Enzyme reduction on solid phase as a tool for the reversible immobilization of yeast β-galactosidase onto a thiol-reactive support

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• 作者：Ovsejevi ; K. ; Grazú ; V. ; Cuadra ; K. ; Batista-Viera ; F.
• 关键词：β ; -Galactosidase ; Enzyme immobilization ; Solid phase reducing agents ; Thiopropyl-supports ; Lactose hydrolysis
• 刊名：Enzyme and Microbial Technology
• 出版年：2004
• 期刊代码：26_01410229
• 类别：ce
• 出版时间：August 5, 2004
• 卷：35
• 期：2-3
• 页码：203-209
• 文件大小：107 K

摘要

Reduction of disulfide bonds in Kluyveromyces lactis β-galactosidase was assessed using solid phase reducing agents synthesized on three different supports (agarose, Toyopearl^® and Eupergit^®), provided with mercaptohydroxypropyl–ether groups. These agents are insoluble supports provided with thiol moieties as the reducing groups. The reduction performed on solid phase allowed a three-fold increase in the enzyme’s initial SH group content; the same result was achieved with dithiothreitol in solution. However, 10-fold less μmoles of SH groups per mg of protein were needed to perform the reduction of β-galactosidase with these solid phase reducing agents than the amount of SH groups required for reduction in solution. After the reduction process, the remaining content of SH groups on the solid phases was determined and found to be 60%for thiopropyl–agarose and thiopropyl–Toyopearl and 40%for thiopropyl–Eupergit. The reduced enzyme was reversibly immobilized on to agarose activated with thiol-reactive structures, thiolsulfinate (TSI) moieties, achieving high immobilization yields: more than 60%(for the enzyme reduced with dithiotreitol or thiopropyl–agarose) and more than 40%(for the enzyme reduced with thiopropyl–Toyopearl or thiopropyl–Eupergit). Half life at 45°C of the resulting derivatives varied from 90min (enzyme reduced with dithiothreitol) to 30min (enzyme reduced with thiopropyl–Toyopearl and thiopropyl–Eupergit). The percentage of lactolysis at 25°C was similar for all the derivatives, 90%after 2h of reaction.