Screening Of A Novel Feruloyl Esterase By The Metagenomic Approach And Its Research On Enzymatic Properties

Feruloyl esterase(FAE;EC 3.1.1.73)can hydrolyze the ester bond in ferulic acid ester,oligosaccharide ferulic acid ester and polysaccharide ferulic acid ester,and release ferulic acid.Ferulic acid,as a kind of phenolic acid,has good physiological functions such as anti-oxidation,anti-bacterial,anti-inflammatory,anti-thrombosis and anti-atherosclerosis,and thus it can be widely used in medicine and cosmetics industries.The traditional screening procedure for FAEs from the wild-active strains based on the soluble hydroxycinnamate compounds as substrates or the ferulic acid-rich agricultural raw materials as enzyme inducers,and then FAEs are obtained in the fermentation product of positive strains.However,this method has many problems,such as complicated operation,long time-consuming,repeated screening,low activity,which greatly affects the application of FAE in industry.Studies showed that more than 99%of microorganisms in the soil cannot be obtained by traditional screening methods,suggesting that there are still huge microbial resources that have not been researched and developed.With the rapid development of molecular biology and high-throughput sequencing technology,there has been a research approach of non-culturable microorganisms-metagenomics technology,which directly uses the total DNA in environmental samples as the research object,selects the appropriate host to express and obtains the desired product.It can bypass the traditional separation and culture,and be used as an effective tool for mining new genes,enzymes and active natural products from non-cultivable microorganisms.In this study,we constructed a metagenomic library,obtained the FAE gene by functional screening method,carried out heterologous expression and characterized the enzymatic properties.The results are listed:(1)Construction of a soil metagenomic library.The soil samples were collected and impurities were removed,then the total DNA was extracted by CTAB extraction.The total crude DNA was purified by gel electrophoresis,electrophoresis in a dialysis bag,and concentrated to obtain high-purity DNA.The DNA was end-repaired,re-recovered,ligated into pCCIFOS vector,phage packaged,infected host Escherichia coli EPI 300,and then estimate the total number of fosmid clones.The metagenomic library was constructed with a capacity of about 120,000 clones and divided into 24 sub-libraries for preservation in which 5000 clones were used as a sub-library.(2)Screening of a FAE gene.The single active clone was screened from the constructed metagenomic library by functional screening method with methyl ferulate as a substrate.With rescreening by HPLC,a highly active FAE clone was obtained.The sequence encoding the FAE gene(bds4)was identified by subcloning,sequencing and ORF Finder analysis.The sequence is 951 base pairs and encodes 317 amino acids and has the highest similarity of 35%to the known carboxylesterase,suggesting that bds4 is a novel FAE gene.The results of multiple sequence alignment analysis indicated that the bds4 sequence contained a conserved pentapeptide motif G-X-S-X-G and a catalytic triad center Ser158-Asp256-His286.The phylogenetic tree showed that BDS4 and C-type FAE were clustered into one clade,indicating that BDS4 belongs to the C-type ferulic acid esterase.(3)Heterologous expression of the FAE gene.The bds4 gene sequence was amplied,digested by restriction enzymes and ligated into the expression vector pET28a,then pET28a-bds4 was constructed and transformed into E.coli BL21(DE3)for heterologous expression.The heterologous expression system of FAE was induced by IPTG and then the crude enzyme was obtained by ultrasonication and centrifugation and purified by nickel column coupled affinity chromatography.The molecular weight of the purified FAE was determined by polyacrylamide gel electrophoresis and determined to be 39 kDa.(4)Characterization of the enzymatic properties of FAE.Enzymatic properties studies showed that BDS4 had a suitable temperature of 37? and an optimum pH of 8.0,also BDS4 has a good stability in the range of pH 7.0-8.5 suggesting it was a weakly alkaline FAE.Substrate specificity experiments showed that BDS4 can hydrolyze methyl ferulate,ethyl ferulate,methyl caffeate,methyl coumarin,methyl vanillate and methyl succinate.BDS4 showed the strongest hydrolysis ability(57.05 U/mg)toward methyl formate.The addition of metal ions and chemical reagents showed that BDS4 was well tolerated by other metal ion types,organic solvents and surfactants except Cu²⁺,Zn²⁺,?-mercaptoethanol and SDS.BDS4 can significantly increase the release of ferulic acid from wheat bran in the presence of xylanase.Compared with BDS4 alone,the release of ferulic acid is 4.3 times higher,up to 28.46 ?g/100 mg,indicating BDS4 can synergize with xylanase to promote the hydrolysis of ferulic acid esters.In addition,BDS4 has certain degradation effects on dimethyl phthalate,diethyl phthalate and dibutyl phthalate,indicating that BDS4 has potential application in degrading phthalates.(5)Construction and screening of a feruloyl esterase mutant library.A random mutation library was constructed by mutating the sequence bds4 using error-prone PCR technology.Taking the enzymatic activity as the screening index,the substrate plate method and the high performance liquid chromatography rescreening method were combined for screening FAEs,finally selected two mutants(EP-BDS4-1,EP-BDS4-2)with improved activities of 15%and 8%respectively.Sequence alignment analysis showed that the arginine at position 147 and the glutamine at position 175 of the mutant EP-BDS4-1 was mutated,the tyrosine at position 221 of EP-BDS4-2 was mutated,suggesting that these residues may be important for improving the enzymatic activities.The BDS4 in the study has the highest enzymatic activity at pH 8.0 and 37?,and can tolerate most of the metal ions,organic reagents and surfactants.BDS4 has the ability to hydrolyze MFA,MCA,MpCA and MSA and also degrade phthalates.Moreover,random mutations can increase enzymatic activity of BDS4 and make it potential to achieve industrialization.These properties make BDS4 as a potential application in degradation of biomass and phthalates.The results of this study demonstrate that metagenomics technology is a powerful tool for mining and discovering novel esterase genes.