Cloning and Expression of a Novel Chitinase Gene and the Analysis of Its Hydrolysis Products

Abstract:

[Objective] A novel chitinase gene was cloned from the genomic DNA of psychrophilic bacterium, Pseudoalteromonas sp. DL-6 and heterologously expressed in Escherichia coli BL21(DE3). The recombinant protein was purified and the enzymatic hydrolysates were analyzed. [Methods] The chitinase gene, chiA, was amplified from Pseudoalteromonas sp. DL-6 by using a PCR protocol, linked to pET28a, and finally expressed in E. coli BL21(DE3). The molecular weight and purity were determined by SDS-PAGE, and enzymatic activity was detected by 4-methylumbelliferyl-β-D-N,N′-diacetylchitobioside (4MU-(GlcNAc)2). The hydrolysis products were identified by electrospray ionisation mass spectrometry (ESI-MS). [Results] The chiA (GenBank: KF234015) was efficiently expressed in E. coli BL21(DE3), and the total activity of purified ChiA was 168.68 U using Ni-NTA resin. ESI-MS analysis showed that the hydrolysis products of 1% colloidal chitin after ChiA digestion consisted of a series of chitin oligomers. [Conclusion] The research provided a reference for endo-type ChiA used in the food, pharmaceutical and agricultural industry.