Biophysical characterization of the chitinase Chi18-5 for biotechnological chitooligosaccharide production

Buenos Aires

Encuentro; Tercer Encuentro y Primer Workshop de la Red de Tecnología Enzimática; 2021

Red Argentina de Tecnología Enzimática

Chitinases are ubiquitous enzymes that have gained recent biotechnological attention due to their potential to transform chitin and chitosan into valued chitooligosaccharides (COS) with wide agricultural, industrial or medical applications. Nevertheless, the obtention of COS with fully defined architecture and desired physicochemical and biological properties requires the use of specific well-characterized chitinases. To address this current problem, we aimed to characterize the biophysical properties of a novel recombinant chitinase Chi18-5. This enzyme, naturally produced by the fungus Trichoderma atroviride, belongs to the GH18 of the Carbohydrate Active Enzyme (CAZy) database and it has 83% identity compared to the well characterized Chi42 of T. harzianum. Several efforts have been made to characterize the biochemical activity of the enzyme and its structure. Herein, we assessed the biophysical properties of the Chi18-5 through different methodologies.We cloned, expressed, and purified recombinant Chi18-5. In order to gain insight into its structure and stability, we performed thermal denaturation studies by Circular Dichroism (CD), Intrinsic Fluorescence (FL), and Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FT-IR) at several pH among 3 and 8. We observed that the conformation of Chi18-5 changes near its pI, and the transitions observed during thermal ramps involve an increment in ꞵ-sheet in detriment of ⍺-helix content. Moreover, we performed amide hydrogen exchange dynamics in selected conditions. Information regarding the different populations of residues involved in the amide HX, showed that at lower pH there exists a higher proportion of fast exchanging residues than at pH ≥6. The conversion of the rate constants to Gibbs free-energy changes for the opening to HX, allowed us to determine that residues bearing a partial accessibility to solvent at pH 7, resulted to be highly exposed when the external milieu is taken to pH 5. Taking into account that the optimum activity is at pH≤5, the results suggest that the more relaxed structure found at pH below pI favors the enzymatic activity, probably by better accommodating the chitin-derivative substrate.The great differences observed in the structure stability as a function of pH represent a starting point for the study of environmental factors that could be critical for the enzymatic activity which is essential for its potential biotechnological applications.