Heterologous Expression Of Ferulic Esterase In Pichia Pastoris And Analysis Of Its Enzymatic Properties

Feruloyl esterase belongs to the subclass of hydroxycinnamate esterase,which can hydrolyze ester bonds in polysaccharide ferulic ester and oligosaccharide ferulic ester.Some types of FAE can also hydrolyze the ester bond in chlorogenic acid,which has a broad application prospect in food and medicine industries.In previous study,a strain of Aspergillus aculeatus was screened in our laboratory,which could produce an enzyme with activity of hydrolyzing chlorogenic acid.The enzyme specific activity was 4.35 U/g,and its fermentation cycle was about 11 days.The whole genome sequencing analysis confirmed that the enzyme was feruloyl esterase.Since E.coli lacked the function of protein processing and modification,inclusion bodies with no biological activity were produced in large numbers when the enzyme was expressed in E.coli,thus inhibiting enzyme activity and expression.In this paper,after the analysis of whole genome sequencing,feruloyl esterase gene was heterologous expressed in pichia,the induced condition was optimized,the re Fae was purified,enzymology properties were characterized and high-density fermentation strategy optimization was carried out.Compared with the amino acid sequences of several feruloyl esterases,the sequence of re Fae in this paper was highly coincident,which indicated that re Fae can be identified as a new type of ferulic esterase.And its advanced structure was predicted.The gene of feruloyl esterase was amplified using genomic sequence of A.aculeatus SD14 as template,and the gene engineering strain was constructed and expressed in the eukaryotic expression system of Pichia pastoris.The recombinant feruloyl esterase expressed by the genetically engineered strain Fae-p PICZαA/Pichia Pastoris GS115 was named as re Fae.The induction conditions of Fae-pPICZαA/Pichia pastoris GS115 were optimized including inoculation quantity,amount of methanol,pH,temperature and fermentation cycle.Accordig to the orthogonal experiment,the optimum induction conditions were determined as follows:inoculation quantity 5%,pH 5.5,methanol concentration 1%,temperature 28℃and fermentation cycle 72 h.The final ferulic esterase specific activity was 3423.71 U/g.The re Fae was isolated and purified by nickel column,and the relative molecular weight of the re Fae was about 56 k Da.The re Fae showed thermal stability at 20℃～50℃and the optimum temperature was 50℃.The re Fae has stability in the pH range of 3.0～10.0 and the optimum pH was 7.0.Monovalent metal ions had no effect on enzyme activity.Mn²⁺and Ca²⁺could activate re Fae to a certain extent,while other divalent metal ions could inhibit enzyme activity to different degrees.Trivalent metal ions would influent enzyme activity,especially when Fe³⁺was used for treatment,the enzyme activity would almost disappear.EDTA treatment did not affect the activity of re Fae.The activity of re Fae was improved by high density fermentation with 3 L system.By selecting the composition of medium,pH and carbon source in the feeding flow,the protein concentration of the final expression strain could reach up to 1.31 g/L and the highest enzyme activity was 42.78 U/m L.The engineered bacteria were successfully constructed and expressed.After optimization of induction conditions and high-density fermentation,the enzyme activity and fermentation cycle were significantly improved.In the future,molecular biological modification can be used to further enhance the activity and expression level for practical application.