Purification, characterization and structural determination of chitinases produced by Moniliophthora perniciosa

Abstract

The enzyme chitinase from Moniliophthora perniciosa the causative agent of the witches' broom disease in Theobroma cacao, was partially purified with ammonium sulfate and filtration by Sephacryl S-200 using sodium phosphate as an extraction buffer. Response surface methodology (RSM) was used to determine the optimum pH and temperature conditions. Four different isoenzymes were obtained: ChitMp I, ChitMp II, ChitMp III and ChitMp IV. ChitMp I had an optimum temperature at 44-73°C and an optimum pH at 7.0-8.4. ChitMp II had an optimum temperature at 45-73°C and an optimum pH at 7.0-8.4. ChitMp III had an optimum temperature at 54-67°C and an optimum pH at 7.3-8.8. ChitMp IV had an optimum temperature at 60°C and an optimum pH at 7.0. For the computational biology, the primary sequence was determined in silico from the database of the Genome/Proteome Project of M. perniciosa, yielding a sequence with 564 bp and 188 amino acids that was used for the three-dimensional design in a comparative modeling methodology. The generated models were submitted to validation using Procheck 3.0 and ANOLEA. The model proposed for the chitinase was subjected to a dynamic analysis over a 1 ns interval, resulting in a model with 91.7% of the residues occupying favorable places on the Ramachandran plot and an RMS of 2.68. Key words:Chitinase, Moniliophthora perniciosa, kinetic characterization, purification, isoenzymes, heat stability, 3D structure, comparative modeling.

Lentinula edodes Genome Survey and Postharvest Transcriptome Analysis

“…These putative chitinases do not have CBM5, but several chitinases without CBM5 have chitindegrading activity in basidiomycetous fungi (46). In particular, the Coprinellus congregatus chitinase Chi2 purified from droplets during cap autolysis, which has a high similarity to that encoded by chi5, can degrade chitin oligosaccharides (47).…”

Application of Doehlert experimental design in the optimization of experimental variables for the Pseudozyma sp. (CCMB 306) and Pseudozyma sp. (CCMB 300) cell lysis

“…Purification of glucanase and chitinase from M. perniciosa was performed according to previous studies by the authors (SENA et al, 2011, GALANTE et al, 2012. On the other hand, in this study, an experimental design was used to evaluate the influence of independent variables on lysis of Pseudozyma sp.…”

Reducing the Standard Deviation in Multiple-Assay Experiments Where the Variation Matters but the Absolute Value Does Not

“…'Repeated measures' consists of an experimental setup very similar to the one used here and described in sec. 2.1, i.e., measuring the same quantity on N systems and repeating the experiment M times, but it contains a fundamental difference: it tackles measurements that are expected to change from repetition to repetition [e.g., a time series, or table II of (Galante et al, 2012) discussed in sec. 2.1].…”

“…As long as we expect different assays to yield the same results, their definition is compatible with what we do here. For example, the different assays in table II of (Galante et al, 2012), where the production of four isoforms of Monilophthora perniciosa chitinase is presented, do not qualify as the setup described here. The reason is simple: they are knowingly carried out at different pH and temperature.…”