Abstract
Chitinolyticbacter meiyuanensis SYBC-H1, a bacterium capable of hydrolyzing chitin and shrimp shell to N-acetyl glucosamine (GlcNAc) as the only product, was isolated previously. Here, the hydrolysis mechanism of this novel strain toward chitin was investigated. Sequencing and analysis of the complete genome of SYBC-H1 showed that it encodes 32 putatively chitinolytic enzymes including 30 chitinases affiliated with the glycoside hydrolase (GH) families 18 (26) and 19 (4), one GH family 20 β-N-acetylglucosaminidase (NAGase), and one Auxiliary Activities (AA) family 10 lytic polysaccharide monooxygenase (LPMO). However, only eight GH18 chitinases, one AA10 LPMO, and one GH20 NAGase were detected in the culture broth of the strain, according to peptide mass fingerprinting (PMF). Of these, genes encoding chitinolytic enzymes including five GH18 chitinases (Cm711, Cm3636, Cm3638, Cm3639, and Cm3769) and one GH20 NAGase (Cm3245) were successfully expressed in active form in Escherichia coli. The hydrolysis of chitinous substrates showed that Cm711, Cm3636, Cm3638, and Cm3769 were endo-chitinases and Cm3639 was exo-chitinase. Moreover, Cm3639 and Cm3769 can convert the GlcNAc dimer and colloidal chitin (CC) into GlcNAc, which showed that they also possess NAGase activity. In addition, NAGase Cm3245 possesses a very high exo-acting activity of hydrolyzing GlcNAc dimer. These results suggest that chitinases and NAGase from SYBC-H1 both play important roles in conversion of N-acetyl chitooligosaccharides to GlcNAc, resulting in the accumulation of the final product GlcNAc. To our knowledge, this is the first report of the complete genome sequence and chitinolytic enzyme genes discovery of this strain.
Highlights
Chitin, a β-1,4-linked N-acetyl glucosamine (GlcNAc) polymer, is the second most abundant polysaccharide in nature with production of 100 billon tons per year (Kaur and Dhillon, 2015)
The degradation of chitin to GlcNAc and (GlcNAc) requires cooperation of chitinolytic enzymes such as chitinase [mainly belonging to glycoside hydrolase (GH) families 18 and 19; hydrolyzes chitin to N-acetyl chitooligosaccharides (N-acetyl COSs)]; β-Nacetylglucosaminidase (GH family 20; hydrolyzes N-acetyl COSs to GlcNAc); and lytic polysaccharide monooxygenase (LPMO) [Auxiliary Activity (AA) families 10 and 11; cleavage of chitin chains with oxidation to enhance the hydrolysis of chitin] (Zhang et al, 2018b)
The genome shows the highest similarity to the reported genome of Chitiniphilus shinanonensis strain SAY3 (95.85%) (Sato et al, 2009), followed by Chitinibacter tainanensis DSM15459 (94.58%) (Chern et al, 2004)
Summary
A β-1,4-linked N-acetyl glucosamine (GlcNAc) polymer, is the second most abundant polysaccharide in nature with production of 100 billon tons per year (Kaur and Dhillon, 2015). Enzymatic production of GlcNAc from chitin using chitinolytic enzymes is a better approach because it is generally environmentally friendly, produces high yields, and generates GlcNAc with high bioactivities (Zhang et al, 2018a). Most reported crude chitinolytic enzymes always produce a mixture of GlcNAc and N-acetyl COSs during enzymatic hydrolysis of chitin. The crude enzymes from Bacillus licheniformis SK-1 chitinase completely hydrolyzed chitin within 6 days, resulting in 75% GlcNAc and 20% (GlcNAc) (Pichyangkura et al, 2002). There are few reports of crude chitinolytic enzymes capable of hydrolyzing chitin to produce GlcNAc as the only product (Sashiwa et al, 2002). Crude chitinolytic enzymes secreted by SYBC-H1 efficient degrade chitin and shrimp shells to GlcNAc without N-acetyl COSs (Zhang et al, 2016b; Wei et al, 2017). This study will help us understand the mechanism by which chitinolytic enzymes from SYBC-H1 convert chitin to GlcNAc as the sole product and advance potential biotechnological applications of SYBC-H1
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