Vidinha Pedro, Augusto Vera, Almeida Miguel, Fonseca Isabel, Fidalgo Alexandra, Ilharco Laura, Cabral Joaquim M S, Barreiros Susana
REQUIMTE/CQFB, Departamento de Química, FCT, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
J Biotechnol. 2006 Jan 2;121(1):23-33. doi: 10.1016/j.jbiotec.2005.06.018. Epub 2005 Aug 10.
Cutinase from Fusarium solani pisi was encapsulated in sol-gel matrices prepared with a combination of alkyl-alkoxysilane precursors of different chain-lengths. The specific activity of cutinase in a model transesterification reaction at fixed water activity in n-hexane was highest for the precursor combination tetramethoxysilane/n-butyltrimetoxysilane (TMOS/BTMS) in a 1:5 ratio, lower and higher chain lengths of the mono-alkylated precursor or decreasing proportions of the latter relative to TMOS leading to lower enzyme activity. Results obtained using combinations of three precursors confirmed the beneficial effect of the presence of BTMS in the preparations. Scanning electron microscopy of the 1:5 TMOS/n-alkylTMS gels showed a direct correlation between the macropore dimensions and the alkyl chain length of the alkylated precursor and revealed that TMOS/n-octylTMS gels suffered extensive pore collapse during the drying process. The specific activity of TMOS/BTMS sol-gel entrapped cutinase was similar to that exhibited by the enzyme immobilized by adsorption on zeolite NaY. However, the incorporation of different additives (zeolites, silica, Biogel, grinded sol-gel, etc.) having in common the capability to react with residual silanol groups of the sol-gel matrix brought about remarkable enhancements of cutinase activity, despite the fact that the global porosity of the gels did not change. The behavior of the gels in supercritical CO 2 (sc-CO 2) paralleled that exhibited in n-hexane, although cutinase activity was ca. one order of magnitude lower (i.e. sol-gel encapsulation did not prevent the deleterious effect of CO 2. The impact that functionalization of some of the additives had on cutinase activity indicates that the enzyme/matrix interactions must play an important role. Some of the best additives from the standpoint of enzyme activity were also the best from the standpoint of its operational stability (ca. 80% retention of enzyme activity at the tenth reutilization cycle). None of the additives that proved effective for cutinase could improve the catalytic activity of sol-gel encapsulated Pseudomonas cepacia lipase.
来自腐皮镰刀菌的角质酶被封装在由不同链长的烷基烷氧基硅烷前体组合制备的溶胶 - 凝胶基质中。在正己烷中固定水活度下的模型酯交换反应中，角质酶的比活性对于四甲氧基硅烷/正丁基三甲氧基硅烷（TMOS/BTMS）比例为1:5的前体组合最高，单烷基化前体链长更低或更高，或者相对于TMOS其比例降低都会导致酶活性降低。使用三种前体组合获得的结果证实了制剂中存在BTMS的有益效果。1:5 TMOS/正烷基TMS凝胶的扫描电子显微镜显示大孔尺寸与烷基化前体的烷基链长之间存在直接相关性，并表明TMOS/正辛基TMS凝胶在干燥过程中遭受广泛的孔塌陷。TMOS/BTMS溶胶 - 凝胶包封的角质酶的比活性与通过吸附固定在NaY沸石上的酶所表现出的比活性相似。然而，尽管凝胶的总体孔隙率没有变化，但加入具有与溶胶 - 凝胶基质的残留硅醇基团反应能力的不同添加剂（沸石、二氧化硅、Biogel、研磨的溶胶 - 凝胶等）会显著提高角质酶活性。凝胶在超临界CO₂（sc-CO₂）中的行为与在正己烷中表现的行为相似，尽管角质酶活性约低一个数量级（即溶胶 - 凝胶包封并未阻止CO₂的有害影响）。一些添加剂的功能化对角质酶活性的影响表明酶/基质相互作用必须起重要作用。从酶活性角度来看，一些最佳添加剂从其操作稳定性角度来看也是最佳的（在第十次重复使用循环时约80％的酶活性保留）。没有一种被证明对角质酶有效的添加剂能够提高溶胶 - 凝胶包封的洋葱伯克霍尔德氏菌脂肪酶的催化活性。
