THE RECOMBINANT CHITINASE CHI42 HAS MORE ACTIVITY IN CONDITIONS OF RELAXED STRUCTURE

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

Buenos Aires

Jornada; Jornadas Virtuales Sociedad Argentina de Biofísica 2020; 2020

Sociedad Argentina de Biofísica

Chitinasesare enzymes that hydrolyze glycosidic bonds in chitin. We purified arecombinant chitinase from Trichodermaharzianum (Chi42).Chi42 has optimal activity at a range of pH 4-5 and between 30-40 ºC.Protein characterization performed by circular dichroism (CD) andintrinsic fluorescence (FL) showed that at pH 6, 7 and 8, the spectraare compatible with 𝛂-helix conformation and have more signalintensity than those obtained at lower pH. FL spectra did not showsignificant differences among pH. We also studied the thermalstability as a function of pH. Protein conformations at pH 3, 4, and5 (low pH) are different from that at pH 6,7 and 8 (high pH).Therefore, the transitions observed at low and high pH representdifferent processes. At pH 5 the calculated Tm was 50 ºC and thefinal spectra was compatible with a fully denatured state. Incontrast, at high pH, the calculated Tm was ~40ºC, but the changes observed do not imply loss of structure. In allcases, the thermal changes were irreversible. By FL, we observed thesame temperature transitions. In contrast to CD, at high pH, the FLwas recovered after returning to the initial temperature. Wealso performed ATR-FT-IR spectroscopy using Fourier Deconvolution(FD) to determine the position of a-helix and β-sheet proteincontributions of Amide I and II signals. We observed that 𝛂-helixand β-sheet structures coexist along all the temperatures testedwithout protein denaturation. Still, it was possible to detectstructural changes: absorbance ratio between 𝛂-helix and β-sheetpeaks suggested a conformational transition (mid-point at 60 ºC).Amide HX kinetics indicated that Chi42 could have a rigid structureand possibly a low degree of solvent accessibility which couldexplain the high thermal stability observed.Altogether,we suggest that Chi42 is more active at low pH, probably due to amore relaxed structure which allows the chitin to fit and exertenzymatic activity in a structured, rigid substrate.p { margin-bottom: 0.1in; direction: ltr; color: #000000; line-height: 115%; text-align: left; orphans: 0; widows: 0 }p.western { font-family: "Arial", serif; font-size: 11pt; so-language: en-US }p.cjk { font-family: "Arial"; font-size: 11pt; so-language: zh-CN }p.ctl { font-family: "Arial"; font-size: 11pt; so-language: hi-IN }