Screening Of Pullulanase-producing Strains And Studies On Its Enzymatic Properties And Molecular Cloning

The pullulanase can specificially degrade alpha-1,6-glucosidic bond which belongs to debranching enzyme and has extensive application in starch processing.In our research,we Screened and Cultivated a pullulanase high-yield strain from soil nearby starch factory.The crude enzyme activity was 5.713 IU/mL.Utilizing squencing 16S rDNA and physiological and biochemical experiment We identified this strain was klebiella variicola strain 7.We verified the optimized fermentation conditions was cornstarch 1.0%,Peptone 1.5%,KH2P04 0.05%,MgSO4.·7H20 0.01%,liquid volume 70mL/250mL,fermentation time 48h,inoculation amount 8%;After optimized we improve the activity 11.87 times.The crude enzyme was salted out by 80%ammonium sulfate and then desalinationized through sephadex column(Sephadex G-25).Finally we obtained the pure pullulanase by using sephadex column(Sephadex G-100).We verified the molecular weight of pullulanase was about 120 kDa by SDS-PAGE.The specific activity of pure pullulanase reach 51.15 U/mg.The recovery rate was 26.9%.After obtaining pure pullulanase we found that the optimized temperture was 45 ? and optimized buffer was Sodium acetate at pH 5.6.The pure pullulanase activity is very under pH 5.6.After 120 min,There was still 80%activity when reaction temperature raised to 55?.Final concentration to 5 mmol·L-1 of Na+ and Ca2+ has a strong role in promoting on enzyme activity,the enzyme was strongly inhibited by Co2+,Cu2+.The specific substrate of our pullulanase was pulullan.After degrading we found the unique product was maltotriose,so this pullulanase was belong to Type I pullulanases.