Ortega Natividad, Perez-Mateos Manuel, Pilar María C, Busto María D
Department of Biotechnology and Food Science, Area of Biochemistry and Molecular Biology, University of Burgos, Plaza Misael Banuelos, Burgos, Spain.
J Agric Food Chem. 2009 Jan 14;57(1):109-15. doi: 10.1021/jf8015738.
Neutrase, a commercial preparation of Bacillus subtilis , was covalently immobilized on alginate-glutaraldehyde beads. Immobilization conditions and characterization of the immobilized enzyme were investigated. Central composite design and response surface methods were employed to evaluate the effects of immobilization parameters, such as glutaraldehyde concentration, enzyme loading, immobilization pH, and immobilization time. Under optimized working conditions (2% alginate, 6.2% glutaraldehyde, 61.84 U mL(-1) Neutrase, pH 6.2, and 60 min) the immobilization yield was about 50%. The immobilized enzyme exhibited higher K(m) compared to the soluble enzyme. The pH-activity profile was widened upon immobilization. The optimum temperature was shifted from 50 to 60 degrees C, and the apparent activation energy was decreased from 47.7 to 22.0 kJ mol(-1) by immobilization. The immobilized enzyme also showed significantly enhanced thermal stability.
中性蛋白酶（一种枯草芽孢杆菌的商业制剂）被共价固定在海藻酸钠-戊二醛珠上。研究了固定化条件及固定化酶的表征。采用中心复合设计和响应面方法来评估固定化参数的影响，如戊二醛浓度、酶负载量、固定化pH值和固定化时间。在优化的工作条件下（2%海藻酸钠、6.2%戊二醛、61.84 U mL(-1)中性蛋白酶、pH 6.2和60分钟），固定化产率约为50%。与可溶性酶相比，固定化酶表现出更高的米氏常数（K(m)）。固定化后pH-活性曲线变宽。最适温度从50℃变为60℃，固定化使表观活化能从47.7 kJ mol(-1)降至22.0 kJ mol(-1)。固定化酶还表现出显著增强的热稳定性。
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