Gene Discovery,Expression And Characterization Of A Novel Feruloyl Esterase From Aspergillus Cristatus

Aspergillus cristatus is the dominant fungus in the process of fermentation of Fuzhuan brick tea in Hunan Province,and it is one of the key factors for the unique quality of the tea.Previous studies showed that Aspergillus cristatus has a variety of benificial effects,such as lipid-lowering,anti-oxidation,immune regulation,anti-tumor and anti-bacterial.However,due to the complexity of secondary metabolites of Aspergillus cristatus and the influences of growth environment,the active components among the metabolites of Aspergillus cristatus are not clear.To better study the functional components of Aspergillus cristatus,we performed a genome-wide sequencing of Aspergillus cristatus and carried out a systematic experiment on it.Ferulic acid is a ubiquitous phenolic acid with the effects of antioxidant,antithrombotic,lipid-lowering,antibacterial and antitumor.In the mature plant cell walls,ferulic acid combines with cellulose and lignin to form ferulic sugar ester,which cannot exert biological activity.Feruloyl esterase is a kind of carboxylic esterase that hydrolyzes the ester bonds formed by phenolic acids such as ferulic acid,p-coumaric acid and dimeric ferulic acid with hemicellulose and lignin in plant cell walls to release free phenolic acid.After genomic analysis,we found that there was a gene sequence in the genome of Aspergillus cristatus,which is 70%similarity with ferulic acid esterase reported before,and contains the common motif GHSLG of type A feruloyl esterase.Hypothetically,it was likely to be a new kind of feruloyl esterase gene,and the catalytic effect of fae may be one of the reasons for the healthy effects of Fuzhuan brick tea and Aspergillus cristatus.Therefore,it is of great significance to further study the gene and its related enzymatic properties.With the method of Augustus and gene blast,we confirmed that the gene sequence is composed of a 92-bp intron and a 846-bp exon.At the same time,using the signal peptide prediction software,we also detected that the protein sequence encoded by the segment contains a signal peptide including 22 amino acids.The whole CDS was was estimated to be 780 bp,encoding a total of 260 amino acids.Therefore,in order to verify the prediction above and to study the characteristics of the enzyme in depth,we amplified the complete coding sequence from the genome and then the expression vector pET22b-Acfae was constructed to express the exogenous protein in E-coli.As a result,the expressed protein was detected in the form of inclusion bodies.After renaturation by dialysis,soluble recombinant protein was obtained.Further,the activity of the enzyme was detected by spectrophotometry,while no feruloyl esterase activity was detested in the refolded protein,which might due to the uncorrect refolding process.Then rationally we switched to Pichia pastoris SMD1168H to express the AcFAE.Firstly,the gene sequence was optimized according to the codon preference of Pichia pastoris.Secondly,the expression vector pGAPZαA-Acfaeo was constructed and electrotransformed into Pichia pastoris SMD1168H cells.The positive transformants were selected and cultured by shake flask fermentation and high-density fermentation.SDS-PAGE and enzyme activity detection showed that the recombinant protein was successfully expressed in pichia pastoris with the catalytic activity of 0.75 U/mL.The recombinant protein was isolated and purified with nickel column and the enzymatic properties were studied.The results implied that the optimum reaction temperature of AcFAE was 55 ℃,and it stayed in a good stability at 40 ℃ and below.However,the enzyme activity disappeared at 55 ℃ for 1 h.The optimal pH value of the recombinant enzyme was 6.6,and had a good stability in the pH range of 5.0-8.0.In summary,we found a new feruloyl esterase gene in the genome of Aspergillus cristatus,and its expression was achieved in Pichia pastoris by rational prediction and heterologous expression verification followed by its enzymatic properties This is the first report on the feruloyl esterase gene in the Aspergillus cristatus,providing a basis for the study on the health mechanism of Fuzhuan brick tea and Aspergillus cristatus.It also offers a theoretical foundation for the industrial production of feruloyl esterase.