The Expression, And Characterization Of A Cellulase From The Mollusca Ampullaria Crossean In Pichia Pastoris

The increasing problem of the CO2emissions besides some energy security concerns has strengthened the interest in alternativ,nonpetroleum-based sources of energy.Cellulose is the most abundant renesable biomass on earth,which can provide enormous energy for human being.To bio-degrade cellulose, cellulase is needed.Under the hydrolyzing of cellulase,the cellulose can be degraded into glucose,which can be easily transformed into bioethanol and biodiesel.Many kinds of orgnisms can produce celuulases,such as fungi,bacteria,plants and protists,even some animals can produce cellulase endogenously.The studies about animal cellulase haven’t started untill1998,when the first animal cellulase gene was cloned.Since then, even though the number of reported cellulase genes is growing,but the recombinant expression of animal cellulase is difficult.In2010,Hiryam etal expressed two termit cellulase (RsEG and NtEG),the maximal yield is26mg/L and31mg/L respectively,which is the maximal production of heterologous expression ever reported.In our previous work,we cloned an endogeous cellulase gene eg27from the Mollusca,Ampullaria crossean. EG27I shows good thermal stability and pH stability(about85%of the activity of EG27could be retained upon incubation at60℃for24h;EG27was acceptably stable at pH3.0-11.0even when the incubation time was prolonged to24h at30℃).which makes it suitable for industrial purpose.In this work,we expressed EG27using pichia pastoris expressing vetor pGAPZaA firstly.After the recombinant strains pGAPZaA-EG27/SMD1168were constructed using molecular biology operations.the response surface method was used to optimize the three compositions of the express medium YPD,tryptone,yeast extract and D-gulucose,and the optimist composition of the express medium is determined.Upon on our above work.we constructed a new expressing vector pGAPH based on the commercial vector pGAPZaA by replacing the a-factor secretion signal with HFBII secretion signal, and a kozark sequence was added right before the initiation codon of HFBII secretion signal.Then the interest gene EG27was cloned into the new vector pGAPH.the recombinant vetor pGAPH-EG27was fisrt transformed into DH5a to amplify the plasmid.then the recombinant plasmid was transformed into pichia pastoris SMD1163.The recombinant strain pGAPH-EG27/SMD1163was cultivated in30L biology reactor, the maximal expression of54.9mg/L was yield.The supernant of fermentation was concentrated by ultrafiltration,then purified by SP-Sepharose and Phenyl-Sepharose.The purified EG27shows similar enzymic character with the wild cellulase.The Western-Blot experiment shows that the recombinant strain pGAPH-EG27/SMD1163posses better ability of secreting EG27,which is5folds higher than pGAPZaA-EG27/SMD1168.