Expression And Characterization Of Pullulanase In Escherichia Coli

Pullulanase,one of the starch debranching enzyme,belongs to family 13 and 57 of amylolytic enzyme.It can cleave ?-1,6-glycosidic linkages in pullulan,starch and related oligosaccharides,and is widely used in the area of food industry,medicine,detergent industry and environmental protection.In earlier study,a pullulanase high-producing strain,Klebiella variicola strain 7,was obtained from the soil near a starch factory.Enzymatic research shows that the pullulanase produced by Klebiella variicola strain 7 is pullulanase type I.The activity of this enzyme is 67.8U/mL.At present domestic pullulan enzyme activity level in 10?30 u/mL,more than the domestic one's current level of pullulanase activity has greatly improved.But Klebiella variicola strain 7 is a opportunistic pathogen,so it cannot be used in food industry.And as its fermentation broth is ropy,it is difficult to extract and purify the enzyme.This study using genetic engineering means,through the heterologous expression to modify pullulanase gene,the results are as follows:1.Cloning of the pullulanase gene.The genomic DNA was extracted from Klebiella variicola strain 7,and the primers,Nde I-P1 Not I-P2,were designed according to the pullulanase gene from Klebiella variicola strain SHN-1 published on NCBI.Then the Pullulanase gene pul(A)(3309bp)was amplificated using the genomic DNA as template.The pul(A)was ligated to pMD19-T vector,and transferred into the host E.coliJM109.The clones were selected,sequenced,and digested with Nde I and Not I.2.Expression of pul(A)in E.coli BL21(DE3).Reconstructed vectors pMD19-pul(A)and plsmid pET-21a were digested with Nde I and Not I,respectively.Then the reconstructed vectors pET-21a-pul(A)were transferred into E.coli BL21(DE3)competent cells.3.Purification of crude Pullulanase.The positive clones were induced by IPTG.The ferment was centrifuged and the cells were obtained.After ultrasonication,SDS-PAGE was carried out.A band was observed at the position of 120KDa and was confirmed as target band.However,there were some other bands in the gel.So we purified the crude enzymes by Co2+ affinity chromatography and moved the salts with G-25 sephadex column.High purified Pullulanase was obtained.4.Enzymatic studies of Pullulanase.In this study,we found the Pullulanse's optimum temperature is 45 ? while the optimum pH is 5.2,similar to the wild strain.However,the thermo stability is less than the wild strain,with 40%activity remained under 45? for 30 minutes.In addition,in the final concentration of 5mmol-L-1,Na+,Mg2+can activate the enzyme activity while Fe3+,Cu2+,2Zn2+,SDS,EDTA inhibit the activity.