Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM, Cantoblanco, Madrid, Spain.
J Biotechnol. 2009 Oct 26;144(2):113-9. doi: 10.1016/j.jbiotec.2009.09.002. Epub 2009 Sep 6.
Immobilized-stabilized aminated lipase from Thermomyces lanuginosus (TLL-A) is not easily reactivated after inactivation by incubation in the presence of organic solvents or chaotropic reagents. To improve the recovered activity of this biocatalyst, immobilized TLL-A has been submitted to different modifications. The best results were obtained when the enzyme was coated with a very hydrophilic and inert polymer: dextran modified with glycine (Dx-Gly). This modification did not reduce enzymatic activity while it increased the stability of this already very stable preparation, in thermal and organic solvent induced inactivation (by a 4-fold factor). Simple incubation in aqueous medium at pH 7 and 25 degrees C permitted to fully recover the activity of the immobilized and modified TLL-A enzyme inactivated by incubation in organic solvents or saturated guanidine during 3 cycles, while the non-modified enzyme only recover some activity. When the inactivation was caused by exposition at high temperatures, the reactivation was higher using the modified biocatalyst, but was far for complete (40% after 3 inactivation-reactivation cycles). The determination of the TLL-A activity in the presence of detergents (that helps the opening of active site of the lipase) allowed, in this case, to significantly improve the results, now near to 90% of the initial activity was recovered (using the non-modified enzyme the recovered activity was around 60%). This very hydrophilic and inert polymer, coating the enzyme surface, seems to help the correct positioning of the hydrophilic and hydrophobic groups of the enzyme, and that way improve both the stability and possibility of reactivation of the enzyme.
固定化稳定化氨基化脂肪酶从Thermomyces lanuginosus（TLL-A）在有机溶剂或变性剂存在下孵育失活后不易重新激活。为了提高这种生物催化剂的回收活性，已对固定化 TLL-A 进行了不同的修饰。当酶用非常亲水和惰性的聚合物：甘氨酸修饰的葡聚糖（Dx-Gly）包被时，得到了最好的结果。这种修饰不会降低酶活性，同时增加了这种已经非常稳定的制剂的稳定性，在热和有机溶剂诱导失活时（增加了 4 倍）。在 pH 7 和 25°C 的水性介质中简单孵育允许在 3 个循环中完全回收在有机溶剂或饱和胍孵育失活的固定化和修饰的 TLL-A 酶的活性，而未修饰的酶仅恢复部分活性。当失活是由于暴露在高温下引起时，使用修饰的生物催化剂的再激活更高，但远未完全（在 3 个失活-再激活循环后为 40%）。在存在洗涤剂（有助于脂肪酶活性部位的打开）的情况下测定 TLL-A 活性，在这种情况下，显著提高了结果，现在接近 90%的初始活性得到了恢复（使用未修饰的酶，回收的活性约为 60%）。这种非常亲水和惰性的聚合物，覆盖在酶表面，似乎有助于酶的亲水和疏水基团的正确定位，从而提高酶的稳定性和再激活的可能性。
