Arana-Peña Sara, Rios Nathalia S, Carballares Diego, Mendez-Sanchez Carmen, Lokha Yuliya, Gonçalves Luciana R B, Fernandez-Lafuente Roberto
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica - CSIC, Campus Universidad Autónoma de Madrid - CSIC Cantoblanco, Madrid, Spain.
Departamento de Engenharia Química, Universidade Federal do Ceará, Fortaleza, Brazil.
Front Bioeng Biotechnol. 2020 Feb 28;8:36. doi: 10.3389/fbioe.2020.00036. eCollection 2020.
The lipase from (PFL) has been immobilized on octyl-agarose beads under 16 different conditions (varying pH, ionic strength, buffer, adding some additives) at two different loadings, 1 and 60 mg of enzyme/g of support with the objective of check if this can alter the biocatalyst features. The activity of the biocatalysts versus -nitrophenyl butyrate and triacetin and their thermal stability were studied. The different immobilization conditions produced biocatalysts with very different features. Considering the extreme cases, using 1 mg/g preparations, PFL stability changed more than fourfolds, while their activities versus NPB or triacetin varied a 50-60%. Curiously, PFL specific activity versus triacetin was higher using highly enzyme loaded biocatalysts than using lowly loaded biocatalysts (even by a twofold factor). Moreover, stability of the highly loaded preparations was higher than that of the lowly loaded preparations, in many instances even when using 5°C higher temperatures (e.g., immobilized in the presence of calcium, the highly loaded biocatalysts maintained after 24 h at 75°c a 85% of the initial activity, while the lowly loaded preparation maintained only 27% at 70°C). Using the highly loaded preparations, activity of the different biocatalysts versus NPB varied almost 1.7-folds and versus triacetin 1.9-folds. In this instance, the changes in stability caused by the immobilization conditions were much more significant, some preparations were almost fully inactivated under conditions where the most stable one maintained more than 80% of the initial activity. Results suggested that immobilization conditions greatly affected the properties of the immobilized PFL, partially by individual molecule different conformation (observed using lowly loaded preparations) but much more relevantly using highly loaded preparations, very likely by altering some enzyme-enzyme intermolecular interactions. There is not an optimal biocatalyst considering all parameters. That way, preparation of biocatalysts using this support may be a powerful tool to tune enzyme features, if carefully controlled.
来自[具体来源未提及]的脂肪酶（PFL）已在16种不同条件（不同的pH值、离子强度、缓冲液、添加一些添加剂）下，以两种不同的负载量（1和60 mg酶/g载体）固定在辛基琼脂糖珠上，目的是检查这是否会改变生物催化剂的特性。研究了生物催化剂对丁酸对硝基苯酯和三醋精的活性及其热稳定性。不同的固定条件产生了具有非常不同特性的生物催化剂。考虑极端情况，使用1 mg/g的制剂时，PFL稳定性变化超过四倍，而其对NPB或三醋精的活性变化了50 - 60%。奇怪的是，使用高酶负载的生物催化剂时，PFL对三醋精的比活性高于低负载的生物催化剂（甚至高出两倍）。此外，高负载制剂的稳定性高于低负载制剂，在许多情况下，即使使用高5°C的温度也是如此（例如，在钙存在下固定，高负载生物催化剂在75°C下24小时后保持初始活性的85%，而低负载制剂在70°C下仅保持27%）。使用高负载制剂时，不同生物催化剂对NPB的活性变化几乎为1.7倍，对三醋精的活性变化为1.9倍。在这种情况下，固定条件引起的稳定性变化更为显著，一些制剂在最稳定的制剂保持超过80%初始活性的条件下几乎完全失活。结果表明，固定条件极大地影响了固定化PFL的性质，部分是由于单个分子的不同构象（在低负载制剂中观察到），但在高负载制剂中更重要得多，很可能是通过改变一些酶 - 酶分子间相互作用。考虑所有参数时不存在最佳生物催化剂。因此，如果仔细控制，使用这种载体制备生物催化剂可能是调节酶特性的有力工具。
