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首页> 外文期刊>Journal of the Chinese Institute of Chemical Engineers >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
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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

机译：N-乙酰基-D-氨基葡萄糖2-表异构酶作为标记蛋白的表达和表征，用于将N-乙酰基-D-氨基葡萄糖转化为N-乙酰基-D-甘露糖胺

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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-m value of 8.9 mM, 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 degrees C.
机译：N-乙酰基-D-葡糖胺2-表异构酶（GlcNAc 2-表异构酶）是工业生产唾液酸（N-乙酰-D-神经氨酸）中使用的关键酶之一。它催化N-乙酰基-D-葡萄糖胺（GlcNAc）相互转化形成N-乙酰基-D-甘露糖胺（ManNAc），后者可与丙酮酸相互作用形成唾液酸。为了用于唾液酸合成，来自集胞藻属（Synechocystis sp。）的GlcNAc 2-表异构酶的基因。使用PCR方法扩增PCC 6803，并克隆以获得N端带有谷胱甘肽S-转移酶（GST）标签的大肠杆菌BL21。通过亲和标签，可以使用GST树脂柱将融合蛋白纯化至同质，并固定在与谷胱甘肽（GSH）结合的颗粒上。标记或非标记形式的过表达蛋白质具有酶促活性，能够催化由GlcNAc产生ManNAc。以ManNAc为底物时，纯化的融合蛋白的Km值为8.9 mM，与先前文献中报道的GlcNAc 2-表异构酶的值接近，这表明添加GST标签可能会略微改变GlcNAc 2-epimerase的亲和力。该酶为底物。用GST标记的融合蛋白获得最高酶活性的最佳pH确定为8.0。当估计pH 7时的酶活性时，最佳温度为50摄氏度。

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