Solitalea canadensis源β-N-乙酰氨基己糖苷酶的基因克

发布时间：2018-07-08 09:26

本文选题：Solitalea + canadensis　；参考：《微生物学报》2017年08期

【摘要】：【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,并将重组质粒导入大肠杆菌BL21(DE3)中进行原核表达。重组蛋白经Ni-NTA纯化,以对硝基苯酚-β-乙酰氨基葡萄糖(pNP-β-Glc NAc)为底物研究其酶学性质,包括最适温度、最适p H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(Gene Bank:WP_014682183.1),全长2586 bp,重组表达所得蛋白表观分子量约为97 k Da,最适pH 6.0,最适温度42°C,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IC_(50)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β1-6连接的乙酰氨基葡萄糖而不能水解β1-4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。
[Abstract]:[Objective] to clone the gene of beta -N- acetaminosidase encoding beta -N- glucosidase in bacterial Solitalea canadensis and obtain recombinant beta -N- acetylglucosidase through prokaryotic expression and study its enzymatic properties. [Methods] the Solitalea canadensis genome DNA was used as a template to clone and encode beta -N- acetaminophen by adding tail PCR. The recombinant expression vector containing the histidine label was constructed, and the recombinant plasmid was introduced into the Escherichia coli BL21 (DE3) for prokaryotic expression. The recombinant protein was purified by Ni-NTA, and its enzymatic properties were studied with p-nitrophenol beta acetaminosamine (pNP- beta -Glc NAc) as the substrate, including the optimum temperature, the optimum P H and the metal ions. [results] [results] the gene fragment of beta -N- acetylhexosidase (Gene Bank:WP_014682183.1) was cloned from strain Solitalea canadensis. The total length of the gene was 2586 BP. The apparent molecular weight of the recombinant protein was about 97 K Da, the optimum pH 6, the optimum temperature 42 degree C, but unstable and the half-life less than 5 min., the enzyme was twelve alkyl. Sodium sulfonate (SDS) is sensitive and the activity is inhibited by Triton X-100 and urea. In addition, two sugar molecules can also inhibit the activity of the recombinant enzyme in varying degrees. The specific inhibitor PugNAc (O- (2-Acetamido-2-deoxy-D-glucopyranosylideneamino) N-phenylcarbamate) has a IC_ (50) of 2 micron to the enzyme and the recombinant protein can hydrolyze p-nitrophenol in addition to the enzyme. Beta acetaminoglucoside and p-nitrophenol - beta acetaminophen galactose (pNP- beta -GalNAc) can also hydrolyze acetyl glucosamine at the end of Core II at the core structure of O- chain. [Conclusion] this paper was first cloned from Solitalea canadensis to hydrolyze the acetylglucosamine linked to the terminal beta 1-6, which could not be hydrolyzed. Beta - -N- acetaminoglycosidase (beta -N-), which is a bonding bond, has been studied and analyzed by substrate specificity, which provides a theoretical basis for the development of a highly efficient and specific sugar chain analysis tool enzyme.
【作者单位】：南京农业大学食品科学技术学院;
【基金】：国家自然科学基金(31371739,31671854) 中央高校基本科研业务费(KYRC201209)~~
【分类号】：Q78;Q936

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