- 期刊
Expression and Characterization of the N-Acetyl-D-Glucosamine 2-Epimerase as a Tagged Protein for the Conversion of N-Acetyl-D-Glucosamine to N-Acetyl-D-Mannosamine
將N-乙醯-D-葡萄糖胺異轉化成N-乙醯-D-甘露醣胺之N-乙醯-D-葡萄糖胺2-表異構酶的融合蛋白質表現及其特性研究
摘要
N-乙醯-D-葡萄糖胺2-表異構酶是工業化生產唾液酸時之關鍵酵素之一，其酵素功能可將N-乙醯-D-葡萄糖胺異構化成N-乙醯-D-甘露醣胺，N-乙醯-D-甘露醣胺再與丙酮酸反應生成唾液酸。本研究將藍綠藻Synechocystis sp. PCC6803中的N-乙醯-D-葡萄糖胺2-表異構酶基因，利用聚合酶連鎖反應法增幅複製，並且基因選殖至BL21型之大腸桿菌中，以融合蛋白質系統表現N端接有麩胱苷肽-S轉移酶蛋白標誌的N-乙醯-D-葡萄糖胺2-表異構酶。由於麩胱苷肽-S轉移酶蛋白(GST)與麩胱苷肽(GSH)之間有強的親和性，因此表現之融合蛋白質可藉由表面含有麩胱苷肽之顆粒，進行純化或固定化。對N-乙醯-D-甘露醣胺基質而言，N-乙醯-D-葡萄糖胺2-表異構酶之K(下標 m)值為8.9mM。此值非常接近該酵素之文獻值，顯示增加麩胱苷肽-S轉移酶蛋白標誌並未顯著地影響酵素對基質的親和性。含麩胱苷肽-S轉移酶蛋白標誌之融合蛋白質，其活性最大時的pH值為8.0；若在pH7之下測活性，溫度為50℃時活性最大。
關鍵字
無資料並列摘要
N-Acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) is one of the key enzymes used in the industrial production of sialic acid (N-acetyl-D-neuraminic acid). It catalyzes the inter-conversion of N-acetyl-D-glucosamine (GlcNAc) to form N-acetyl-D-mannosamine (ManNAc), which can interact with pyruvate to form sialic acid. For application in sialic acid synthesis, the gene of GlcNAc 2-epimerase from Synechocystis sp. PCC 6803 was amplified using the PCR method and cloned to obtain Escherichia coli BL21 with a glutathione S-transterase (GST) tag in the N-terminus. By means of affinity tag, the fusion protein could be purified to homogeneity using a GST-resin column and immobilized on particles bound with glutathione (GSH). The overexpressed protein in either tagged or non-tagged form was enzymatic active and able to catalyze the production of ManNAc from GlcNAc. With ManNAc used as the substrate, the purified fusion protein showed a K(subscript m) value of 8.9mM, which is close to the values previously reported in the literature for GlcNAc 2-epimerase, suggesting that the addition of the GST tag could marginally change the affinity of this enzyme for the substrate. The optimal pH for attaining the highest enzyme activity with the GST-tagged fusion protein was determined to be 8.0. When the enzymatic activity was estimated at pH 7, the optimal temperature was 50℃.