Biophysical characterization of the recombinant chitinase chi18-5 with potential biotechnological interest

VILLANUEVA, MARTÍN EDUARDO; DA SILVA, MARÍA ANGEL; BARRA, JOSÉ LUIS; MONTICH, GUILLERMO GABRIEL; BIANCO, ISMAEL DARIO; SALINAS, SILVINA R.

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

SPRINGER

Año: 2022 vol. 106 p. 1185 - 1197

0175-7598

Abstract: Chitinase chi18-5 is an enzyme able to hydrolyze chitin and chitosan producing chitooligosaccharides (COS) of potential technological interest. chi18-5 is produced naturally by the fungus Trichoderma atroviride. It belongs to the glycosyl hydrolase (GH) family 18 of the Carbohydrate Active Enzyme (CAZy) database and it has 83% identity compared to the well-characterized chi42 of Trichoderma harzianum. Several efforts have been made to characterize the biochemical activity of the enzyme and its structure. Here, we studied the biophysical properties of recombinant chi18-5. In order to gain insight into its structure and stability, we studied thermal denaturation by Circular Dichroism (CD), Intrinsic Fluorescence (FL), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR) at several pH between 3 and 8. We observed that the conformation of chi18-5 changes near its pI, and the transitions as a function of the temperature involved an increment in β-sheet secondary structure at the expenses of ⍺-helix. We also performed amide hydrogen exchange dynamics in selected conditions. At pH ≤ 6, the proportion of fast exchanging residues are larger than at pH ≥ 6. Our results suggest that at pH below pI, chi18-5 is in a less compact structure which may have influence in the interaction with substrate and enzyme activity. Graphical abstract: [Figure not available: see fulltext.] Key Points: ? Characterization of enzyme behavior is critical for their wide applications ? We produced and characterized biophysically a chitinase as a function of pH ? The pH of optimum activity correlates with a less compact structure of chi18-5