[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 1,6-1inked GlcNAc in Core Ⅱ structure,but not the β 1,4-1inked 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 1,6-1inked but not β 1,4-1inked 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.%[目的]对细菌Solitalea canadensis中编码β-N-乙酰氨基己糖苷酶的基因进行克隆,通过原核表达获得重组β-N-乙酰氨基己糖苷酶,并研究其酶学性质.[方法]以Solitalea canadensis基因组DNA为模板,使用加尾PCR的方法克隆编码β-N-乙酰氨基己糖苷酶的基因,构建含有组氨酸标签的重组表达载体,并将重组质粒导人大肠杆菌BL21(DE3)中进行原核表达.重组蛋白经Ni-NTA纯化,以对硝基苯酚-p-乙酰氨基葡萄糖(pNP-β-GlcNAc)为底物研究其酶学性质,包括最适温度、最适pH以及金属离子和抑制剂的影响.[结果]从菌株Solitalea canadensis克隆得到了β-N-乙酰氨基己糖苷酶基因片段(GeneBank:WP_014682183.1),全长2586 bp,重组表达所得蛋白表观分子量约为97 kDa,最适pH 6.0,最适温度42℃,但不稳定,半衰期小于5 min.该酶对十二烷基磺酸钠(SDS)敏感,活性受Triton X-100和尿素的抑制.此外二糖分子也能不同程度地抑制该重组酶的活性,特异性抑制剂PugNAc (O-(2-Acetamido-2-deoxy-D-glucopyranosylideneamino) N-phenylcarbamate)对该酶的IC50为2μmol/L.该重组酶蛋白除能水解对硝基苯酚-β-乙酰氨基葡萄糖苷和对硝基苯酚-β-乙酰氨基半乳糖(pNP-β-GalNAc)外,还能对O-链聚糖核心结构Core Ⅱ末端的乙酰氨基葡萄糖进行水解.[结论]本文首次从Solitalea canadensis中克隆得到能水解末端β 1-6连接的乙酰氨基葡萄糖而不能水解β 1-4连接键的β-N-乙酰氨基己糖苷酶,并对其进行了酶学性质研究和底物特异性分析,为开发高效特异性强的糖链分析工具酶提供理论基础.
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