Feruloyl Esterase: Strain Screening,heterologous Expression And Its Structural Analysis

Biomass resources are widely and abundantly distributed all over the world.In order to improve the utilization efficiency of biomass resources,enzymatic degradation is a common method.It can also increase the yields of fermentable sugars,bioethanol or highly valuable by-products.Among them,ferulic acid is a natural antioxidant,which plays a significant role in the treatment of diseases,such as Alzheimer’s disease,diabetes,cardiovascular and cerebrovascular diseases.Feruloyl esterase（FAE）is a key enzyme in the process of the degradation of biomass resources and production of ferulic acid,which catalyzes the breakage of ester or ether bonds between ferulic acid and polysaccharides or lignin.However,the applications of FAE are limited by its lower enzyme activity.Therefore,it is crucial to obtain a strain secreting FAE with high catalytic activity.In this study,a strain producing FAE was screened and identified.Subsequently,the methods including optimizing fermentation conditions and constructing recombinant strain were used to improve the expression level and simplify the purification process.Last,the structure of Bp FAE-His was investigated.（1）A strain SK52.001 secreting FAE was isolated and screened.The enzyme reaction products were analyzed by HPLC and LC-MS and identified as ferulic acid.SK52.001 was identified as Bacillus pumilus by physiological and biochemical tests,molecular biological analysis and colony and cell morphology.The phylogenetic tree was constructed based on the gene encoding 16S r RNA.（2）The fermentation conditions of the enzyme production were optimized in shaking flask.By one-factor-at-a-time method,the compositions of fermentation culture were optimized containing the sorts and concentrations of carbon sources,different nitrogen sources and initial p H of fermentation culture.The results indicated the optimal fermentation culture was composed of 45 g/L wheat bran,5 g/L tryptone,and the initial p H value of 6.0.When the inoculation quantity was controlled in 5%（v/v）,the highest activity of SK52.001 during fermentation reached 421 U/L at 30°C and 200 r/min for 26 h,which was enhanced by 115%compared to the initial strain.When the strains were preincubated in fermentation medium with45 g/L glucose as the carbon source for 8 h and induced by 45 g/L wheat bran for 44 h,the highest enzyme activity was up to 808 U/L,suggesting that FAE from B.pumilus is an inducible enzyme.（3）After the extraction of whole genome of SK52.001,PCR products were sequenced after the primers encoding FAE from B.pumilus were designed.Biological information analysis showed that the gene of Bp FAE-His possessed 912 base pair and encoded 303 amino acids.The theoretical molecular weight and isoelectric point are 33736.22 Da and 5.22,respectively.Bp FAE-His without signal peptide is a hydrophilic protein and its conserved domain belongs toα/βhydrolase superfamily,ranging from 67 to 272 residues.Moreover,Bp FAE-His is rooted in type A according to the relationship of phylogenetic tree.（4）A recombinant plasmid p MA5/fae-His was constructed and successfully transformed into B.subtilis WB800.The purified enzyme was acquired,in turn,by fermentation for 14 h,collection of cells and purification of Ni²⁺affinity chromatography.It was used to determinate physicochemical properties and hydrolytic capacity of de-starched wheat bran.The enzyme exists as a monomer with a molecular mass of 33.6 k Da.It displays an optimal alkaline p H of9.0,an optimal temperature of 50°C,and half-lives of 1434 min,327 min,235 min,and 68 min at 50°C,55°C,60°C,and 65°C,respectively.The melting temperature was 61.3°C.Furthermore,the kinetic parameters of Bp FAE-His was in accordance with Michaelis-Menten equation,the catalytic efficiency(K_cat/K_m)of which was 542.01 mmol/（L·s）.The results of synergetic action between Bp FAE-His and commercial xylanase showed the amount of ferulic acid reached 60%of the total ferulic acid in de-starched wheat bran,increasing 12 times compared to treatment with Bp FAE-His alone.（5）The structure of Bp FAE-His was studied including secondary structure,surface hydrophobicity,the contents of free thiol and disulfide bonds,and crucial residues of amino acid.The secondary structure of the purified enzyme consists of 18%α-helix,33%β-sheet,19%β-turn and 30%random coli.The surface hydrophobicity was 455.79.Bp FAE-His possesses a disulfide bond and two free thiols.Combined with homologous modeling,chemical modification showed a possible catalytic triad of Ser-Asp-His played a core role in catalysis and the amino acids of tryptophane and cysteine are important for catalytic activity.