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

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

Buenos Aires

Workshop; Tercer encuentro y primer workshop de la red Argentina de Tecnología Enzimática; 2021

Red Argentina de Tecnología Enzimática

Chitinasesare ubiquitous enzymes that have gained recent biotechnologicalattention due to their potential to transform chitin and chitosaninto valued chitooligosaccharides (COS) with wide agricultural,industrial or medical applications. Nevertheless, the obtention ofCOS with fully defined architecture and desired physicochemical andbiological properties requires the use of specific well-characterizedchitinases. To address this current problem, we aimed to characterizethe biophysical properties of a novel recombinant chitinase Chi18-5.This enzyme, naturally produced by the fungus Trichodermaatroviride,belongs to the GH18 of the Carbohydrate Active Enzyme (CAZy) databaseand it has 83% identity compared to the well characterized Chi42 ofT.harzianum.Several efforts have been made to characterize the biochemicalactivity of the enzyme and its structure. Herein, we assessed thebiophysical properties of the Chi18-5 through differentmethodologies. Inorder to gain insight into its structure and stability, we performedthermal denaturation studies by Circular Dichroism (CD), IntrinsicFluorescence (FL), and Attenuated Total Reflection Fourier TransformInfrared Spectroscopy (ATR-FT-IR) at several pH among 3 and 8. Weobserved that the conformation of Chi18-5 changes near its pI, andthe transitions observed during thermal ramps involve an increment inꞵ-sheet in detriment of ⍺-helixcontent. Moreover, we performed amide hydrogen exchange dynamics inselected conditions. Information regarding the different populationsof residues involved in the amide HX, showed that at lower pH thereexists a higher proportion of fast exchanging residues than at pH ≥6.The conversion of the rate constants to Gibbs free-energy changes forthe opening to HX, allowed us to determine that residues bearing apartial accessibility to solvent at pH 7, resulted to be highlyexposed when the external milieu is taken to pH 5. Taking intoaccount that the optimum activity is at pH≤5, the results suggestthat the more relaxed structure found at pH below pI favors theenzymatic activity, probably by better accommodating thechitin-derivative substrate. Thegreat differences observed in the structure stability as a functionof pH represent a starting point for the study of environmentalfactors that could be critical for the enzymatic activity which isessential for its potential biotechnological applications.p { margin-bottom: 0.1in; direction: ltr; color: #000000; line-height: 115%; text-align: left; orphans: 2; widows: 2 }p.western { font-family: "Calibri", serif; font-size: 11pt; so-language: en-US }p.cjk { font-family: "Calibri"; font-size: 11pt; so-language: en-US }p.ctl { font-family: ; font-size: 11pt; so-language: ar-SA }