Cloning,Expression And Enzymatic Properties Of Thermophilic Pullulanase

Pullulanase is one of the starch-debraching enzymes which is widely used in lots of industries,while there is no pullulanase of domestic self-independent production.So,developing pullulanase of self-independent property rights has been becoming the current research focus.In this study,thermophiles Thermotoga petrophila?DSM13995?and Thermosipho melanesiensis?DSM12029?which encod the gene of pullulanase were selected from database,in order to obtain pullulanase of excellent enzymatic properties.The results are as following:?1?Construction of recombinant plasmids.The genes of TP-pul A?acession no.WP₀04082389.1?and TM-pul A?acession no.WP₀12056687.1?were obtained from the genome of Thermotoga petrophila and Thermosipho melanesiensis.And the recombinant plasmids TP-pul A-p ET21 a and TM-pul A-p ET21 a were constructed.?2?After being induced in Escherichia coli BL21?DE3?,analysis of hydrolysis product and the properties of crude enzyme.The recombinant plasmids TP-pul A-p ET21 a and TM-pul A-p ET21 a were transformed into E.coli BL21.After being induced,the production of both TP-pul A and TM-pul A hydrolysing pullulan was maltotriose,so,TP-pul A and TM-pul A were both type ?pullulanase.The optimum p H of TP-pul A and TM-pul A were both 6.6,the optimum temperature were both 80 ?.The half life period at 70 ? of the enzymes TP-pul A and TM-pul A were 24.16 h and 5.24 h,respectively.?3?TP-pul A and TM-pul A rare codon analysis and host replacement.TP-pul A and TM-pul A rare codons were analyzed because of the low expression quantity of TP-pul A and TM-pul A.The results showed that there were many rare codons in the sequences of TP-pul A and TM-pul A.So,the expression quantity of TP-pul A and TM-pul A increased by 9 times and 7 times in Escherichia coli Rosetta?DE3?,respectively.?4?Crude enzyme purification and properties analysis of the purified enzymes.The recombinant plasmids TP-pul A-p ET21 a and TM-pul A-p ET21 a were transformed into E.coli Rosetta?DE3?.And the purified enzymes can be used for properties analysis after purification.The results showed that the optimum p H of TP-pul A and TM-pul A were 6.4 and 5.8,respectively.The optimum temperature of TP-pul A and TM-pul A were 85 ? and 80 ?,respectively.The half life period at 70 ? of the enzymes TP-pul A and TM-pul A were 26.28 h and 4.75 h,respectively,which showed TP-pul A and TM-pul A were thermostable.The activity of TP-pul A and TM-pul A was increased by Na+,K+,Ca2+ and Mg2+,while the activity of TP-pul A and TM-pul A was inhibited by Mn2+,Co2+,AL3+,Fe3+,SDS and EDTA.?5?Kinetics determination.The Km,Vmax,Kcat and Kcat/Km of TP-pul A were 0.72 mg/m L,0.0049 ?mol·m L-1·s-1,154.63 s-1 and 214.76,respectively.The Km,Vmax,Kcat and Kcat/Km of TM-pul A were 4.68 mg/m L,0.0085 ?mol·m L-1·s-1,71.18 s-1 and 15.21,respectively.The results showed that compared with TM-pul A,TP-pul A had a higher substrate affinity and catalytic efficiency.?6?Analyzing optimum temperature,half-life and kinetic parameters of TP-pul A and TM-pul A,compared with TM-pul A,the results showed that TP-pul A was superior pullulanase of enzymatic properties.