Construction Of High-throuput Screening Method For Ketones And Its Application In Mining New Enzymes And Directed Evolution Of High-yield Raspberry Ketones

Catalytic oxidation of alcohols to ketones is an important reaction in organic synthesis.Its products are very important as basic materials for synthetic organic chemistry and biocatalysis,and are important building blocks of some valuable chemicals.In the bioenzymatic synthesis of ketones,screening is the main constraint bottleneck.Therefore,the construction of high-throughput screening methods for ketones is crucial for bioenzymatic synthesis(ie,directed evolution of enzymes).By using the fluorescent dye p-methoxy-2-aminobenzylamine oxime(PMA),we can quickly determine various structures of ketones.This topic studies the applicable conditions,performance characterization and application of the PMA high-throughput screening method.This PMA-based fluorescence detection method has four major advantages.First,it has high sensitivity and can be accurate to μM;second,it has a wide linear range and high HPLC/GC-fluorescence fitting,which can reach more than 0.9,and can be qualitatively and quantitatively.The third is a wide spectrum of detectable carbonyl compounds,including most of the aldehydes,and four ketones such as hydroxyketones,aromatic ketones,cyclic ketones,and fatty ketones;the fourth is low background noise,which can also be worked in cells.Because of its sensitive and stable fluorescence signal transformation,it has the potential to further construct fluorescence-activated cell sorting(FACS)and droplet microfluidic technologies.We successfully applied it to the mining of alcohol dehydrogenase libraries and the directed evolution of alcohol dehydrogenase(TbSADH)from Thermoanaerobacter brockii.1-Hydroxy-2-butanone is a precursor of the synthetic antituberculosis drug ethambutol.Through the PMA fluorescence detection method,an alcohol dehydrogenase E99,which catalyzes the conversion of 1,2-butanediol to 1-hydroxy-2-butanone to 100%,was dug from the alcohol dehydrogenase ADH library,and the GC-fluorescence fitting degree reached 0.83.Raspberry ketones can be used as flavors and fragrances,and have whitening and weight loss effects.The biological enzymatic synthesis is generally obtained by oxidizing rhododendrol,of which screening is the bottleneck.Using the PMA fluorescence detection method,directed evolution of the alcohol dehydrogenase TbSADH that catalyzed the reaction was successfully screened for mutants with a conversion rate from wild type 39%to 75%,and the GC-fluorescence fit reached 0.97.Through further calculation and analysis,it was found that the W110 site of TbSADH is the "transmission" that controls the oxidation reaction.When it is mutated to a,highly rigid and highly sterically hindered amino acid,its oxidative activity is almost lost;when it is mutated to a highly flexible and small sterically hindered amino acid,its oxidative activity is greatly improved.This subject constructed a high-throughput screening method based on the fluorescent dye PMA,which provided a universal tool for the detection of carbonyl compounds in the development of biocatalysts,and applied it to the discovery of new enzymes and directed evolution of enzymes,greatly improving the screening efficiency.By integrating and sorting out the general ideas of screening methods based on fluorescent dyes to construct a well plate,it will provide some reference for the development of other fluorescent dyes.The high-throughput screening method was successfully applied in the screening of high-quality mutants by enzyme-directed evolution,which is helpful for the industrial application of raspberry ketones.By analyzing the relationship between the amino acid changes in the enzyme-catalyzed pocket and their catalytic properties,it is helpful to understand the enzyme’s catalytic mechanism and lay the foundation for efficient rational design.