Functional Investigation of Mutant Vespa tropica Hyaluronidase Produced in Pichia pastoris: In Silico Studies and Potential Applications
2
Issued Date
2025-09-01
Resource Type
eISSN
23115637
Scopus ID
2-s2.0-105017419014
Journal Title
Fermentation
Volume
11
Issue
9
Rights Holder(s)
SCOPUS
Bibliographic Citation
Fermentation Vol.11 No.9 (2025)
Suggested Citation
Janpan P., Schmelzer B., Klamrak A., Nabnueangsap J., Saengkun Y., Rahman S.S., Rungsa P., Mattanovich D., Daduang S. Functional Investigation of Mutant Vespa tropica Hyaluronidase Produced in Pichia pastoris: In Silico Studies and Potential Applications. Fermentation Vol.11 No.9 (2025). doi:10.3390/fermentation11090497 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/112467
Title
Functional Investigation of Mutant Vespa tropica Hyaluronidase Produced in Pichia pastoris: In Silico Studies and Potential Applications
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Abstract
The hyaluronidase enzyme derived from Vespa tropica (VesT2a) venom contains two putative catalytic residues. Herein, a double mutation was introduced into VesT2a at its catalytic sites by substituting Asp107 and Glu109 with Asn and Gln, respectively, to assess their essential roles in enzymatic function. We used Pichia pastoris to produce the mutated version of the VesT2a (mVesT2a) protein, and the process was more efficient when employing the methanol-inducible promoter (P<inf>AOX1</inf>) compared to the constitutive promoter (P<inf>GAP</inf>). In bioreactor scale-up, P. pastoris harboring the pAOX1-αMF-mVesT2a plasmid secreted 34.03 ± 2.31 mg/L of mVesT2a, with an apparent molecular mass of 46.6 kDa, retaining only 2.9% of hyaluronidase activity, thus indicating successful mutation. The newly developed indirect ELISA-based method using mVesT2a demonstrated its potential as an alternative approach for measuring hyaluronic acid (HA) at low concentrations and was also used to confirm HA-binding capacity. In silico docking and molecular dynamics simulations further supported the stable interaction of the mVesT2a–HA complex while suggested other surrounded acidic amino acid residues, which may play a minor role in HA degradation, supporting the remaining activity observed in the in vitro experiments.