Publication: Expression and functional analysis of recombinant serine protease from beauveria bassiana in baculovirus
Date
2026-01
Authors
Azali, Muhammad Azharuddin
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Serine proteases from entomopathogenic fungi represent a promising source of industrially relevant biocatalysts, yet their recombinant production and biochemical characteristics remain underexplored. In this study, a recombinant serine protease (SP) from Beauveria bassiana was designed, assembled, and functionally characterised using the Baculovirus Expression Vector System (BEVS) in Spodoptera frugiperda (Sf9) cells. The SP gene was synthetically constructed using assembly PCR, whereby overlapping oligonucleotides were progressively extended and fused to generate a full-length, codon-optimised open reading frame containing the native signal peptide and catalytic domains for efficient expression in insect cells. Structural prediction using AlphaFold followed by refinement of the modelled tertiary structure confirmed the presence of conserved S8A subtilisin motifs, correct catalytic triad geometry, and well-defined substrate-binding pockets. To further explore substrate recognition, molecular docking simulations were performed in HADDOCK using α-, β-, and κ-casein as ligand substrates. The HADDOCK results demonstrated favourable interaction energies and stable protein–substrate interfaces, with β-casein forming the most stable complex within the catalytic cleft. Experimentally, recombinant expression was successfully achieved, with maximal secretion at 72 hours post-infection (MOI 10), while intracellular expression peaked at 96 hours. SDS-PAGE, Western blotting, and LC-MS/MS analyses verified the size, integrity, and identity of the recombinant enzyme. Enzymatic assays showed distinct catalytic profiles depending on expression pathway: secreted SP exhibited higher proteolytic activity under dilute substrate conditions (10×–100×), whereas intracellular SP demonstrated optimal activity with concentrated substrate (5×). The enzyme displayed an optimal pH of 6.8 and an optimal temperature of 40 °C for β-casein hydrolysis. Cytotoxicity evaluation showed only mild reductions in Sf9 viability, indicating that SP expression does not induce substantial cellular damage. Overall, this study establishes a complete workflow from assembly PCR-based synthetic gene construction, AlphaFold structural modelling, and HADDOCK molecular docking, to BEVS-mediated expression and biochemical characterisation of B. bassiana serine protease. The findings deepen current understanding of fungal subtilases and support their potential application in food protein hydrolysis, detergent formulations, and broader industrial biocatalysis.