The recombinant chitinase Chi42 has more activity i n conditions of relaxed structure

GUILLERMO G. MONTICH; MARTÍN E. VILLANUEVA; SILVINA R. SALINAS; MARÍA A. DA SILVA ; ISMAEL D. BIANCO

Buenos Aires

Jornada; Primeras jornadas virtuales de la Sociedad Argentina de Biofísica; 2020

UBA (Universidad de Buenos Aires)

Chitinases are enzymes that hydrolyze glycosidic bonds in chitin. We purified a recombinant chitinase from Trichoderma harzianum (Chi42). Chi42 has optimal activity at a range of pH 4-5 and between 30-40 ºC. Protein characterization performed by circular dichroism (CD) and intrinsic fluorescence (FL) showed that at pH 6, 7 and 8, the spectra are compatible with 𝛂-helix conformation and have more signal intensity than those obtained at lower pH. FL spectra did not show significant differences among pH. We also studied the thermal stability as a function of pH. Protein conformations at pH 3, 4, and 5 (low pH) are different from that at pH 6,7 and 8 (high pH). Therefore, the transitions observed at low and high pH represent different processes. At pH 5 the calculated Tm was 50 ºC and the final spectra was compatible with a fully denatured state. In contrast, at high pH, the calculated Tm was ~40 ºC, but the changes observed do not imply loss of structure. In all cases, the thermal changes were irreversible. By FL, we observed the same temperature transitions. In contrast to CD, at high pH, the FL was recovered after returning to the initial temperature. We also performed ATR-FT-IR spectroscopy using Fourier Deconvolution (FD) to determine the position of a-helix and β-sheet protein contributions of Amide I and II signals. We observed that 𝛂-helix and β-sheet structures coexist along all the temperatures tested without protein denaturation. Still, it was possible to detect structural changes: absorbance ratio between 𝛂-helix and β-sheet peaks suggested a conformational transition (mid-point at 60 ºC). Amide HX kinetics indicated that Chi42 could have a rigid structure and possibly a low degree of solvent accessibility which could explain the high thermal stability observed.Altogether, we suggest that Chi42 is more active at low pH, probably due to a more relaxed structure which allows the chitin to fit and exert enzymatic activity i n a structured, rigid substrate.