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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2012, Vol. 32 Issue (09): 28-33    
研究报告     
人己糖胺酶D的原核表达、纯化及酶学特性研究
刘琳1, 徐2, 蔡春梅3, 李静2, 蔡玉梅1
1. 山东农业大学动物科技学院 泰安 271018;
2. 南开大学药学院药物化学生物学国家重点实验室 天津市分子药物研究重点实验室 天津 300071;
3. 德州市第二人民医院 德州 253024
Expression, Purification and Enzymatic Characteristics of Human Hex D in E.coli
LIU Lin1, XU Yan2, CAI Chun-mei3, LI Jing2, CAI Yu-mei 1
1. College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, China;
2. College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China;
3. Dezhou Second People’s Hospital, Dezhou 253024, China
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摘要: 糖基化修饰是一种重要的蛋白质翻译后修饰,参与生物体中的信号传导、细胞识别等多种细胞活动,糖基缀合物的正常水解是生物体代谢的必需途径。人己糖胺酶D(Hexosaminidase D)是新发现的一种存在于人细胞质中的切除GalNAc糖基化修饰的外切酶,但该酶的酶学特性尚不清楚。利用PCR的方法,将Hex D的cDNA序列构建到质粒pET3C中,重组质粒转化大肠杆菌BL21(DE3)plysS后,通过优化异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度(0.1 mmol/L)和诱导时间(10 h)获得了高可溶性表达的重组蛋白酶。采用Ni-NTA亲和层析对重组蛋白进行了纯化,SDS-PAGE检测分子量的大小(58 kDa)和纯度(95%以上)。以4-甲基伞形酮-2-乙酰氨基-2-脱氧半乳糖(4-MU-O-GalNAc)为荧光底物,测定该酶的最适反应pH值为5.5,最适反应温度为37℃,且该酶的热稳定性较好,在50℃下放置半小时仍有较高活性,1mmol/L的金属离子(CuSO4、FeSO4·7H2O、MgCl2·6H2O、CaCl2、NiSO4·6H2O、AlCl3·6H2O、ZnSO4·7H2O、MnCl2)及EDTA对该酶活性影响不大,10mmol/L AlCl3、CuSO、FeSO4·7H2O对该酶有不同程度的抑制。在最适条件下(pH 5.5,37℃)下,该酶的Km为0.16mmol/L,最大反应速率为3.06 μmol/(min·mg)。
关键词: 己糖苷酶D表达纯化酶学特性    
Abstract: Glycosylation is an important protein post-translational modification, which is involved in many cellular processes, including signal transduction and cell recognition. Properly hydrolysis of glycoconjugates is essential for organism metabolism. Human hexosaminidase D (Hex D) is a newly discovered glycosidase which cleaves GalNAc ( O-linked N-Acetyl-β-D-glatosamine) modification. However, the enzymatic characteristics of Hex D remains unknown. Here, the cDNA sequence was amplified by PCR and cloned into plasmid pET3C. After transformed the recombinant plasmids into Escherichia coli BL21 (DE3) plys, the expression of Hex D was optimized with 0.1 mmol/L IPTG in 10 hours and purified it with the Ni-NTA affinity chromatography. SDS-PAGE verified the molecular weight (58 kDa) and the purity (> 95%). Using 4-MU-GalNAc (4-Methylumbelliferyl 2-acetamido-2-deoxy-β-D-galctopyranoside)as substrate, the optimal pH and temperature for Hex D is 5.5 and 37℃ respectively. Assay of Hex D pre-incubated for 30 min at different temperature indicated that it had high thermal stability and retain activity at 50℃. 1mmol/L metal ions(CuSO4、FeSO4·7H2O、MgCl2·6H2O、CaCl2、NiSO4·6H2O、AlCl3·6H2O、ZnSO4·7H2O、MnCl2)and EDTA has no different effect on Hex D enzymic activity. However,10mmol/L AlCl3,CuSO4 or FeSO4·7H2O decreased the activity of Hex D to different extent. Using the optimum pH and temperature the Km value and Vmax of Hex D were 0.16mmol/L and 3.06 μmol/(min·mg) respectively.
Key words: Hexosaminidase D    Expression    Purification    Enzymatic characteristics
收稿日期: 2012-04-27 出版日期: 2012-09-25
ZTFLH:  Q786  
基金资助: 国家自然科学基金资助项目(31000371)
通讯作者: 蔡玉梅     E-mail: caiyum@163.com
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引用本文:

刘琳, 徐, 蔡春梅, 李静, 蔡玉梅. 人己糖胺酶D的原核表达、纯化及酶学特性研究[J]. 中国生物工程杂志, 2012, 32(09): 28-33.

LIU Lin, XU Yan, CAI Chun-mei, LI Jing, CAI Yu-mei. Expression, Purification and Enzymatic Characteristics of Human Hex D in E.coli. China Biotechnology, 2012, 32(09): 28-33.

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https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I09/28

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