Biochemical Characterization Of Extracellular Novel Feruloyl Esterase From Penicillium Piceum And Its Application In Biomass Bioconversion

Feruloyl esterase is a part of hemicellulose.Feruloyl esterases(FAEs,EC3.1.1.73)hydrolyze the ester bond between hydroxycinnamic acids and sugars in plant cell wall.and the three-dimensional structure of the polymer to form a stable Feruloyl esterase cancut down the ferulic acid residues from polymerized substance.Therefore,the structure of the polymerized substancefinally brokeand long chain become shorter.Therefore,in addition to the application of food pulping,paper making and biosynthesis,it can also have a wide application value in biomass biorefinery.The enzymatic activity and secretion of feruloyl esterase have great space for modification,and there is no large-scale application in industrial production.So the study of feruloyl esterase in efficient expression would have great value.We report the discovery of a novel feruloyl esterase from Penicillium piceum through the use of its genome sequence(unpublished data).The characteristics and functions of feruloyl esterase have been studied.Moreover there is no feruloyl esterase in the extracellular protein of T.reesei.The novel feruloyl esterase as a synergistic enzyme will supplement with cellulase from T.reesei.In this study,the novel feruloyl esterase gene from P.piceum was amplified by PCR.The expression casstte p SIMPLE-19-Pp FAE has been constructed.The expression casstte p SIMPLE-19-Pp FAE was transferred to A.niger protoplasm.The enzymatic activity and SDS-PAGE of the transformant supernatant were analyzed.Finally,the novel feruloesterase was found to be successfully expressed in Aspergillus Niger.The enzymatic properties of new feruloesterase and its new functions in biomass degradation were studied.Secondly,the optimum combination of inducers was screened by fluorescence quantitative PCR.Thus,novel feruloesterase was expressed efficiently in Aspergillus Niger,and the hydrolysis efficiency of the existing enzyme system of Trichoderma edulis was greatly improved.The research contents :1.p SIMPLE-19-Pp FAE expression caste construction in Aspergillus Niger.This study successfully cloned the whole fragment of the FAE-encoding gene from the chromosomal DNA of P.piceum using designed primers.The generated PCR products and plasmid were double digested and ligated by T4.Finally the expressioncaste p SIMPLE-19-Pp FAE has been constructed.2.Screening and identification of allogeneic recombinants.The expression casstte p SIMPLE-19-Pp FAE was transferred to A.niger protoplasm.The positive colony was identified by PCR.The positive colony would have the same size land with target strip.The novel feruloyl esterase was successfully transferred in A.niger.3.Expression of ferulic acid esterase in Aspergillus Niger.This study with ferulic acid methyl ester as the substrate,the enzymatic activity of feruloyl esterasefwas up to 22.02 IU/mg.There is an apparent band of 53 k Da in SDS-PAGE of the fermentative supernatant.The band in SDS-PAGE was identified by MALDI-TOF.The results showed that the novel feruloyl esterase has been successfully expressed in A.niger.4.The optimum combination of inducers was screened by fluorescence quantitative PCR of ferulic acid esterase.The optimum inducer screening by real-time PCR The novel feruloyl esterase would be induced expressed via different inducers.The best inducer would be screened by real-time PCR.5.Cocktail with cellulase from T.reesei.There is no feruloyl esterase in the extracellular protein of T.reesei.The successful expressed feruloyl esterase was supplemented with cellulase from T.reesei.The biomass conversion and hydrolysis efficiency was greatly improved.To sum up,based on the above research results,the structure and properties of feruloyl esterase are studied in the near step,which is helpful for the further study.