Departamento de Biocatalisis, ICP-CSIC, Campus UAM-CSIC, Madrid 28049, Spain; Departamento de Química, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago, Ñuñoa 7800003, Chile.
Departamento de Biocatalisis, ICP-CSIC, Campus UAM-CSIC, Madrid 28049, Spain; Agri-food Engineering Laboratory (GENIAAL), Institute of Food, Nutrition and Agri-Food Technologies (INATAA), University of Brothers Mentouri Constantine 1, Algeria.
Enzyme Microb Technol. 2024 Dec;181:110517. doi: 10.1016/j.enzmictec.2024.110517. Epub 2024 Sep 21.
Ficin extract has been immobilized using different supports: glyoxyl and Aspartic/1,6 hexamethylenediamine (Asp/HA) agarose beads. The latter was later submitted to glutaraldehyde modification to get covalent immobilization. The activities of these 3 kinds of biocatalysts were compared utilizing 4 different substrates, casein, hemoglobin and bovine serum albumin and benzoyl-arginine-p-nitroanilide at pH 7 and 5. Using glyoxyl-agarose, the effect of enzyme-support reaction time on the activity versus the four substrates at both pH values was studied. Reaction time has been shown to distort the enzyme due to an increase in the number of covalent support-enzyme bonds. Surprisingly, for all the substrates and conditions the prolongation of the enzyme-support reaction did not imply a decrease in enzyme activity. Using the Asp/HA supports (with different amount of HA) differences in the effect on enzyme activity versus the different substrates are much more significant, while with some substrates the immobilization produced a decrease in enzyme activity, with in other cases the activity increased. These different effects are even increased after glutaraldehyde treatment. That way, the conformational changes induced by the biocatalyst immobilization or the chemical modification fully altered the enzyme protein specificity. This may also have some implications when following enzyme inactivation.
乙二醛和天冬氨酸/1,6-己二胺（Asp/HA）琼脂糖珠。后者后来经过戊二醛修饰，以获得共价固定化。利用 4 种不同的底物（酪蛋白、血红蛋白和牛血清白蛋白以及苯甲酰-精氨酸-对硝基苯胺），在 pH7 和 5 下比较了这 3 种生物催化剂的活性。使用乙二醛琼脂糖，研究了酶-载体反应时间对两种 pH 值下四种底物活性的影响。研究表明，由于共价支持-酶键数量的增加，反应时间会使酶变形。令人惊讶的是，对于所有的底物和条件，延长酶-载体反应时间并不意味着酶活性的降低。使用 Asp/HA 载体（具有不同量的 HA），对不同底物的酶活性的影响差异更为显著，而在某些情况下，固定化会降低酶活性，而在其他情况下，酶活性会增加。在经过戊二醛处理后，这些不同的效果甚至会进一步增加。这样，固定化生物催化剂或化学修饰所诱导的构象变化完全改变了酶蛋白的特异性。当后续进行酶失活研究时，这也可能产生一些影响。
