Performance of an immobilized recombinant leucine aminopeptidase after storage in ethanol–water solution

Abstract

Immobilized enzymes offer different benefits such as the feasibility to be reused for reproducible bioprocesses. The challenge is to establish the appropriate storage conditions that allow the maintenance of their properties for long periods. In this study, we immobilized a recombinant leucine aminopeptidase (I-rLAP) on a siliceous support synthesized from tetraethyl orthosilicate (TEOS) activated with glutaraldehyde to evaluate its residual activity after storage in 20% v/v ethanol and sodium azide solutions at 4 and 25 °C. The characterization of the support by X-ray diffraction (XRD), diffuse reflectance infrared Fourier transform (DRIFT) and field emission probe microanalyzer (EPMA) was consistent with previous characterization reports of silica gel matrices. Particle size ≤420 μm exhibited a suitable performance that avoided high backpressure into the columns and increased the amount of immobilized enzyme. I-rLAP recovered up to 90% of the applied activity after 64 days of storage at 4 and 25 °C in 20% v/v ethanol. Conversely, no effect was observed when the insoluble enzyme was stored in sodium azide. Activity recovery of I-rLAP after storage in ethanol solution could be related to the formation of disulfide bonds as suggested by free thiol analyses. Reverse phase-ultra performance liquid chromatography (RP-UPLC) and Mass Spectrometry confirmed that the immobilized enzyme maintained its specificity to remove N-terminal methionine from a recombinant hormone. The obtained results indicate that this methodology constitutes an alternative for bioprocesses involving long-term storage of immobilized enzymes.

Keywords:

• Enzyme immobilization
• structural change
• storage stability

Acknowledgements

The authors thank to Araceli Mauricio Sánchez, Martín Hernández Landaverde and Eleazar Urbina Alvarez from CINVESTAV Querétaro, for their support in the sol-gel process, DRIFT, XRD and EPMA techniques. We also thank National Laboratory for Research and Technological Development of Advanced Coatings (LIDTRA) for performing the support characterization analyses and Carlos E. Espinosa de la Garza for his valuable contribution in improving the manuscript.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Additional information

Funding

Mario Eduardo Abad Javier thanks CONACYT for the fellowship (FINNOVA 174104) granted during his Master’s thesis.