Structural engineering and truncation of α-amylase from the hyperthermophilic archaeon Methanocaldococcus jannaschii

• Published in: International journal of biological macromolecules 2024-01, Vol.256 (Pt 1), p.128387-128387, Article 128387
• Main Authors: Shad, Mohsin, Sajjad, Muhammad, Gardner, Qurratulann Afza, Ahmad, Saira, Akhtar, Muhammad Waheed
• Format: Article
• Language: English
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2023.128387

Alpha amylases catalyse the hydrolysis of α-1, 4-glycosidic bonds in starch, yielding glucose, maltose, dextrin, and short oligosaccharides, vital to various industrial processes. Structural and functional insights on α-amylase from Methanocaldococcus jannaschii were computationally explored to evaluate a catalytic domain and its fusion with a small ubiquitin-like modifier (SUMO). The recombinant proteins' production, characterization, ligand binding studies, and structural analysis of the cloned amylase native full gene (MjAFG), catalytic domain (MjAD) and fusion enzymes (S-MjAD) were thoroughly analysed in this comparative study. The MjAD and S-MjAD showed 2-fold and 2.5-fold higher specific activities (μmol min  mg ) than MjAFG at 95 °C at pH 6.0. Molecular modelling and MD simulation results showed that the removal of the extra loop (178 residues) at the C-terminal of the catalytic domain exposed the binding and catalytic residues near its active site, which was buried in the MjAFG enzyme. The temperature ramping and secondary structure analysis of MjAFG, MjAD and S-MjAD through CD spectrometry showed no notable alterations in the secondary structures but verified the correct folding of MjA variants. The chimeric fusion of amylases with thermostable α-glucosidases makes it a potential candidate for the starch degrading processes.