Gene cloning,expression,purification and characterization of a beta-N-acetylhexosaminidase from Solitalea canadensis

Abstract:

[Objective] We intended to discover and characterize a novel beta-N-acetylhexosaminidase from Solitalea canadensis. [Methods] Genomic DNA extracted from Solitalea canadensis was used as the template for gene cloning of the beta-N-acetylhexosaminidase using PCR reaction. The PCR product was digested with restriction endonucleases Nde I and Xho I, then ligated to pET-30a vector. After plasmid was transformed into E.coli BL21 (DE3) cells, the recombinant enzyme was expressed by IPTG induction and purified with nickel affinity chromatography. Characterization of recombinant SoCaHexNAc including optimal pH and temperature, metal ions dependency and inhibitor was done using pNP-β-GlcNAc as the substrate. Effect of different chemical compounds and disaccharides on the enzyme activity was also measured. [Results] A beta-N-acetylhexosaminidase gene with an open reading fragment of 2586 bp was successfully obtained, which encodes 856 amino acids with a putative molecular size of 97 kDa. The results of SDS-PAGE revealed that the recombinant SoCaHexNAc (GeneBank accession number:WP_014682183.1) was expressed and purified successfully. Characterization of the enzyme showed that the optimum pH of SoCaHexNAc is 6.0, and the optimum temperature is 42℃ with a half-life being less than 5 minutes. The recombinant SoCaHexNAc was sensitive to SDS and could be partly inhibited by Trition X-100 and urea. Different concentrations of lactose, maltose and cellobiose could also inhibit the activity of SoCaHexNAc to different extends. The IC₅₀ of a specific β-N-acetylhexosaminidase inhibitor, PugNAc, was 2 μmol/L. The substrate specificity result showed that the recombinant SoCaHexNAc was active to pNP-GlcNAc and pNP-GalNAc. When being used for the hydrolysis of GlcNAc from natural glycans, the recombinant SoCaHexNAc exhibited linkage specificity evidenced by the fact that only β 1,6-linked GlcNAc in Core Ⅱ structure, but not the β 1,4-linked GlcNAc in NGA2 structure, was removed, although the terminal GlcNAc was the exceptional terminal sugar in both substrates. [Conclusion] A beta-N-actylhexosamindase with activity specifically towards β 1,6-linked but not β 1,4-linked GlcNAc was discovered and characterized from Solitalea canadensis for the first time. The results of characterization and substrate specificity showed it might be a potential novel tool enzyme which could be used in structure analysis of glycans.