Protein Engineering Of The Monoamine Oxidase From Aspergillus Niger

Optically active amines and amino acids play an important role in the pharmaceutical, agrochemical and chemical industry. They are frequently used as synthons for the pre-paration of various pharmaceutically active substances and agrochemicals, but also as resolving agents to obtain chiral carboxylic acids. Inspired by the structure of natural products, chemists finetune and optimize their physiological effects by synthesizing simi-lar or related compounds. For their preparation, optically pure amines, such as achiral primary amines, are valuable synthons and hence chemical as well as enzymatic methods for their synthesis are ongoing research topics in today's laboratories. Consequently, there is a need for efficient methods to obtain the desired enantiomer of a given target structure in optically pure form.Enzymatic syntheses have developed as an environmentally benign alternative to chemical methods, because they operate under mild conditions, are usually not air-or water-sensitive and avoid the need for highly flammable metal-organic reagents or heavy metal contamination. Additionally, if an efficient heterologous overexpression in a microbial host is pro-vided, enzymes can be produced very easily and inexpensively compared to many of their chemical counterparts.Based on the molecular docking results of the (-)-tetrabenazine with MAO-D5,we introduced the stop codon at W463to constructed W463*muant,in the present of50mM phosphate buffer (pH7.4) and1mM racemic tetrabenazine, after24hours reaction at28℃the (-)-tetrabenazine was converted16.7%.Constructed the CASTing Saturation Mutagenesis Library (Leu207and Phe204)based on the molecular docking results of (S)-3-amino-l-phenyl-butane with MAO-D5, the saturation mutagenesis library was screening with plate and96-well plates,13clones were picked to expression and purification, after sequencing they contains six kinds of mutants, the most effective mutant was F204L/L207V which the specific activity was0.71U/mg,the specific activity of wild-type MAO-D5was0.15U/mg. L207V mutant was used as the catalyst to kinetic resolution3-amino-1-phenyl-butane racemic, after reaction at30℃for12hours, the enantiomeric excess (ee) of the wild-type MAO-D5reactive group reached29%,the L207V reaction group was78.44%.