Cloning And Expression Of The Feruloyl Esterase Gene From Cochliobolus Lunatus In Pichia Pastoris

In this paper,the strains which can produce feruloyl esterase was screened by the transparent zone method of plate containing method,which was collected from the Changqing group mountain.7 strains with clear transparent ring were seleceted.The high enzymatic activity strain was named QU108.The colony surface of strain QU108 is uneven,there are radial wrinkles,and the early mycelium is white.With the extension of the culture time,the color gradually deepens,and the final is black.Under the microscope,the spores of the spore stem are less than 3,the spores bend like crescent,and the septum is separated into 3 septum.The 18S rDNA gene of the strain was amplified by PCR,and the product size was 1638bp.Blast homology analysis showed that the identity of Cochliobolus sp.was 99%.It was preliminarily determined that strain QU108 belonged to the genus Curvularia.The ITS sequence of strain QU108 was amplified by universal primers,and the PCR product was 336bp.The homology comparison showed that the Cochliobolus lunatus identity was as high as 100%.According to the morphological observation and molecular biological identification,we can determine the isolated strain belongs to fungi,epiphyte,genera,and genus conidia.Using the protein sequence of ferulic acid esterase A from Aspergillus niger as the target sequence,the candidate sequence of ferulic acid esterase A was obtained by BlastP alignment with the total genome sequence of Cochliobolus lunatus.PCR technique was used to clone the gene of ferulic acid esterase A of QU108,the length of the gene was 1022bp,the ratio of GC content was 53.7%,containing 2 exons and 1introns.Encoding 317 amino acids,the theoretical protein molecular weight is 34.75KD,and the theoretical isoelectric point is 7.29.The signal peptide sequence is MKFTAIALAALAAPLAQA.In secondary structure of FAE protein,alpha helix accounted for 29.02%,and beta fold accounted for 18.3%,and irregular coil accounted for 52.68%.Phylogenetic tree analysis showed that the identity of ferulic acid esterase A protein with ferulic acid esterase from Stemphylium lycopersici sequence was 52%.The intron of the faeA from strain QU108 was removed by overlapping extension method,and the cDNA gene without signal peptide was obtained.The length of the cDNA gene was 900bp,encoding 299 amino acids,and the theoretical molecular weight was 33.0 kDa.The recombinant expression vector of pPIC9K-ClfaeA was transformed into Pichia pastoris GS115.From 23 transformants,5 transformants with larger diameter of transparent circle were selected on the ethyl ferulate ethyl plate.Ferulic acid esterase activity assay showed that 15#transformant had the strongest activity,which reached 8.7 U/mL.The analysis of SDS-PAGE of the fermentation supernatant from15#transformant was carried out.The results showed that there were 1 distinct bands at the size of 32 kDa,indicating that the ferulic acid ferulic acid esterase A gene was successfully expressed in Pichia pastoris.The enzymatic properties of the recombinant ferulic acid ester enzyme were studied.The results showed that the optimum temperature of the recombinant ferulic esterase was60~oC,the optimum pH was about 6.0,and the enzyme activity was still above 70%after incubation at 70 C for 30min.In the range of pH4.5-6.0,the enzyme stability is good,and the residual enzyme activity is higher than 92%.