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

Alex G. Taranto,Gonçalo A.G. Pereira,Aristóteles Góes-Neto,Maria G.B. Koblitz,Júlio C.M. Cascardo,Sandra H. Cruz,Sandra A. de Assis,Carlos P. Pirovani,Rafaela S. Galante

doi:10.1590/s0001-37652012000200016

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 44-73ºC and an optimum pH at 7.0-8.4. ChitMp II had an optimum temperature at 45-73ºC and an optimum pH at 7.0-8.4. ChitMp III had an optimum temperature at 54-67ºC and an optimum pH at 7.3-8.8. ChitMp IV had an optimum temperature at 60ºC 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.

Highlights

Chitin, a homopolymer of N-acetyl-D-glucosamine (Glc-NAc) residues linked by β-1,4 bonds, is a common constituent in cocoons, exoskeletons and peritrophic membranes of insects, crustacean shells, and fungal cell walls (Wang et al 2002, Schrempf 2001, Nicol 1991, Gooday 1977)
Chitinase was extracted from M. perniciosa as described in Materials and Methods
Chitinase was eluted on a Sephacryl S-200 column equilibrated with a 50 mM sodium phosphate buffer at pH 7.0

Summary

Introduction

A homopolymer of N-acetyl-D-glucosamine (Glc-NAc) residues linked by β-1,4 bonds, is a common constituent in cocoons, exoskeletons and peritrophic membranes of insects, crustacean shells, and fungal cell walls (Wang et al 2002, Schrempf 2001, Nicol 1991, Gooday 1977). All organisms that contain chitin have chitinases (EC 3.2.1.14) and other chitinolytic enzymes, as these enzymes are presumably required for the morphogenesis of cell walls and exoskeletons (Patil et al 2000, Dahiya et al 2006). Chitinases are glycosidases; they are found in organisms that do not contain chitin, such as plants, bacteria, and viruses. The physiological roles of these enzymes remain unresolved, it is clear that plant enzymes are involved in the defense against fungal pathogens and in regulating development (Merzendorfer 2006, Dahiya et al 2006, Duo-Chuan 2006). Filamentous fungi occupy a delicate balance between biosynthesis and hydrolysis of the cell wall during the remodeling for hyphal growth, hyphal branching and septum formation, thereby requiring the involvement of chitinolytic enzymes in this process (Dahiya et al 2006, Selvaggini et al 2004).

Methods

Results

Conclusion