Fermentation Optimization Of Feruloyl Esterases By Aspergillus Terreus

Feruloyl esterase,also known as cinnamate esterase,which is one kind of the microbial cell wall degrading enzymes,and belongs to a subclass of carboxylesterase.It combines cellulose hydrolase to crack insoluble cell walls and release ferulic acid.Therefore,the enzyme has a good application prospect in the food industry,paper industry,feed production,bioenergy,and other fields.The enzyme extraction methods mainly include plant extraction,chemical synthesis,and biosynthesis.Plant extraction and chemical synthesis are expensive,with poor economic benefits.Biosynthesis has gradually become a research hotspot due to its low energy consumption and low pollution.However,the current wild strain fermentation cannot meet the needs of industrial production.In this paper,filamentous fungi were screened out from nature samples,and the ferulic acid esterase producing strains were obtained by using the ethyl ferulate plate method.One of the strains W2 with high feruloyl esterase yield was selected,and DNA was extracted to identify its species.The fermentation medium was optimized.Cloning its feruloyl esterase gene,using Pichiap astoris as host,the recombinant strain GS115-pPIC9K-FAE was successfully constructed,and its culture medium was preliminarily optimized.High-density fermentation was carried out in a 10-L fermentator to obtain high-level of enzyme production.The main research contents are as follows:(1)Forty three strains of filamentous fungi were screened from natural soil samples,and 8 strains of filamentous fungi with ferulic acid esterase production were re-screened.Morphology and microstructure observation showed that the 8 strains had different colony and spore morphology.After fermentation in the basic fermentation medium,it was found that one filamentous fungus numbered W2 produced the highest ferulic acid esterase activity.PCR amplification was carried out using chromosome DNA of strain W2 as a template.The 5.8S ITS-rDNA sequence obtained by sequencing,homology comparison(Blastn),and phylogenetic tree analysis showed that strain W2 was Aspergillus terreus.(2)After a single factor and climbing test,the liquid fermentation medium of the strain was optimized by response surface design.The optimal medium formula for ferulic acid esterase production of the strain was obtained as follows:glucomannan 3%,beer lees 2.45%,bean dregs 8.1%,FeSO4.7H2O 0.001%,KCl 0.05%,MgSO4.7H2O 0.05%,K2HPO40.1%,pH 5.0.Under shaking flask fermentation conditions,the highest enzyme activity obtained by using the above medium was 205 U/L,which was about 3 times of that before optimization.(3)Primers were designed to clone the feruloyl esterase gene,through DNAMAN software design optimization,introns and signal peptides were removed,and the recognition sites of restriction enzymes EcoR I and SnaB I were introduced upstream and downstream of the gene respectively.The gene was synthesized by the company and constructed based on the expression vector of Ppastoris.The pPIC9K-FAE expression vector was used to express the feruloyl esterase gene in P pastoris GS115.The single-factor experiment determined that the optimal induction conditions were the initial cell OD600=2,the induction pH of 6,and the induction temperature of 28℃.Under the above conditions,the highest yield of feruloyl esterase obtained in shake flask and 10 L fermentor was 3025 U/L and 20918 U/L.