Substrate Recognition And Catalytic Mechanisms Of Feruloyl Esterase LP?0796 From Lactobacillus Plantarum

Xylan is a common dietary fiber,which is widely present in various plant foods.Enzymatic hydrolysis of xylan can obtain functional components such as xylo-oligosaccharides,xylose and ferulic acid,and can be widely used in food,pharmaceuticals,cosmetics and other fields.Ferulic acid is an antioxidant that can neutralize oxygen free radicals.It also has anti-cancer,anti-allergic,antiinflammatory and neuroprotective and cardiovascular effects.Feruloyl esterase is an indispensable biocatalyst in the food processing and pharmaceutical industries.It catalyzes the cleavage of the crosslinked ester bond between the polysaccharide side chain of hemicellulose and ferulic acid in the plant cell wall,releasing free ferulic acid which can exert biological activity.LP?0796 is a feruloyl esterase derived from Lactobacillus plantarum,which have ideal potential applications in the food industry.However,its utilization and application are limited due to the lack of catalysis and substrate recognition mechanisms.This thesis aims to explore the molecular mechanism of substrate specificity,as well as the substrate recognition and catalysis mechanisms,by studying the enzymatic properties and crystal structure of LP?0796,combined with molecular docking simulation and site-directed mutagenesis.In this study,LP?0796 was firstly cloned and expressed to obtain a homogeneity and high-purity protein,which exists as a monomer in solution.Methyl caffeate(MCA)was used as the substrate to characterize the optimal temperature of 40?,optimum pH value of 6.5,thermal stability of 20-30? and pH stability of 5-7.We determined the crystal structure of LP?0796 at a resolution of 2.5 (?).LP?0796structure contains two molecules in an asymmetric unit.Each molecule includes a smaller cap subdomain and a larger ?/? hydrolase folding subdomain.The catalytic triad of Ser94,Asp195 and His225 constitutes the center of the active site.One phenomenon that distinguishes LP?0796 from other CE1 homologues is the flexible loop where Asp195 of LP?0796 located appears to orientate in an opposite direction,forming a relatively open substrate binding pocket,which allows it to bind more different types of substrates.In order to further clarify the substrate recognition and catalytic mechanism of LP?0796,we constructed a structural model of LP?0796 in complex with MCA through molecular docking and investigated the importance of the amino acids at the substrate binding pocket by site-directed mutagenesis.The results showed that in addition to the catalytic triad,Arg125 and Val128 played a key role in the substrate binding and catalysis.Interestingly,the aspartic acid mutations of Ala23 and Ile198 increased the enzyme activity by 4 and 5 times,respectively,indicating that they can serve as ideal rational design sites for the enzyme.In summary,this study clarifies the molecular basis of LP?0796's broad substrate specificity,reveals the substrate recognition and catalytic mechanism of this enzyme,and provides important theoretical basis for the subsequent directed evolutionary modification and industrial application based on catalytic activity and thermal stability.