Bolivar Juan M, Wilson Lorena, Ferrarotti Susana Alicia, Fernandez-Lafuente Roberto, Guisan Jose M, Mateo Cesar
Departamento de Biocatalisis, Instituto de Catalisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
Biomacromolecules. 2006 Mar;7(3):669-73. doi: 10.1021/bm050947z.
Formate dehydrogenase (FDH) is a stable enzyme that may be readily inactivated by the interaction with hydrophobic interfaces (e.g., due to strong stirring). This may be avoided by immobilizing the enzyme on a porous support by any technique. Thus, even if the enzyme is going to be used in an ultra-membrane reactor, the immobilization presents some advantages. Immobilization on supports activated with bromocianogen, polyethylenimine, glutaraldehyde, etc., did not promote any stabilization of the enzyme under thermal inactivation. However, the immobilization of FDH on highly activated glyoxyl agarose has permitted increasing the enzyme stability against any distorting agent: pH, T, organic solvent, etc. The time of support-enzyme reaction, the temperature of immobilization, and the activation of the support need to be optimized to get the optimal stability-activity properties. Optimized biocatalyst retained 50% of the offered activity and became 50 times more stable at high temperature and neutral pH. Moreover, the quaternary structure of this dimeric enzyme becomes stabilized by immobilization under optimized conditions. Thus, at acidic pH (conditions where the subunit dissociation is the first step in the enzyme inactivation), the immobilization of both subunits of the enzyme on glyoxyl-agarose has allowed the enzyme to be stabilized by hundreds of times. Moreover, the optimal temperature of the enzyme has been increased (even by 10 degrees C at pH 4.5). Very interestingly, the activity with NAD(+)-dextran was around 60% of that observed with free cofactor.
甲酸脱氢酶（FDH）是一种稳定的酶，但与疏水界面相互作用（例如由于强烈搅拌）时可能容易失活。通过任何技术将酶固定在多孔载体上可避免这种情况。因此，即使该酶将用于超滤膜反应器，固定化也具有一些优势。用溴化氰、聚乙烯亚胺、戊二醛等活化的载体上固定化并不能促进酶在热失活下的任何稳定化。然而，将FDH固定在高度活化的乙醛酸琼脂糖上可提高酶对任何变形剂（如pH、温度、有机溶剂等）的稳定性。需要优化载体与酶的反应时间、固定化温度以及载体的活化，以获得最佳的稳定性-活性特性。优化后的生物催化剂保留了50%的活性，并且在高温和中性pH下稳定性提高了50倍。此外，这种二聚体酶的四级结构在优化条件下通过固定化而变得稳定。因此，在酸性pH下（亚基解离是酶失活的第一步的条件下），酶的两个亚基固定在乙醛酸琼脂糖上使酶的稳定性提高了数百倍。此外，酶的最适温度也有所提高（在pH 4.5时甚至提高了10摄氏度）。非常有趣的是，与NAD(+)-葡聚糖的活性约为游离辅因子活性的60%。
