Dudek Hanna M, Fink Michael J, Shivange Amol V, Dennig Alexander, Mihovilovic Marko D, Schwaneberg Ulrich, Fraaije Marco W
Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Appl Microbiol Biotechnol. 2014 May;98(9):4009-20. doi: 10.1007/s00253-013-5364-1. Epub 2013 Nov 19.
Baeyer-Villiger monooxygenase-catalysed reactions are attractive for industrial processes. Here we report on expanding the substrate scope of phenylacetone monooxygenase (PAMO). In order to introduce activity on alicyclic ketones in PAMO, we generated and screened a library of 1,500 mutants. Based on recently published structures of PAMO and its mutants, we selected previously uncharacterised positions as well as known hot-spots to be targeted by focused mutagenesis. We were able to mutate 11 positions in a single step by using the OmniChange method for the mutant library generation. Screening of the library using a phosphate-based activity detection method allowed identification of a quadruple mutant (P253F/G254A/R258M/L443F) active on cyclopentanone. The substrate scope of this mutant is extended to several aliphatic ketones while activity on aromatic compounds typical for PAMO was preserved. Moreover, the mutant is as thermostable as PAMO. Our results demonstrate the power of screening structure-inspired, focused mutant libraries for creating Baeyer-Villiger monooxygenases with new specificities.
拜耳-维利格单加氧酶催化的反应在工业过程中具有吸引力。在此，我们报告了扩大苯丙酮单加氧酶（PAMO）底物范围的研究。为了使PAMO对脂环族酮具有活性，我们构建并筛选了一个包含1500个突变体的文库。基于最近发表的PAMO及其突变体的结构，我们选择了以前未表征的位点以及已知的热点区域进行定点诱变。通过使用OmniChange方法构建突变体文库，我们能够一步对11个位点进行突变。使用基于磷酸盐的活性检测方法对文库进行筛选，鉴定出了对环戊酮有活性的四重突变体（P253F/G254A/R258M/L443F）。该突变体的底物范围扩展到了几种脂肪族酮，同时保留了对PAMO典型的芳香族化合物的活性。此外，该突变体与PAMO一样具有热稳定性。我们的结果证明了筛选受结构启发的定点突变体文库以创建具有新特异性拜耳-维利格单加氧酶的强大能力。
