Protein engineering of arabidopsis thaliana hydroxynitrile lyase AtHNL improves promiscuous nitroaldolase and retro nitroaldolase activity

Abstract

ABSTRACT newlineApplication of enzymes as catalyst is emerging in academics and industries due to their high newlineselectivity, environment friendly nature, and sustainable reaction conditions. Being not evolved to newlinecatalyze non-natural reactions, they often require modifications for industrial and research newlineapplications. Altering wild type enzymes usually produces potential biocatalysts with a wide range newlineof desirable properties, suitable for industrial application. Hydroxynitrile lyases (HNLs) are a class newlineof enzymes emerged as potent biocatalysts in the global market for the synthesis of various newlinevaluable optically pure compounds, i.e., and#945;-cyanohydrins, and and#946;-nitroalcohols. Based on the protein newlinestructure, enantioselectivity and presence of cofactor, HNLs are diverse in nature; yet their newlineapplication in biotechnology industry is restricted with several limitations such as poor newlineenantioselectivity, limited yield and not being evolved to accept unnatural substrates. In the current newlinestudy, a plant HNL was engineered to achieve desired biocatalytic properties, such as increased newlineenantioselectivity, conversions, stability, selectivity and broad substrate selectivity, while newlinecatalyzing a promiscuous reaction. newlineHere, we engineered a HNL and employed the variants to synthesize/prepare enantiopure and#61538;- newlinenitroalcohols using the promiscuous Henry (nitroaldol) and retro-Henry (retro-nitroaldol) newlinereactions. Enantiopure and#61538;-nitroalcohols are chiral drug intermediates that have diverse applications newlinein pharmaceutical, agrochemical, and cosmetic industries. HNL catalyzed stereoselective synthesis newlineof chiral and#61538;-nitroalcohols is considered to be one of the most predominant methods among other newlinebiocatalytic approaches as it involves an atom economy approach, and does not require additional newlinesteps of substrate synthesis. So far, Arabidopsis thaliana hydroxynitrile lyase (AtHNL) is the only newlineHNL reported for both stereoselective cleavage (retro-nitroaldolase activity) and synthesis newline(nitroaldolase activity) of chiral and#946;-nitro alcohols. Unlike (R)-selective GtHNL