Process-integrated matrix engineering and drying strategy for stabilization of fibrinolytic protease from Bacillus tequilensis toward biomanufacturing applications

Abstract

Stabilizing enzyme functionality during downstream processing remains a central challenge in scalable biomanufacturing. This study presents a process-integrated evaluation of microencapsulation strategies to preserve the activity of a fibrinolytic protease derived from Bacillus tequilensis HSFI-5, with an emphasis on the interplay among the encapsulant matrix, drying method, and enzymatic accessibility. Encapsulation systems based on maltodextrin, Arabic gum, chitosan, carrageenan, and alginate (5%–15% w/v) were processed by freeze-drying and spray-drying and assessed for activity retention and microencapsulation yield. Results demonstrated that both formulation and processing conditions significantly influenced functional performance (P < 0.05). Lower polymer concentrations favored higher apparent enzymatic activity, whereas higher concentrations improved powder recovery, revealing a trade-off between catalytic accessibility and process efficiency. A moderate negative correlation between activity retention and yield (r = − 0.62, P = 0.018) supports a matrix-dependent diffusion constraint. A comparative analysis of crude, diluted, and microencapsulated systems showed that the reduced apparent activity in encapsulated formulations may be associated with matrix-related accessibility constraints. This interpretation is supported by the increased activity observed after mechanical disruption, suggesting that the decrease was not solely due to complete enzyme inactivation. Among the evaluated systems, maltodextrin-based formulations exhibited a favorable balance between activity retention and yield, particularly under spray-drying conditions, indicating compatibility with scalable processing. Qualitative clot degradation assays confirmed preservation of fibrinolytic functionality after encapsulation, although with a delayed response consistent with controlled enzyme release. Morphological analysis revealed spherical, relatively smooth microcapsules that may facilitate improved hydration and substrate diffusion. Collectively, these findings establish a process-level framework that links matrix composition, drying strategy, and mass-transfer behavior to enzymatic performance. This work highlights the importance of designing encapsulation systems that balance structural protection with functional accessibility, thereby providing a rational basis for developing stable, scalable enzyme formulations for biomanufacturing applications.

Graphical abstract

Keywords

Bacillus tequilensis / Enzyme stabilization / Fibrinolytic protease / Freeze-drying / Microencapsulation / Spray-drying

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