Local Fungal Endophytes as Rich Sources of Chitinase Genes
Zabrina Bernice L. Malto, Christine Jurene O. Bacal,
Mark Jeffrey S. Diaz, and Eizadora T. Yu*
Institute of Chemistry, College of Science, University of the Philippines
Diliman, Quezon City 1101 Philippines
*Corresponding Author: This email address is being protected from spambots. You need JavaScript enabled to view it.
ABSTRACT
The ability of three fungal endophytes (JB10, JB11, and D12 isolates) to degrade chitin, and their potential as microbial sources of chitinases was investigated. Amplification and sequencing of the ITS regions revealed the identity of the fungal isolates: JB10 (Fomitopsis sp.), JB11 (Aspergillus tubingensis), and D12 (Daldinia eschscholzii). All three fungi were able to grow on minimal media with colloidal chitin as sole carbon source, albeit at different rates. Isolates JB11 and D12 are observed to have comparable or faster growth rates in chitin as compared to the simpler potato dextrose carbon source. Turbidimetric measurements show that the fungal cultures are able to degrade chitin with 3–5 d of incubation. While the crude, secreted proteins from these three fungi show comparable total chitinolytic activities (~0.35 U/mL), JB11 was found to have the highest exochitinase activity (~0.25 U/mL). Bioinformatic analysis of the chitinase (GH18) genes for A. tubingensis (JB11) and D. eschscholzii (D12) revealed variability in the GH18 chitinase sequences in terms of the amino acid sequences of the canonical DXXDXDXE catalytic motif as well as the presence of additional domain architectures, which make these fungi ideal sources for chitinases for both biotechnology applications and chitinase enzyme mechanistic studies.
INTRODUCTION
The processing of crustacean products generates a lot of shell waste as crustacean meat accounts for less than 50% of the animal’s body mass. Efforts to valorize and refine these crustacean shells for chemicals (e.g., amino acids, calcium carbonate, and N-acetyl glucosamine or NAG) are being explored to create a high-value supply chain for what is normally just discarded as waste (Yan and Chen 2015). While the complete breakdown of chitin to NAG is desirable, depolymerization to lower molecular weight chitin oligosaccharides (CTOS) can also yield high-value products with biomedical applications. Enzymes such as chitinases can degrade chitin into CTOS or NAG monomers by hydrolyzing the glycosidic bonds under physiological conditions. These chitin-degrading enzymes can be endochitinases, which cleave the polymer internally producing CTOS or exochitinases, which cleave the reducing end of the polymer producing NAG [for N-acetyl glucosaminidases (NAGase)] or (NAG)2 (for chitobiosidases) (Hamid 2013). These enzymes can, therefore, be used in the processing of crustacean waste to produce CTOS or NAG precursors. . . . read more
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