中文摘要：

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）。

英文摘要：

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.