Font Size: a A A

High Efficient Expression Of The Aspergillus Niger Fae Gene In Pichia Pastoris And Aspergillus Niger

Posted on:2015-01-08Degree:MasterType:Thesis
Country:ChinaCandidate:Y Y ZhouFull Text:PDF
GTID:2250330428473336Subject:Food Science
Abstract/Summary:PDF Full Text Request
Hemicellulose is the most abundant component in plant cell walls except cellulose.Xylan comprises about50%of the hemicellulose. The biodegradation of the xylanbackbone depends on two classes of enzymes: endoxylanases and beta-xylosidases.Endoxylanases (EC3.2.1.8) cleave the xylan backbone into smaller oligosaccharides,which can be further degraded to xylose by beta-xylosidases (EC3.2.1.37). In additionto the two types of enzymes play a leading role, other enzymes involved in thedegradation of xylan include, for example, feruloyl esterase, acetylxylan esterase,arabinase, alpha-glucuronidase, and p-coumaric acid esterase. Feruloyl esterase, a keyenzyme, is capable of hydrolyzing ester bonds forming by ferulic acid or diferulic acidbetween plant cell wall macromolecules, resulting in destruction of complex networkstructure of the plant cell wall and access to the substrate of other hydrolases such ascellulase, hemicellulase and pectinase. Feruloyl esterases had great potentials in the feed,food, bio-energy, cosmetic and pharmaceutical industries fields.Based on the sequence of feruloyl esterase of Aspergillus niger from GenBank, twoamplified primers were designed and used to clone the total feruloyl esterase gene (faeA)by polymerase chain reaction (PCR), with genomic DNA from A. niger h408as thetemplate. DNA sequence analysis showed that the length of A. niger faeA (AnfaeA)gene was903bp, containing two exons, an one intron. AnfaeA cDNA sequence of A.niger h408was successfully cloned by overlap extension PCR technique, and theaccession number of GenBank was KF911349. Sequence analysis showed that AnfaeAcDNA gene was783bp, containing an open reading frame (ORF), which encode260amino acids. Blast analysis revealed that the cloned cDNA gene was99%identity to thefaeA gene from A. niger. The deduced amino acids contained a typical active lid andcatalytic triad of lipase.The expression vector pPIC9K-Anfae was constructed by the ligation of the AnfaeA gene into the shuttle vector pPIC9K. The plasmid pPIC9K-Anfae was linearized bySacI and then electrotransformed into P. pastoris GS115. Five recombinant strains withrelative high level of FAE activity were obtained through plate screening, and thetransformant with highest actvitity (24.72U/mL)was named pPIC9K-Anfae5. Thespecific activity of recombinant feruloyl esterase was40.84U/mg. Compared with A.niger h408, the recombinant enzyme activity increased about to1100times. SDS-PAGEresult indicated that molecular weight of the recombinant FAE from pPIC9K-Anfae5 was about33kDa, which is different with the expected molecular weight (28kDa). Theoptimal temperature and pH for recombinant FAE was50°C and5.0, respectively.Recombinant FAE showed stability in the temperature40-50°C and was active in thepH range4.0-9.0.The fermentation conditions of recombinant pPIC9K-Anfae5were optimized. Theoptical inoculation was5%-10%. The initial pH of culture growth and induction wasabout pH6.0. The methanol concentration and induction time were1%and84h,respectively. Based on the parameters optimized by flask experiments, recombinantstrain were fermented in50L and500L fermenter. The results showed that the ferulicacid esterase activity was detected in the early stage of exponential phase, and theactivity rapidly increased with the extension of fermentation time and. reached highestlevel(102.1U/mL and121.8U/mL, respectively) when the fermention time was84h.Compared the activity of recombiant FAE cultivated by shake flask fermentation, theactivity of fermentor increased about to5times. After84h, FAE activity began todecline, due to choose84h (methanol-induced56h) as the end point of fermentation.Pilot of recombinant Pichia strain could accumulate data and experience for theindustrialization of feruloyl esterase.Although the expression of feruloyl esterase by Pichia fermentation was strong, therecombinant enzyme was unique. The application invitro of recombinant FAE fromPichia strain was not as good as those enzymes produced by filamentous fungi,therefore feruloyl esterase was overexpressed in A. niger. The promoter of enolase gene(Peno) from A.niger and terminator (Tpdc) of pyruvate decarboxylase were selected toconstruct the Peno-fae-Tpdc (PFT) expression cassette. The PFT expression cassette andlinearized pAN7-1were co-transfomed into protoplasts prepared from A.niger h408,and the constitutive expression in A.niger was achieved.19transformants withhygromycin resistance were obtaind by rescreening in selection medium containinghygromycin. By transparent flat circle method, seven transformants with relative highferuloyl activity were selcted and verified by PCR amplification. PCR amplificationrevealded that PFT expression cassette had been integrated into the genome of A. niger.Compared the FAE activity of strain h408, transformant with the highest activity(567.46mU/mL) incresed251times. In particular, the peak time of enzyme productionfrom recombinant A. niger had shorten to48h, whereas the peak time of original strianh408was7d. Further researchs were required to identify the integration location andcopy number. of transformant may be the target gene in Aspergillus niger integratedifferent chromosomes caused by the need for further validation of molecular hybridization. The enolase promoter was proved to be a a strong promoter in A. niger.The successful expression of ferulic acid esterase in A. niger provided strains for theefficient degradation of cellulase and a new regulatory elements for expression offoreign genes in A. niger.
Keywords/Search Tags:feruloyl esterase, gene cloning, expression, Pichia pastoris, Aspergillus niger
PDF Full Text Request
Related items