Date of Award
Master of Science
Biochemistry, Biophysics and Molecular Biology
Olga A Zabotina
Plants are an important form of life. They are required for atmospheric oxygen, chemical energy (Food), medicine, fossilized fuels, and understanding cellular processes critical for survival. Plants have systems of defense composed of physical barriers, chemical messengers, and proteins that detect and respond to pathogen attack. These defenses can be eluded by some specialized fungal pathogens and cause diseases such as anthracnose, rots, and rusts. Plants express chitinases which degrade chitin, a major cell wall component of fungi, and shells of crustaceans, and cuticles of insects. Chitinase and chitinase-like proteins (CLPs) expressed in plants belong to the glycosyl hydrolases family 18 and 19, which exhibit varied functions. Maize chitinases belonging to the GH19 family have been recognized as important in the plant defense against fungal pathogens. They are composed of a small, hevein-like domain attached to a carboxy-terminal chitinase domain. The abundance and the diversity of oligo- and polysaccharides provide a wide range of biological roles attributed either to these carbohydrates or to their relevant enzymes, i.e., the glycoside hydrolases (GHs). The biocatalysis by these families of enzymes is highly attractive for the generation of products used in potential applications, e.g., pharmaceuticals and food industries. It is thus very important to extract and characterize such enzymes, particularly from plant tissues. In this study, two chitinases, EndochitinaseA and Basic-EndochitinaseA of the GH19 family from the roots of Zea mays were cloned, expressed, and characterized. Here, we report an analysis of the activity of the recombinant form of these chitinases under different conditions of salt, temperature, and pH. A modeling analysis of the proteins was also performed. The recombinant maize roots chitinases were purified from both Escherichia coli and HEK293 cells by Ni-NTA column purification. Enzyme activity was evaluated by the DNS method for reducing sugars, using colloidal chitin as substrate. Protein structure prediction was performed using the Swiss-Model and I-TASSER servers. Basic-EndochitinaseA is active in the hydrolysis of colloidal chitin and maintains its activity at medium temperatures (20 - 40°C) and close to neutral pH (pH 6 – 8, with optimum at pH 7), while EndochitinaseA showed lower activity, with optimum activity at high temperatures (50-60°C) and a broad pH optimum range (pH 6-8). Analysis of predicted structures show EA to be a class IV GH19 family chitinase while BEA was determined to be a class I GH19 family chitinase. Homology modelled structure show that Glu143, Glu152 and Ser 184 of EA, and Glu147 and Glu169 of BEA are directly involved in catalyzing chitin degradation. The results obtained make it possible to invest in these chitinases to be used as antifungal and other applications. Multiple isoforms of chitinases participate in plant defense against outside invaders, however, the functions of hydrolase family 19 (GH19) chitinases on pest control remain largely unknown. CLPs in the GH18 family have been structurally and functionally characterized; however, there are only few structures available for the GH19 family. Further analysis of the ligand binding to these chitinases will improve understanding of the substrate-recognition mechanism of GH-19 enzymes.
Samuel Oluwatoyin Shobade
Shobade, Samuel Oluwatoyin, "Preparation and characterization of recombinant plant chitinases" (2020). Graduate Theses and Dissertations. 18228.
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