Reetz Manfred T, Soni Pankaj, Fernández Layla
Max-Planck-Institut für Kohlenforschung, Mülheim, Germany.
Biotechnol Bioeng. 2009 Apr 15;102(6):1712-7. doi: 10.1002/bit.22202.
In rare but nevertheless important cases it is of practical interest to decrease the thermostability of an enzyme, that is, to increase thermolability in a controlled manner. In the present model study, this unconventional goal has been reached by applying directed evolution to the lipase from Pseudomonas aeruginosa (PAL). By utilizing the B-factor iterative test (B-FIT), previously developed to increase the thermostability of enzymes, it was possible to reduce the T(15)(50) value from 71.6 degrees C in the case of wild type (WT-PAL) to 35.6 degrees C (best mutant) without affecting the catalytic profile in terms of substrate acceptance or enantioselectivity at room temperature. Accordingly, saturation mutagenesis was performed at sites in PAL, which on the basis of its X-ray structure, have the lowest B-factors indicative of high rigidity. Focused mutations were introduced which can be expected to decrease rigidity, the ensuing increased flexibility leading to higher thermolability without changing the actual catalytic profile.
在罕见但却重要的情况下，降低酶的热稳定性，即以可控方式提高热不稳定性具有实际意义。在当前的模型研究中，通过对铜绿假单胞菌脂肪酶（PAL）进行定向进化实现了这一非传统目标。利用先前开发的用于提高酶热稳定性的B因子迭代测试（B-FIT），能够将野生型（WT-PAL）的T(15)(50)值从71.6℃降至35.6℃（最佳突变体），且不影响室温下底物接受度或对映选择性方面的催化特征。因此，基于PAL的X射线结构，在具有最低B因子（表明高刚性）的位点进行饱和诱变。引入了预期可降低刚性的集中突变，由此增加的灵活性导致更高的热不稳定性，同时不改变实际的催化特征。
