Hrydziuszko Zofia, Dmytryk Agnieszka, Majewska Paulina, Szymańska Katarzyna, Liesiene Jolanta, Jarzębski Andrzej, Bryjak Jolanta
Wrocław University of Technology, Faculty of Chemistry, Department of Bioorganic Chemistry, Poland.
Silesian University of Technology, Department of Chemical Engineering, Poland.
Acta Biochim Pol. 2014;61(1):1-6. Epub 2014 Mar 17.
In bioprocesses lipases are typically used in immobilized form, irrespective of type of reaction systems, to ensure an even distribution of catalysts in water restricted media and/or to facilitate separation and reuse. In these studies we report on the selection of appropriate enzyme-carrier preparation for hydrolysis reaction in aqueous and biphasic systems and transesterification in organic solvent. For this Candida rugosa lipase was bound by adsorption or covalent attachment onto various carriers to give 24 preparations. Selection of proper preparation was based on reactivity, thermal stability (4 h at 60°C), possibility of drying and operational stability in 17-23 successive batch processes of 4-nitrophenyl palmitate hydrolysis in water. Activity of preparations varied from 20 to 5100 U∙mL(-1) but the most stable preparations were those of moderate activity: bound by adsorption or covalent attachment to NH₂-Kieselgel or acrylic carrier (retained activity over 90%). Selected preparations were used for hydrolysis of ethyl (1-butyryloxyethyl)-phenylphosphinate in biphasic system, and, after drying, in ethyl (1-hydroxyethyl)-phenyl-phosphinate transesterification. In this study operational stability was the principal criterion of selection. In water system, lipase covalently bound to NH₂-Kieselgel was the best - preserved 50% of initial activity in consecutive batch processes. In biphasic system and lipase covalently bound to acrylic and NH₂-Kieselgel the values were 90 or 77%, respectively, whereas in organic solvent, when lipase was immobilized on NH₂-Kieselgel by adsorption, it was 50%. Thus, NH₂-Kieselgel appears to be an universal matrix for investigated lipase immobilization and can be used in all reaction systems.
在生物过程中，无论反应体系类型如何，脂肪酶通常以固定化形式使用，以确保催化剂在水受限介质中均匀分布和/或便于分离与重复使用。在这些研究中，我们报告了为水相和双相体系中的水解反应以及有机溶剂中的酯交换反应选择合适的酶-载体制剂。为此，通过吸附或共价连接将皱褶假丝酵母脂肪酶固定在各种载体上，得到24种制剂。合适制剂的选择基于反应活性、热稳定性（60°C下4小时）、干燥可能性以及在17-23次连续批次的水相中4-硝基苯基棕榈酸酯水解过程中的操作稳定性。制剂的活性从20到5100 U∙mL(-1)不等，但最稳定的制剂是那些活性适中的制剂：通过吸附或共价连接固定在NH₂-硅胶或丙烯酸载体上（保留活性超过90%）。所选制剂用于双相体系中(1-丁酰氧基乙基)-苯基次膦酸乙酯的水解反应，干燥后用于(1-羟乙基)-苯基次膦酸乙酯的酯交换反应。在本研究中，操作稳定性是选择的主要标准。在水体系中，共价连接到NH₂-硅胶上的脂肪酶表现最佳——在连续批次过程中保留了50%的初始活性。在双相体系中，共价连接到丙烯酸和NH₂-硅胶上的脂肪酶的值分别为90%或77%，而在有机溶剂中，当脂肪酶通过吸附固定在NH₂-硅胶上时，该值为50%。因此，NH₂-硅胶似乎是用于所研究脂肪酶固定化的通用基质，可用于所有反应体系。
