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Cloning And Expressing The Fae Gene Of Aspergillus Terreus In Pichia.pastoris

Posted on:2014-07-07Degree:MasterType:Thesis
Country:ChinaCandidate:J ChenFull Text:PDF
GTID:2250330425952201Subject:Fermentation engineering
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Feruloy esterase (FAE) as hydrolase can release monomeric and dimeric ferulic acidsfrom arabinoxylans making the cell wall more susceptible to further enzymatic attack. It canbe widely used in pulp and paper production, textiles, feedstuff, and food industries.In this paper, the contribution and induced expression of gene engineering strain wereinvestigated.3strains producing FAE were isolated from Cell Bank and soil samples, namedSD01, SD02and SD03respectively. These strains were identified by colonial morphologyand the analysis of18S rDNA and ITS sequence.The strain SD01has a amazing growth rate, dark green moist colonies spread the plate intwo days. Its spores curved to crescent, and each spore stems has no more than three conidias.Every spore has three compartment, and they are enlarged gradually from inside to outside.On the basis of the colonial morphology and its sequence of18S rDNA and ITS, the strain isidentified as Cochliobolus lunatus.The strain SD02grows fast, you can find white mycelia at first, then they will turn greenfrom golden yellow. Spores are loose and lightweight, easily to fall off. The head of the sporeenlarged, conidia arranged along the second small stems evenly in to two rows.There are fluffin the lateral of the mycelia and the diaphragm in the mycelia. It has the typical characteristicsof yellow-green Aspergillus. The strain was identified as Aspergillus flavus.The strain SD03grows very solwly, and mature spores were produced in two weeks. Themycelium are white and gradually become earthy brown. Finally earthy-brown colonies withtypical screw thread and Jagged edges were observed. This strain was identified asAspergillus terreus according to sequences of18S rDNA and ITS.On the basis of feruloyl esterase activity and strain safety, A. terreus SD03was selectedas the research materia. Then, a pair of nested primers were designed by Software Primer5.0and used to clone the fae gene of A. terreus. The fae DNA gene were cloned byoverlapped ligation PCR. DNA sequencing shows that fae gene has an open readingframe of723-bp, which encodes a putative polypeptide of341amino acids. Comparedwith the protein sequnce of ferulic acid esterase reported by NCBI, alanine andthreonine located in the position of120and121respectively are substituted by valineand serine. The differece in two amino acids don’t affect the activity of feruloylesterase.The expression vector pPIC9K-fae was successfully constucted by restrictionendonuclease digestion of EcoR I and Avr II. Pichia pastoris GS115and KM71strains were transformed by Electrotransformation and chemical transformation with linearizedpPIC9K-fae digested by Sal I or Bgl II. The recombinant p.pastoris strains were grown at30°C in MD solid medium till they are all coming out. The recombinant strains were washeddown by sterile saline, then spreaded in MD solid medium supplemented with6mmol/L G418.64high-resistant strains from more than1000recombinant p.pastoris strains were obtained.However, only16recombinant p.pastoris strains have feruloyl esterase activity.16recombinants strains DNA were prepared using glass beads, and amplified by PCR with thepPIC9K expression cassette primer and specific primers of fae. Finaly,6strains werecorrectly amplified to obtain the expected length of PCR fragments.The6recombinant P.pastoris strains’s fermentation performance have been researched.After two days cultured in seed medium, we transfered the recombinant strains intofermentation medium supplemented with0.5%methanol. After inducing cultured for4days,the fermentation broth was centrifuged. The supernatant was crude enzyme solution. On theone hand we tested the activity of the crude enzyme solution by UV method using4-nitrophenyl ferulate (4NPF) as substrate, on the other hand we analyzed the protein in thecrude enzyme solution by SDS-PAGE. The results show that the protein molecular size is32KDa, and the highest enzyme was observed in G3transformant and the activity is up to12U/L.
Keywords/Search Tags:feruloy esterase, gene engineering strain, pichia pastoris, expression, SOE-PCR(gene splicing by overlap extension PCR)
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