Purification of p-coumaric acid esterase by immobilized metal ion affinity chromatography

Importance of the topic

p-Coumaric acid esterase (p-CAE) plays a key role in liberating hydroxycinnamic acids from plant cell walls, facilitating biomass deconstruction and enabling production of value-added compounds. Its broad substrate specificity makes it attractive for applications in food processing, pharmaceuticals and renewable resource conversion.

Objectives and overview of the study

This study aimed to express 6xHis-tagged p-CAE in Pichia pastoris and to develop an efficient purification protocol using immobilized metal ion affinity chromatography (IMAC). Four IMAC configurations were compared: Ni-IDA and Ni-NTA resins at pH 6 and pH 8, to determine optimal conditions for yield and activity retention.

Methodology and instrumentation

p-CAE was recombinantly produced in P. pastoris with a C-terminal His tag. Crude lysate was loaded onto 1 mL IDA or NTA IMAC columns using sodium phosphate buffers with imidazole gradients at pH 6 or pH 8. Protein concentration was measured by Bradford assay and enzyme activity assessed via an optimized assay coupled to reverse-phase HPLC. Purification runs were performed on an AZURA® bio-purification system equipped with high-pressure pumps, a UV detector, fraction collector and selection valve.

Main results and discussion

The Ni-IDA column at pH 8 yielded the highest recovery of active p-CAE, with 35.4% of total protein identified as active enzyme and 78.6% activity recovery, corresponding to a 21.4% activity loss. Ni-NTA and pH 6 systems were less effective, showing lower adsorption capacity and additional elution peaks. An alkaline pH proved essential to avoid protonation of histidine residues and to achieve sharp, single-peak elution.

Benefits and practical applications of the method

This optimized IMAC protocol enables rapid one-step purification of recombinant p-CAE with high purity and activity retention. It provides a scalable approach for producing this enzyme for industrial use in biomass processing, food ingredient modification and pharmaceutical formulations.

Future trends and potential applications

Future work may focus on scaling up the protocol using preparative IMAC columns, integrating continuous purification processes, and engineering p-CAE variants for enhanced stability and activity. Exploration of alternative metal ions and mixed-mode resins could further improve selectivity and throughput.

Conclusion

The study demonstrates that Ni-IDA resin at pH 8 offers a robust and efficient platform for purifying recombinant p-CAE with high yield and functional integrity, supporting its deployment in various biotechnological applications.

Reference

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