Abstract
Chitinases catalyze the hydrolysis of chitin, a linear homopolymer of β-(1,4)-linked N-acetylglucosamine. The broad range of applications of chitinolytic enzymes makes their identification and study very promising. Metagenomic approaches offer access to functional genes in uncultured representatives of the microbiota and hold great potential in the discovery of novel enzymes, but tools to extensively explore these data are still scarce. In this study, we develop a chitinase mining pipeline to facilitate the comprehensive search of these enzymes in environmental metagenomic databases and also to explore phylogenetic relationships among the retrieved sequences. In order to perform the analyses, UniprotKB fungal and bacterial chitinases sequences belonging to the glycoside hydrolases (GH) family-18, 19 and 20 were used to generate 15 reference datasets, which were then used to generate high quality seed alignments with the MAFFT program. Profile Hidden Markov Models (pHMMs) were built from each seed alignment using the hmmbuild program of HMMER v3.0 package. The best-hit sequences returned by hmmsearch against two environmental metagenomic databases (Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis—CAMERA and Integrated Microbial Genomes—IMG/M) were retrieved and further analyzed. The NJ trees generated for each chitinase dataset showed some variability in the catalytic domain region of the metagenomic sequences and revealed common sequence patterns among all the trees. The scanning of the retrieved metagenomic sequences for chitinase conserved domains/signatures using both the InterPro and the RPS-BLAST tools confirmed the efficacy and sensitivity of our pHMM-based approach in detecting putative chitinases sequences. These analyses provide insight into the potential reservoir of novel molecules in metagenomic databases while supporting the chitinase mining pipeline developed in this work. By using our chitinase mining pipeline, a larger number of previously unannotated metagenomic chitinase sequences can be classified, enabling further studies on these enzymes.
Highlights
Enzymes are catalysts that support the development of environmental-friendly industrial processes
The results confirmed the efficacy of our pipeline in detecting chitinase sequences and highlighted the power of Profile Hidden Markov Models (pHMMs)-based strategies to identify remote homologues
The InterPro search inferred the occurrence of chitinase signatures in 81.7%, 89.4% and 98.8% of the metagenomic sequences belonging to glycoside hydrolases (GH) family-18, 19 and 20, respectively (Figures 2(d)-(f))
Summary
Enzymes are catalysts that support the development of environmental-friendly industrial processes. Chitinases are enzymes responsible for the hydrolysis of chitin, a linear homopolymer of β-(1,4)-linked N-acetylglucosamine, which is the second most abundant biopolymer in nature. GH family-19 contains plant, bacteria and some Streptomyces chitinases, and GH family includes the β-N-acetylhexosaminidases from bacteria, fungi, Streptomyces and humans [4] [7]. These enzymes have widespread applications, such as in bioremediation [8], biological control [9]-[11], production of chitooligosaccharides [12]-[14], preparation of single-cell protein [15] and isolation of protoplasts from fungi [16]
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