The Design Of A Monoamine Oxidase MAO-N-D5by Two Directed Evolution Methods

The value of chiral amine in the pharmaceutical, agrochemical and chemical industry is gradually increased. They are often used as resolving agents, various pharmaceutically active substances and pesticides synthon. For their preparation, chemical as well as enzymatic methods for their synthesis are ongoing research topics in today’s laboratories.Compared with the chemical method, enzymatic synthesis become the hot spot because of its mild operating environments and environment-friendly. One mutated monoamine oxidase (MAO-N-D5) from Aspergillus niger was screened with high activity, high Enantioselectivity and broad substrate spectrum after three directed evolution screened.Here, on the base, we used two different directional evolutionary strategy including CASTing and error-prone PCR to enginerred MAO-N-D5again. In the CASTing process, by analyzing the docking result of MAO-N-D5and rac-mexilitine,6amino acid sites were selected in the vicinity of the substrate pockets and were divided into three groups to conducted saturation mutagenesis(A:Phe210/Leu213, B:Met242/Met246, C:Tyr365/Ile367).In the first round screening, two mutant enzyme were screended:A-7(F210V) with the activity of21%and A-1(F210V/L213C) with the activity of100%, which is5times higer than that of A-7. In the second round, after combination of A-1and A-7with library C, another mutation was screened, which was A7Cl (F210V/I367T) with the activity of80%.The optimal mutant A-1obtained in CASTing process was choosed as the starting point to conducted futher directed evolution by error-prone PCR. After screending of the random mutant library, one positive mutant enzyme ep-1(F210V/L213C/T162A) was obtained with the activity of189%, which is higer than A-1. As for the substrate spectrum, compaired with the unmutated MAO-D5, the catalytic activities of A-1and eP-1to some measured amines improved significantly. What’s important, the two mutant enzymes retained excellent enantioselectivity of MAO-D5. In the resolution of mexiletine, A-1showed the conversion rate of50%and ee92%, A7C1with the conversion of52%and ee95.6%, ep-1with the conversion of41.8%and ee99%.