| Heparinase is one of the polysaceharide lyase which acting on heparin and heparan sulfate.It can specifically breaking different sequence with specific modifications on heparin chain,thus produce different oligosaccharide fragments.Heparinase exist in animal tissue cell widely,various kinds of bacterium and bacteroid also.People have made a lot of researchs after discovering heparinase in Flavobacterium heparinum.It's most important application is producing low molecular weight heparin.Furthermore,it also used to eliminate heparin in extracorporeal blood circulation,determinate precise structural of heparin,research on anticoagulant mechanism of heparin as well as it's structure and function,apply to obstetric field.Afterwards,researchers found that there is a close relationship between heparinase and invasion and metastasis of the tumor.Heparinase involved in tumor angiogenesis.Therefore heparinase has become a promising target of anti-tumor therapy.However,commercial heparinase mostly come from Flavobacterium heparinum at present.Common people cannot accept because of the exorbitant price.So,doing researchers on heparinase has especially significance.At present,most of researchs on expression of heparinase are in Escherichia Coli.The expression product likely to form inclusion body and enzyme activity decreased after protein renaturation.Furthermore,it is hard to separate and purify which limited the widely application of heparinase.However,Pichia pastoris has been utilized widely as an excellent heterologous gene expression system recently.It has been reported that there are a lot of heterologous gene expressed in the Pichia pastoris successfully.For all the reasons above,we aim to construct recombinant yeast to express Hpa I and through response surface optimization and high density cultivation in a 5 L fermentor to enhance the yield of Hpa I.The research result was listed as follows:In this study,the Hpa I gene was from Flavobacterium heparinum and ligated into the plasmid pPIC9K to generate the recombinant expression vector pPIC9K-Hpa I.Linearized by the restriction enzyme BamH I,this constructed expression vector was transformed into the genome of Pichia pastoris GS115 by electroporation method.High copy transformant was distinguished by G418,and though the method of azure A measuring enzyme activity to screen the highest yield strains called 2 recombinant yeast,which enzyme activity was 210.26 U/L.SDS-PAGE show that the molecular weight of the recombinant Hpa I was about 43 kDa.Explored the effection of 2 recombinant yeast expressing Hpa I by seven important factors,induced time,induced concentration,medium volume,oleic acid,PTM1,Tween-80 and cell concentration before induced.On that basis,by 2-level Factorial and Box-Behnken design,the optimal culture medium components obtained were as follows:1%yeast extract,2.00%tryptone,1.34%YNB,100 mM potassium phosphate,0.96%methanol,0.071%oleic acid,0.04%PTM1,4.00×10-5%biotin and 0.5%Tween-80;the optimum conditions were:inoculum concentration 7%,medium volume 34.5 mL in 250 mL flask,began to induce by adding methanol when OD600 come to 7,and after 96 hours induced,the highest Hpa I activity was obtained with yield of 322.95 U/L.The protein content was 1.03 g/L.On the basis of the above studies,high density cultivation condition of recombinant yeast was explored in a 5 L fermenter.The whole fermentation period included four stages,that was accumulate biomass,glycerin fed,carbon starvation and induction.The highest Hpa I yield and protein level were obtained at 398.5 U/L and 1.29 g/L at 110 h,only increased 4 U/L and 0.02 g/L comparing to 96 h.Therefore,we think the 96 h is the best induction time.The research lay a good foundation of industrialization cultivation about Hpa I. |
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