Molecular Characterization of a Novel Chitinase Chi1 from SYBC-H1 and Its Use in -acetyl-d-glucosamine Production

Background: -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.

Results: A novel chitinase gene () cloned from SYBC-H1 and expressed in BL21(DE3) cells. The recombinant enzyme (Chi1) contains a glycosyl hydrolase family 18 catalytic module that shows low identity (12-27%) with the corresponding domain of the well-characterized chitinases. Chi1 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. Chi1 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, Chi1 exhibited / values of 7.8 ± 0.11 mL/s/mg and 239.1 ± 2.6 mL/s/μmol toward CC and 4-nitrophenol -diacetyl-β-d-chitobioside [-NP-(GlcNAc)], respectively. Analysis of the hydrolysis products revealed that Chi1 exhibits exo-acting, endo-acting and -acetyl-β-d-glucosaminidase activities toward -acetyl chitooligosaccharides (-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 Chi1 alone with a yield of nearly 100% and separated simply from the hydrolysate with a high purity of 98%.

Conclusion: The hydrolytic properties and good environmental adaptions indicate that Chi1 has excellent potential in commercial GlcNAc production. This is the first report on exo-acting, endo-acting and -acetyl-β-d-glucosaminidase activities from species.