Tee Boon L, Kaletunç Gönül
Food Agricultural and Biological Engineering Dept., The Ohio State University, 590 Woody Hayes Drive, Columbus, OH 43210, USA.
Biotechnol Prog. 2009 Mar-Apr;25(2):436-45. doi: 10.1002/btpr.117.
Thermostable alpha-amylase was covalently bound to calcium alginate matrix to be used for starch hydrolysis at liquefaction temperature of 95 degrees C. 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDAC) was used as crosslinker. EDAC reacts with the carboxylate groups on the calcium alginate matrix and the amine groups of the enzyme. Ethylenediamine tetraacetic acid (EDTA) treatment was applied to increase the number of available carboxylate groups on the calcium alginate matrix for EDAC binding. After the immobilization was completed, the beads were treated with 0.1 M calcium chloride solution to reinstate the bead mechanical strength. Enzyme loading efficiency, activity, and reusability of the immobilized alpha-amylase were investigated. Covalently bound thermostable alpha-amylase to calcium alginate produced a total of 53 g of starch degradation/mg of bound protein after seven consecutive starch hydrolysis cycles of 10 min each at 95 degrees C in a stirred batch reactor. The free and covalently bound alpha-amylase had maximum activity at pH 5.5 and 6.0, respectively. The Michaelis-Menten constant (K(m)) of the immobilized enzyme (0.98 mg/mL) was 2.5 times greater than that of the free enzyme (0.40 mg/mL). The maximum reaction rate (V(max)) of immobilized and free enzyme were determined to be 10.4-mg starch degraded/mL min mg bound protein and 25.7-mg starch degraded/mL min mg protein, respectively. The high cumulative activity and seven successive reuses obtained at liquefaction temperature make the covalently bound thermostable alpha-amylase to calcium alginate matrix, a promising candidate for use in industrial starch hydrolysis process.
将耐热α-淀粉酶共价结合到海藻酸钙基质上，用于在95℃的液化温度下进行淀粉水解。使用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐（EDAC）作为交联剂。EDAC与海藻酸钙基质上的羧基和酶的氨基反应。采用乙二胺四乙酸（EDTA）处理以增加海藻酸钙基质上可用于EDAC结合的羧基数量。固定化完成后，将珠子用0.1 M氯化钙溶液处理以恢复珠子的机械强度。研究了固定化α-淀粉酶的酶负载效率、活性和可重复使用性。在搅拌间歇式反应器中，在95℃下连续进行七个10分钟的淀粉水解循环后，共价结合到海藻酸钙上的耐热α-淀粉酶每毫克结合蛋白总共产生53克淀粉降解产物。游离的和共价结合的α-淀粉酶分别在pH 5.5和6.0时具有最大活性。固定化酶的米氏常数（K(m)）（0.98毫克/毫升）是游离酶（0.40毫克/毫升）的2.5倍。固定化酶和游离酶的最大反应速率（V(max)）分别测定为10.4毫克淀粉降解/毫升·分钟·毫克结合蛋白和25.7毫克淀粉降解/毫升·分钟·毫克蛋白。在液化温度下获得的高累积活性和七次连续重复使用使得共价结合到海藻酸钙基质上的耐热α-淀粉酶成为工业淀粉水解过程中有前景的候选物。
