Enzymatic characterization and structure-function relationship of two chitinases, LmChiA and LmChiB, from Listeria monocytogenes

• Published in: Heliyon 2020-07, Vol.6 (7), p.e04252-e04252, Article e04252
• Main Authors: Churklam, Wasinee, Aunpad, Ratchaneewan
• Format: Article
• Language: English
• Subjects: Biochemistry; Biotechnology; Characterization; Chitinase; Homology modeling; Infectious disease; Listeria monocytogenes; LmChiA; LmChiB; Microbiology; Molecular biology; Mutagenesis
• ISSN: 2405-8440; 2405-8440
• DOI: 10.1016/j.heliyon.2020.e04252

Listeria monocytogenes possesses two chitinases (LmChiA and LmChiB) belonging to glycoside hydrolase family 18 (GH18). In this study, two chitinase genes (lmchiA and lmchiB) from L. monocytogenes 10403S were cloned and their biochemical characteristics were studied. Using colloidal chitin as substrate, both chitinases exhibited maximum catalytic activity at pH 6–7 with optimum temperature at 50 °C. Their activities were stable over broad pH (3–10) and temperature (10–50 °C) ranges. Kinetic analysis using [4NP-(GlcNAc)2] as substrate indicated that LmChiB had an approximately 4-fold lower Km and 2-fold higher kcat than LmChiA, suggesting that the catalytic specificity and efficiency of LmChiB were greater than those of LmChiA. LmChiA and LmChiB showed the same reactivity toward oligomeric substrates and exhibited both non-processive endo-acting and processive exo-acting (chitobiosidase) activity on colloidal chitin, chitin oligosaccharides and 4-nitrophenyl substrates. Structure-based sequence alignments and homology modeling of the catalytic domains revealed that both chitinases consisted of an (α/β)8 TIM barrel fold with a conserved DXDXE motif. The key residues involved in the substrate hydrolysis were conserved with other bacterial chitinases. The site-directed mutagenesis of conserved Asp and Glu residues in DXDXE motif of both chitinases significantly reduced the chitinolytic activity toward colloidal chitin substrate and revealed their critical role in the catalytic mechanism. LmChiA and LmChiB might have potential in chitin waste utilization and biotechnological applications. Microbiology; Biotechnology; Biochemistry; Molecular Biology; Infectious Disease; Chitinase; Listeria monocytogenes; Characterization; LmChiA; LmChiB; Homology modeling; mutagenesis