Gene cloning,expression,purification and characterization of a beta-N-acetylhexosaminidase from Solitalea canadensisChinese Full Text
Meng Wang;Shuang Wei;Ting Wang;Josef Voglmeir;Li Liu;College of Food Science and Technology,Nanjing Agricultural University;
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-30 a 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 p H and temperature, metal ions dependency and inhibitor was done using p NP-β-Glc NAc 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 k Da. The results of SDS-PAGE revealed that the recombinant SoCaHexNAc（Gene Bank accession number: WP014682183.1） was expressed and purified successfully. Characterization of the enzyme showed that the optimum p H of SoCaHexNAc is 6.0, and the optimum temperature is 42 °C 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, Pug NAc, was 2 μmol/L. The substrate specificity result showed that the recombinant SoCaHexNAc was active to p NP-Glc NAc and p NP-Gal NAc. When being used for the hydrolysis of Glc NAc from natural glycans, the recombinant SoCaHexNAc exhibited linkage specificity evidenced by the fact that only β 1,6-linked Glc NAc in Core Ⅱ structure, but not the β 1,4-linked GlcNAc in NGA2 structure, was removed, although the terminal Glc NAc 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 Glc NAc 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.
- DOI：
10.13343/j.cnki.wsxb.20170109
- Series：
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- Classification Code：
Q78;Q936
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