Abstract
Humans have exploited natural resources for a variety of applications. Chitin and its derivative chitin oligosaccharides (CHOS) have potential biomedical and agricultural applications. Availability of CHOS with the desired length has been a major limitation in the optimum use of such natural resources. Here, we report a single domain hyper-transglycosylating chitinase, which generates longer CHOS, from Enterobacter cloacae subsp. cloacae 13047 (EcChi1). EcChi1 was optimally active at pH 5.0 and 40 °C with a Km of 15.2 mg ml−1, and kcat/Km of 0.011× 102 mg−1 ml min−1 on colloidal chitin. The profile of the hydrolytic products, major product being chitobiose, released from CHOS indicated that EcChi1 was an endo-acting enzyme. Transglycosylation (TG) by EcChi1 on trimeric to hexameric CHOS resulted in the formation of longer CHOS for a prolonged duration. EcChi1 showed both chitobiase and TG activities, in addition to hydrolytic activity. The TG by EcChi1 was dependent, to some extent, on the length of the CHOS substrate and concentration of the enzyme. Homology modeling and docking with CHOS suggested that EcChi1 has a deep substrate-binding groove lined with aromatic amino acids, which is a characteristic feature of a processive enzyme.
Highlights
Chitin [(C8H13O5N) n»1], is a linear homopolymer of N-acetyl glucosamine (GlcNAc) units linked through β (1 → 4) glycosidic bonds
We show that EcChi[1] has structural features of a hyper-TG chitinase similar to SpChiD of Serratia proteamaculans
Extracellular protein was isolated from the induced E. coli Rosetta-gami culture pellet and purified EcChi[1] through Ni-nitrilotriacetic acid (Ni-NTA) affinity chromatography
Summary
Chitin [(C8H13O5N) n»1], is a linear homopolymer of N-acetyl glucosamine (GlcNAc) units linked through β (1 → 4) glycosidic bonds. It is an abundant renewable natural resource next to cellulose in the biosphere. Chitin oligosaccharides (CHOS) generated from polymeric chitin with specific composition and length have potential applications. Chitinases could be possible alternatives for production of long chain CHOS, especially for biological applications. Chitinases, classified into two glycoside hydrolase (GH) families i.e. GH18 and GH19 could be exochitinases that act terminally and endochitinases that cleave randomly at internal sites of the chitin, eventually producing a variety of low molecular mass or short length CHOS4. Detailed understanding of chitinases at the biochemical or functional level may help in understanding their role in pathogenicity
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