Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase
Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase
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DOI：
10.1007/s10847-006-9163-5
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发表时间：
2007-01
影响因子：
2.3
通讯作者：
M. Prousoontorn;Supranee Pantatan
中科院分区：
文献类型：
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作者：
M. Prousoontorn;Supranee Pantatan
以双官能团戊二醛为载体，通过共价键合固定化了从Paenibacillus.A11中分离纯化的环糊精糖基转移酶(CGTase)。在所测试的载体中，氧化铝是最好的固定化载体。优化了几个参数对载体活化和固定化酶的影响。固定化CGTase的最佳制备工艺保持了31.2%的酶活力。对固定化后的酶性质进行了研究，并与游离酶进行了比较。固定化CGTase的最适pH从6.0移至7.0，最适温度保持不变(60℃)。固定化CGTase和游离CGTase的pH稳定性相似，但固定化CGTase的热稳定性比固定化CGTase高20%。由β-CD的形成速率测定了游离酶和固定化酶的动力学数据(KMandVmax)，发现固定化形式具有较高的KM和较低的Vmax。固定化CGTase在4℃和25℃下保存2个月也表现出较高的稳定性。将固定化于氧化铝上的酶进一步用于以抗坏血酸和α-环糊精为原料批量生产2-O-β-葡萄糖-L-抗坏血酸(AA-2G)。AA-2G的产率为2.92%，固定化CGTase的活性在3次循环后仍保持初始活性的74.4%。固定化CGTase在制备各种转糖基化化合物和淀粉水解法生产环糊精方面具有广阔的应用前景。
Cyclodextrin glycosyltransferase (CGTase) isolated and purified fromPaenibacillussp.A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers, alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher. Kinetic data (KMandVmax) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized form had higherKMand lowerVmax. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by the hydrolysis of starch.