Viswanath S, Wang J, Bachas L G, Butterfield D A, Bhattacharyya D
Department of Chemical & Materials Engineering, University of Kentucky, Lexington, Kentucky 40506, USA.
Biotechnol Bioeng. 1998 Dec 5;60(5):608-16.
Kinetic comparisons have been made between a randomly immobilized and a site-specifically immobilized subtilisin BPN' on microfiltration membranes of varying hydrophilicities in both aqueous and organic media. Site-directed mutagenesis was employed to introduce a single cysteine into the amino acid sequence of subtilisin at a location away from the active site. Immobilization of this mutant enzyme was then carried out using the single cysteine residue to orient the active site of the enzyme away from the membrane surface. Kinetic comparison of the immobilized mutant enzyme with the randomly immobilized wild-type enzyme in aqueous media showed an activity enhancement on both hydrophilic silica-containing and hydrophobic poly(ether)sulfone membranes. Higher loading efficiencies were observed for the site-directed enzyme on immobilization. Optimal enzyme loading values were calculated for the randomly immobilized enzyme. An enhancement of activity was also observed for the site-directed immobilized systems using nearly anhydrous hexane as the solvent.
在水性和有机介质中，对固定在不同亲水性微滤膜上的随机固定化枯草杆菌蛋白酶BPN'和位点特异性固定化枯草杆菌蛋白酶BPN'进行了动力学比较。采用定点诱变技术，在远离活性位点的位置将单个半胱氨酸引入枯草杆菌蛋白酶的氨基酸序列中。然后利用这个半胱氨酸残基将突变酶固定化，使酶的活性位点远离膜表面。在水性介质中，将固定化突变酶与随机固定化野生型酶进行动力学比较，结果表明，在含亲水性二氧化硅的膜和疏水性聚醚砜膜上，突变酶的活性均有所提高。定点固定化酶的负载效率更高。计算了随机固定化酶的最佳酶负载值。在以几乎无水的己烷为溶剂的定点固定化体系中，也观察到了活性的增强。
