Presentation Information
[P02-212]Biochemical Characterisation and Immobilisation-Based Stabilisation of Recombinant Carbonic Anhydrase from Acropora digitifera
○Fahmi Ihza Alghiffary1, Shuto Yamaoka1, Tomoko Matsuda1 (1. Institute of Science Tokyo (Japan))
Keywords:
Carbonic anhydrase,Immobilisation,Carbon dioxide capture,Corals,Enzymes
Carbonic anhydrases (CAs) are Zn-dependent metalloenzymes that catalyse the reversible hydration of CO2 to bicarbonate and proton. In α-CAs the catalytic Zn2+ is coordinated by three histidine residues and a water/OH- ligand. CAs are of interest both biologically and biotechnological applications, especially in CO2-capture technologies. They are essential in the lives of corals, yet many coral-derived CAs remain uncharacterised. In this study, a CA gene from Acropora digitifera (AdCA) was heterologously expressed in Eschericia coli and subjected to biochemical characterisation. Immobilisation was then attempted as a non-genetic strategy to improve enzyme durability.
A putative CA type II gene sequence from NCBI database was selected and was then codon-optimised for E. coli expression. The recombinant protein was then purified using metal affinity chromatography. Amino acid sequence alignment was carried out to examine conservative residues. AdCA activity was evaluated using CO2 hydration and p-nitrophenyl acetate hydrolysis assays. In efforts to enhance the enzyme's durability, AdCA was immobilised on Co2+-phosphate nanoflowers.
Recombinant AdCA was successfully obtained as a soluble protein in E. coli and purified successfully. Amino acid sequence alignment with other carbonic anhydrases indicated that the gene provides the conservation of residues required for Zn2+ binding in the active site. Thermal characterisation exhibited that the free enzyme possessed limited temperature durability. To address this drawback, immobilisation of AdCA in Co2+-phosphate nanoflowers was attempted, resulting in a modest improvement in thermal endurance compared to free enzyme.
Previous work on coral CAs, particularly in Stylophora pistillata (Bertucci et al., 2011), linked CAs to coral calcification and inorganic carbon supply, highlighting the importance of biochemical characterisation of coral enzymes. In that context, present results show that a CA from A. digitifera can be produced in a bacterial host and retains activity after purification, and biochemically characterised using standard enzymatic assays. Despite showing limited thermal robustness, its successful expression and measurable catalytic activity establish a basis for further studies. The stabilisation observed after nanoflower incorporation show that immobilisation is a practical route for improving coral CA durability without protein engineering for possible biotechnological use.
This work provides an inital charcterisation of recombinant AdCA and give the feasibility of its enhancement through immobilisation-based approaches. These findings contribute to the limited body of biochemical information on coral CAs and provide a foundation for future studies on their roles in coral physiology, biomineralisation, and CO2-related biotechnology.
Reference
Bertucci, A., Tambutté, S., Supuran, C.T. et al. A New Coral Carbonic Anhydrase in Stylophora pistillata. Mar Biotechnol 13, 992-1002 (2011).
A putative CA type II gene sequence from NCBI database was selected and was then codon-optimised for E. coli expression. The recombinant protein was then purified using metal affinity chromatography. Amino acid sequence alignment was carried out to examine conservative residues. AdCA activity was evaluated using CO2 hydration and p-nitrophenyl acetate hydrolysis assays. In efforts to enhance the enzyme's durability, AdCA was immobilised on Co2+-phosphate nanoflowers.
Recombinant AdCA was successfully obtained as a soluble protein in E. coli and purified successfully. Amino acid sequence alignment with other carbonic anhydrases indicated that the gene provides the conservation of residues required for Zn2+ binding in the active site. Thermal characterisation exhibited that the free enzyme possessed limited temperature durability. To address this drawback, immobilisation of AdCA in Co2+-phosphate nanoflowers was attempted, resulting in a modest improvement in thermal endurance compared to free enzyme.
Previous work on coral CAs, particularly in Stylophora pistillata (Bertucci et al., 2011), linked CAs to coral calcification and inorganic carbon supply, highlighting the importance of biochemical characterisation of coral enzymes. In that context, present results show that a CA from A. digitifera can be produced in a bacterial host and retains activity after purification, and biochemically characterised using standard enzymatic assays. Despite showing limited thermal robustness, its successful expression and measurable catalytic activity establish a basis for further studies. The stabilisation observed after nanoflower incorporation show that immobilisation is a practical route for improving coral CA durability without protein engineering for possible biotechnological use.
This work provides an inital charcterisation of recombinant AdCA and give the feasibility of its enhancement through immobilisation-based approaches. These findings contribute to the limited body of biochemical information on coral CAs and provide a foundation for future studies on their roles in coral physiology, biomineralisation, and CO2-related biotechnology.
Reference
Bertucci, A., Tambutté, S., Supuran, C.T. et al. A New Coral Carbonic Anhydrase in Stylophora pistillata. Mar Biotechnol 13, 992-1002 (2011).
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