysis showed the presence of CaO (72%) and carbon (18%) indicated role of carbon concentrating mechanisms of Serratia sp., amember of Enterobacteriaceae isolated from marble mines. (C)2013 Elsevier Ltd. All rights reserved.