Single substitution in α-helix of active center enhanced thermostability of Aspergillus awamori exo-inulinase

• Published in: Journal of molecular graphics & modelling 2023-03, Vol.119, p.108381-108381, Article 108381
• Main Authors: Dotsenko, Anna, Denisenko, Jury, Zorov, Ivan, Wasserman, Luybov, Semenova, Margarita, Korolev, Andrei, Rozhkova, Aleksandra, Sinitsyn, Arkady
• Format: Article
• Language: English
• Subjects: Aspergillus - genetics; Aspergillus awamori; Enzyme Stability; exo-inulinase; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Molecular dynamics; Protein Conformation, alpha-Helical; Rational design; Thermostability
• ISSN: 1093-3263; 1873-4243
• DOI: 10.1016/j.jmgm.2022.108381

Exo-inulinases are applied in inulin hydrolysis and production of feed additives and need to be stable at temperatures of 60–95 °C. Aspergillus awamori exo-inulinase Inu1 is considerably thermostable, with a Tm of 73.2 °C. However, the thermostability of the enzyme should be improved. A single substitution G338A in α-helix in the active center of the enzyme provided a 3.5 °C improvement in Tm. The time of half-life at 70 °C and 80 °C was increased in 5.7- and 2.7-times, respectively, compared to wild-type. Molecular dynamics simulations demonstrated that the substitution G338A caused a decrease in RMSF not only for the α-helix 337-YAANI-341, but also for the catalytically active residues D41 and E241 and the amino acid residues forming the cleft of the active center. Calculations with Constraint Network Analysis for the variant G338A showed the increase in the stability of intramolecular clusters. [Display omitted] •Tm was raised by 3.5 °C and 3.9 °C according to DSC and DSF, respectively.•Half-life time at 70 °C and 80 °C was improved in 5.7- and 2.7-times, respectively.•RMSF was decreased for amino acid residues forming the cleft of the active center.•Stability of intramolecular clusters in CNA was increased.