Di Lorenzo Mirella, Hidalgo Aurelio, Molina Rafael, Hermoso Juan A, Pirozzi Domenico, Bornscheuer Uwe T
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, D-17487 Greifswald, Germany.
Appl Environ Microbiol. 2007 Nov;73(22):7291-9. doi: 10.1128/AEM.01176-07. Epub 2007 Sep 21.
A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.
为提高米根霉脂肪酶原（proROL）对脂质氧化产物（如醛类）的稳定性，从而改善其在油脂化学工业中的性能，对其进行了工程改造。在22个易于与醛反应的氨基酸残基（15个赖氨酸和7个组氨酸）中，选择了6个赖氨酸残基和所有组氨酸残基（催化组氨酸除外）进行饱和诱变。为了在所得文库中快速可靠地鉴定出稳定性突变体，通过琼脂平板上的活性染色法对活性变体进行预筛选。活性突变体在大肠杆菌Origami中以96孔微量滴定板形式表达，并以辛醛作为模型失活剂进行稳定性测试。最稳定的组氨酸突变体（H201S）稳定性提高了60%，与赖氨酸突变体（H201S/K168I）组合后，稳定性进一步提高到100%。其他醛类也证实了这种稳定性的提高。有趣的是，这些突变不影响比活性，因为其仍与野生型酶相似。
