Construction And Application Of A Rapid Assay Method For Fingerprinting The Substrate Specificity Of Esterases/Lipases

Esterases and lipases are considered as an important group of biocatalysts for industrial applications, such as pharmaceuticals, foods and detergents. So far, there have been a range of high-throughput screening methods developed to screen lipases and esterases. Currently, the general trend in screening is to construct a library of structurally diverse substrates for providing an analytical tool for characterizing the reactivity fingerprint of certain enzymes.A series of structurally diverse chromogenic esters of 4-nitrophenol, including a new compound (4'-nitrophenyl 2-methylpentanoate), have been synthesized in this study, constituting an array of 17 substrates which could be applied to rapidly assay the activity of lipases or esterases for revealing their substrates specificity and functional characteristics. Combining with the genetic technologies of new generaton, such as "data mining", metagenomics and directed evolution, such an assay of reactivity fingerprints of an enzyme against diverse substrates might become a promising method as a platform for rapid discovery of industrially useful enzymes. The fingerprint of commercially available Candida antarctica lipase B (CALB) as a model enzyme was firstly measured to confirm the reliability of this method. Then three new enzymes mined from genomic database were successfully fingerprinted, revealing the functional characteristics of those enzymes and potentials for industrial application. Among them, the enzyme SrfAD was found to have a special activity fingerprint, making it more promising in industrial synthesis of aromatic acids with methyl substitution on a-position.