The recombinant chitinase Chi42 has more activity in conditions of relaxed structure

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

Buenos Aires

Jornada; Primeras Jornadas Virtuales SAB 2020.; 2020

Sociedad Argentina de Biofísica

Chitinases are enzymes that hydrolyze glycosidic bonds i n 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 i ntrinsic fluorescence (FL) showed that at pH 6, 7 and 8, the spectra are compatible with 𝛂-helix conformation and have more signal i ntensity than those obtained at l ower 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 i mply l oss of structure. In all cases, the thermal changes were i rreversible. By FL, we observed the same temperature transitions. In contrast to CD, at high pH, the FL was recovered after returning to the i nitial 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, i t 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 i ndicated that Chi42 could have a rigid structure and possibly a l ow degree of solvent accessibility which could explain the high thermal stability observed. Altogether, we suggest that Chi42 i s more active at l ow pH, probably due to a more relaxed structure which allows the chitin to fit and exert enzymatic activity i n a structured, rigid substrate.