Effect Of Removing Terminal Disordered Amino Acids On Pullulanase Enzymatic Property

Pullulanase is one of the starch debranching enzymes,which can cleave ?-1,6-glycosidic linkages.Pullulanase belongs to glycoside hydrolase family13 and 57 of amylolytic enzyme.At present,it has been widely applied to the feed,brewing,medicine and other related fields.Because of the limitation of technology,we do not have the independent property rights of pullulanase.In the present study,a pullulanase high-producing strain,Klebiella variicola strain 7,was obtained from the soil near a starch factory.The gene encoding pullulanas from Klebiella variicola strain 7 was overexpression in Escherich coli BL21(DE3).However,there are some problems in the expression of pullulanase,such as: the lower expression,intracellular enzyme,instability.So,it can not achieve industrial production needs.In this study,we use genetic engineering mean to heterologous expression pullulanase gene in Bacillus subtilis.At the same time,we use the method of protein engineering to remove terminal disordered amino acids of pullulanase.The results showed that:(1)Expression of pul A in Bacillus subtilis.Firstly,we designed a pair of primers Xba?-F1?Aat?-R1.Then pullulanase gene was amplificated using recombinant plasmid p ET21A-plu A-signal as a template,which do not have the signal peptide.Plsmid p HT-43 and plu A were digested with Xba?and Aat?,respectively.Then we constructed p HT43-plu A recombinant plasmid,which was transformed into E.coli JM109 competent cells.After getting positive transformation of recombinant plasmid p HT43-plu A,we transformed the recombinant plasmid into Bacillus subtilis WB600 competent cells.The extracellular enzyme activity is 0.6 U/m L.(2)Removing terminal disordered amino acids of pullulanaseThrough bioinformatic methods,we analysised the sequences of gene and protein secondary structures.And we found thirty-two and three disordered amino acids respectively in N-terminal and C-terminal,which do not form the regular secondary structure.We think that it is an adverse impact to the stability of pullulanase.Through genetic engineering methods,we remove these amino acids respectively.Finally,we evaluate the enzymology properties of the mutant enzymes.The enzymatic studies results showed that: 1)The optimum p H of plu A-N31 drop from 6.0 to 5.6,but the optimum p H of other mutants do not change.2)The Topt of plu A-N35 elevated from 45 ?C to 48 ?C,but the other mutants do not change.The half-life of the mutants were improved greatly.The half-life of plu A-N31,plu A-N35 and plu A-C3 is 6.17-,5-and 4.16-fold that of plu A,respectively.The half-time of plu A-N4 and plu A-N4-C3 is 1.66-and 1.66-fold that of plu A,respectively.3)Compared with plu A,the specific activity of plu A-N31 and plu A-N35 elevated 1.6times and 1.06 times,but other than mutant enzyme activity decreases.4)The Km of plu-N31,plu A-N4,plu A-N35,plu A-C3 and plu A-N4-C3 is 0.45-,1.49-,1.31-,1.35-and 1.35-fold that of plu A,respectively.The Kcat of plu A-N31,plu A-N4,plu A-N35,plu A-C3 and plu A-N4-C3 is 0.83-,0.67-,1.1-,0.38-and 0.92-fold that of plu A,respectively.So,Removing terminal disordered amino acids have the positive role to improving the thermal stability of pullulanase.