Font Size: a A A

Study On Xylanase Process Produced By Microorganism And Plants

Posted on:2009-06-20Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y C SuFull Text:PDF
GTID:1100360275981511Subject:Crop biotechnology
Abstract/Summary:PDF Full Text Request
Xylan, a kind of poly-pentose, which is the major component of hemicelluloses account forone-third of the total plant carbohydrate, is the second rich renewable biological resources runafter cellulose in nature. Xylanase is the enzyme for xylan oligomer and xylan depolymerization, whichwill degrade xylan into Xylooligo- saccharides, xylose and oligomer. Xylanase is widely used infeed, paper, food, pharmaceutical and energy industries. The mainly studies of xylanaseproduced by Aspergillus niger in this paper as follows:1. Optimization of Aspergillus niger culture medium. The optimum liquid fermentationmedium for Aspergillus niger that produce xylanase were determianed by single factorexperiment and orthogonal experiment as follow: determined that the optimum liquidfermentation medium for Aspergillus niger that produce xylanase is: bran 80g/L, Tween802g/L, inorganic salt K2HPO4 2.6%, CaCl2 0.5%, MgSO4.7H2O 1%, medium volume 50ml/250 ml, initial pH value of cultivation is 5.0, cultivation temperature is 28℃, cultivationtime is 72h, rotating speed of shaker is 150r/min. Under these conditions, the xylanaseactivity of the strain is higher; it is up to 85.3U/ml.2. ATPE purification of xylanase. In this paper use ATPE to purify xylanase and determined theoptimum conditions for purification is: concentration of PEG 4000 is 19% (w/w),concentration of K2HPO4 is 10% (w/w), concentration of NaCl is 0(w/w). Under theseconditions, it has a better extraction efficiency of xylanase; K and Yt are 29.34, 88.67%respectively.3. Study on the properties of xylanase. The properties of xylanase that produced fromAspergillus niger were studied in this paper, the results indicated that: the optimum reactiontemperature of the xylanase is 50℃, and the optimal pH value is 5.0, the xylanase has abetter stability under acidic conditions within pH3.0~6.0 and is a typical acid xylanase, it isthermal stable between 40~50℃. As to metal ions, Fe2+,Mg2+,Zn2+ have differentlypromotion on the xylanase activity; Al3+ has a little inhibition effect on the xylanase activity;and most of others metal ions have little impact on the xylanase activity.4. This paper took a strain of genomic DNA from Aspergillus niger as the template, obtainedapproximately 750bp and 680bp fragment by amplified PCR. Sequencing results indicatedthat the total length of this gene is 746bp, which contain 68bp intron, and the coding gene is678bp. At present, the gene had been submitted to Gene Bank, the login number isEU423881. Connected the correct gene xynB, which was obtained from sequence, to expression vector pPIC9K from secretary yeast, transformed into E.coli DH-5α, constructedexpression vector pPIC9K-xynB. Then recombinant plasmid into Pichia pastoris (PichiapastorisGS115) by electroporation method, screening by the MM / MD speed and slow spot,picking a few slow spots for methanol inducing cultivation, spot it on RBB-xylan plate after4 to 6 days, got a clear and transparent circle. Xylanase harvest of recombinant strainSMD-xylB-2 up to 216U/ml, increased 2.53 times to initial strain. The expression producthas biological activity, excrete outside cell, the xylanase is soluble and its expression hasgenetic stability. The xylanase properties of recombinant xylanase is similar to originalxylanase, the optimal reaction temperature is 50℃, optimal pH value is 5.0, thermalstability between 40℃-70℃does not change obviously, xylanase activity decrease 0.2%compared to original strain.5. Effect of culture mediums, hormones and their concentrations for embryogenic callusinduction, and differential mediums on embryoid differentiation in vitro were investigated,which took hypocotyl, cotyledon, leaf and petiole of alfalfa as explants. The results indicatedthat the hypocotyl embryogenic callus induction rate was the highest; optimal medium forembryogenic callus induction was the modified SH + 2.4-D 2.0 mg/L + 6-BA 0.5 mg/L;optimal medium for embryoid induction was MSO + 2.0 mg/L6-BA + 0.5 mg/L NAA;optimal medium for plant development was 1/2MS + 1% sucrose + 0.7% agar. Expressionvector PBI121-xynB was constructed, and determined by enzyme examination successfully.The vector was transformed into alfalfa, transformation rate was lower, and the xylanaseactivity isolated from leaf was 10.5U/g.
Keywords/Search Tags:Aspergillus niger, xylanase, fermentation process, Pichia pastoris, gene cloning, alfalfa
PDF Full Text Request
Related items