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

• Published in: Applied microbiology and biotechnology Vol. 106; no. 3; pp. 1185 - 1197
• Main Authors: Villanueva, Martín Eduardo, da Silva, María Angel, Barra, José Luis, Montich, Guillermo Gabriel, Bianco, Ismael Dario, Salinas, Silvina R.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.02.2022; Springer; Springer Nature B.V
• Subjects: Analysis; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Carbohydrates; Chitin; Chitinase; Chitinases - genetics; Chitinases - metabolism; Chitosan; Circular dichroism; Conformation; Dichroism; Enzymatic activity; Enzyme activity; Enzymes; Exchanging; Fluorescence; Fourier transform infrared spectroscopy; Fourier transforms; Fungi; Glycosidases; Glycosyl hydrolase; Hydrogen exchange; Hydrogen-Ion Concentration; Hydrolase; Identification and classification; Infrared reflection; Infrared spectroscopy; Life Sciences; Microbial Genetics and Genomics; Microbiology; pH effects; Properties; Protein structure; Protein Structure, Secondary; Secondary structure; Spectroscopy, Fourier Transform Infrared; Structural stability; Structure; Substrates; Temperature; Thermal denaturation; Argentina; Chitinase; Characterization; Infrared spectroscopy; Biophysical characterization; Chi18-5; Circular dichroism
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-022-11782-9

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 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