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Improvement of thermostability by increasing rigidity in the finger regions and flexibility in the catalytic pocket area of Pseudoalteromonas porphyrae κ-carrageenase.
- Source :
-
World journal of microbiology & biotechnology [World J Microbiol Biotechnol] 2024 May 28; Vol. 40 (7), pp. 216. Date of Electronic Publication: 2024 May 28. - Publication Year :
- 2024
-
Abstract
- Poor thermostability reduces the industrial application value of κ-carrageenase. In this study, the PoPMuSiC algorithm combined with site-directed mutagenesis was applied to improve the thermostability of the alkaline κ-carrageenase from Pseudoalteromonas porphyrae. The mutant E154A with improved thermal stability was successfully obtained using this strategy after screening seven rationally designed mutants. Compared with the wild-type κ-carrageenase (WT), E154A improved the activity by 29.4% and the residual activity by 51.6% after treatment at 50 °C for 30 min. The melting temperature (T <subscript>m</subscript> ) values determined by circular dichroism were 66.4 °C and 64.6 °C for E154A and WT, respectively. Molecular dynamics simulation analysis of κ-carrageenase showed that the flexibility decreased within the finger regions (including F1, F2, F3, F5 and F6) and the flexibility improved in the catalytic pocket area of the mutant E154A. The catalytic tunnel dynamic simulation analysis revealed that E154A led to enlarged catalytic tunnel volume and increased rigidity of the enzyme-substrate complex. The increasing rigidity within the finger regions and more flexible catalytic pocket of P. porphyrae κ-carrageenase might be a significant factor for improvement of the thermostability of the mutant κ-carrageenase E154A. The proposed rational design strategy could be applied to improve the enzyme kinetic stability of other industrial enzymes. Moreover, the hydrolysates of κ-carrageenan digested by the mutant E154A demonstrated increased scavenging activities against hydroxyl (OH) radicals and 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radicals compared with the undigested κ-carrageenan.<br /> (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)
- Subjects :
- Bacterial Proteins genetics
Bacterial Proteins chemistry
Bacterial Proteins metabolism
Kinetics
Temperature
Circular Dichroism
Protein Conformation
Carrageenan metabolism
Enzyme Stability
Mutagenesis, Site-Directed
Glycoside Hydrolases genetics
Glycoside Hydrolases chemistry
Glycoside Hydrolases metabolism
Pseudoalteromonas enzymology
Pseudoalteromonas genetics
Molecular Dynamics Simulation
Catalytic Domain
Subjects
Details
- Language :
- English
- ISSN :
- 1573-0972
- Volume :
- 40
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- World journal of microbiology & biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 38802708
- Full Text :
- https://doi.org/10.1007/s11274-024-04029-4