Abstract
BackgroundN-acetyl-d-glucosamine (GlcNAc) possesses many bioactivities that have been used widely in many fields. The enzymatic production of GlcNAc is eco-friendly, with high yields and a mild production process compared with the traditional chemical process. Therefore, it is crucial to discover a better chitinase for GlcNAc production from chitin.ResultsA novel chitinase gene (Cmchi1) cloned from Chitinolyticbacter meiyuanensis SYBC-H1 and expressed in Escherichia coli BL21(DE3) cells. The recombinant enzyme (CmChi1) contains a glycosyl hydrolase family 18 catalytic module that shows low identity (12–27%) with the corresponding domain of the well-characterized chitinases. CmChi1 was purified with a recovery yield of 89% by colloidal chitin affinity chromatography, whereupon it had a specific activity of up to 15.3 U/mg. CmChi1 had an approximate molecular mass of 70 kDa after the sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its optimum activity for colloidal chitin (CC) hydrolysis occurred at pH 5.2 and 50 °C. Furthermore, CmChi1 exhibited kcat/Km values of 7.8 ± 0.11 mL/s/mg and 239.1 ± 2.6 mL/s/μmol toward CC and 4-nitrophenol N,N′-diacetyl-β-d-chitobioside [p-NP-(GlcNAc)2], respectively. Analysis of the hydrolysis products revealed that CmChi1 exhibits exo-acting, endo-acting and N-acetyl-β-d-glucosaminidase activities toward N-acetyl chitooligosaccharides (N-acetyl CHOS) and CC substrates, behavior that makes it different from typical reported chitinases. As a result, GlcNAc could be produced by hydrolyzing CC using recombinant CmChi1 alone with a yield of nearly 100% and separated simply from the hydrolysate with a high purity of 98%.ConclusionThe hydrolytic properties and good environmental adaptions indicate that CmChi1 has excellent potential in commercial GlcNAc production. This is the first report on exo-acting, endo-acting and N-acetyl-β-d-glucosaminidase activities from Chitinolyticbacter species.
Highlights
N-acetyl-d-glucosamine (GlcNAc) possesses many bioactivities that have been used widely in many fields
Detection of hydrolysis products Reaction mixtures containing purified CmChi1 (50 μg) and various substrates [colloidal chitin (CC) and N-acetyl CHOSs (DP 2–6)] at a final concentration of 10 g/L were incubated in 100 mL of 50 mM sodium citrate buffer at 50 °C for various time intervals
CmChi1 can be purified with a recovery yield of 89% and a specific activity of 15.3 U/mg by colloidal chitin affinity chromatography
Summary
N-acetyl-d-glucosamine (GlcNAc) possesses many bioactivities that have been used widely in many fields. About 6–8 million tonnes of crab, shrimp and lobster shell wastes are produced globally annually. This causes severe environmental pollution because it is not utilized effectively [2]. N-acetyl-d-glucosamine (GlcNAc), the monomeric unit of the polymer chitin, exhibits many bioactivities that have been used widely in many fields, such as the food, pharmaceutical, biomedical and fine chemicals industries [3]. It would be economically and environmentally significant if a way could be found to efficiently produce GlcNAc from normally discarded chitin resources
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