Identification And Characterization Of A Feruloyl Esterase Screened By Metagenomic Technology

Feruloyl esterase(EC 3.1.1.73,ferulic acid esterase,FAE)can hydrolyze ester bonds between ferulic acid and polysaccharides in plant cell walls to release ferulic acid.The released ferulic acid exhibited excellent biological activities,e.g.,antioxidant,anti-tumor,anti-atherosclerosis,antimicrobial and anti-inflammatory.In addition,FAEs are also widely used in food,paper pulp and feed industry.The traditional FAE screening method was mainly based on the culturable technology of microorganisms.Although this method can screen FAEs,as deepening of research and the number of FAE increasing,some known FAEs are discovered repeatedly.Moreover,studies have shown that culturable microorganisms account for less than 1%of the total number of microorganisms.Up to 99%of non-culturable microorganisms are the subject of microbial research,suggesting that there are richer FAEs in them.Recently,with the development of molecular biology and bioinformatics,more interest has been fueled on the culture-independent metagenomic methods.This method avoided the isolation and cultivation of microorganisms,directly extracting total DNA(eDNA)from the environment,constructing and screening DNA libraries to obtain target monoclonals,heterologously expressing to obtain the target product.This method has proved to be a powerful tool in mining new FAEs.In this study,FAE monoclonal was screened from the soil metagenomic library by functional screening method and the gene sequence encoding FAE was found by ORF Finder.The sequence was analyzed by bioinformatics and heterologously expressed using E.coli expression system.The enzymatic properties of the purified FAE were characterized.1.The positive clone was screened by functional screening method using FAE activity as an index.The sequence named fae-xuan which encoded FAE was found by ORF Finder and a BLASTp analysis showed that the corresponding amino sequence displayed the highest identity(49%)with the known FAE,which indicating that the sequence encoded a new FAE,trivially named FAE-Xuan.The multi-alignment analysis suggested that FAE-Xuan contained a classical conserved pentapeptide signature motif G-X-S-X-G.The serine(S,84)residue was within the motif,together with aspartate(D,203)and histidine(H,224)residues constitute a typical catalytic triad of FAE.The phylogenic relationships showed that our new FAE was clearly a member of type A FAE.The FAE-Xuan tertiary structure was predicted by the SWISS-MODEL program.A typical ?/? hydrolase fold is observed in the three-dimensional structure of FAE-Xuan,which consists of a cap domain and a catalytic domain.2.Heterologous expression of FAE monoclonal.The putative FAE gene(fae-xuan)was ligated into the pET28a expression vector to form the recombinant plasmid called pET28a-fae-xuan and the recombinant plasmid was transformed into E.coli BL21(DE3).The recombinant enzyme was purified by NI-NTA-Sefinose.The purified recombinase was detected by polyacrylamide gel electrophoresis(PAGE)and found to have a molecular weight of 29 kDa,which was in agreement with the predicted value,and there was only one electrophoresis band,indicating that a pure FAE was obtained.3.Characterization of FAE enzymatic properties.Determining the optimal conditions of the enzyme,including reaction temperature,pH value by optimizing experiments.The substrate specificity of FAE was determined using four methyl hydroxycinnamate and the kinetic constants for the enzyme reaction were determined.The effect of different metal ions and organic solvents on enzyme activity was studied.The purified FAE-Xuan showed an optimal temperature and pH of 30? and 5.0,respectively,suggesting it was an acidic enzyme.Additionally,the residual enzyme activity remained more than 70%when the enzyme was incubated for 4h in the range of pH 3.0-10.0,indicating that it had a high stability of pH.The substrate specificity test showed that the maximum activity was as high as 40 U/mg when using MFA as a substrate.FAE-Xuan activity was strongly inhibited by the presence of Ba2+,Zn2+,Cu2+,isopropanol and DMF,wherase K+,Na+and Mg2+can enhance the enzyme activity.Other metal ions and chemical reagents displayed little effect on enzyme activity.FAE-Xuan can hydrolyze DSWB to release ferulic acid and higher yields of FA were obtained by joint action of FAE-Xuan with xylanase.The FAE-Xuan activity in this study is 2-3 times higher than that of most FAEs from culturable microorganisms,and it is a medium enzyme activity compared to FAEs derived from metagenomics.The enzyme is stable in the pH range of 3.0-10.0.The broad pH range of FAE is relatively rare in reports.The FAE-Xuan described here has excellent properties and is expected to be a potential candidate enzyme in industrial applications.In addition,The results of the current study suggested that the functional metagenomic strategy can be a powerful tool to mine novel enzymes.