Molecular Modification And Thermal Stability Of Alteromonas Sp. Y-389 Pullulanase

Pullulanase is a kind of starch debranched enzyme,which can specifically hydrolyze the alpha-1,6-glycoside bond of amylopectin to form amylose.It can interact with alpha-amylase to hydrolyze starch completely,so it is important in starch processing,beer brewing and industrial production of functional medicine polysaccharide.Exocrine ability of strain is a vital index to evaluate the performance of engineering strain.The saccharification of starch is generally carried out under the condition of 60? and p H 4.5-p H 6.0,and the saccharification time is generally 48-60 hours.Therefore,it is of great significance to improve the acid-base adaptability,extracellular secretion ability and thermal stability of the recombinant strain for reducing production cost and improving production technology during starch processing.In this study,we constructed five domain-truncated variants and four site-directed variants based on multiple sequence alignment and domain analysis with Alteromonas sp.Y-389 Pul A as the research object.Meanwhile,the effects of excising different domains of pullulanase and constructing different mutations on its catalytic activity and enzymatic properties were studied.The main findings are as follows:(1)On the basis of sequence comparison and domain analysis of Alteromonas sp.Y-389 pullulanase,one N-terminal and four C-terminal variants strains were successfully constructed using the recombinant natural enzyme plasmid as template(Pul A+p ET-28a).Except Puld4,all the other 4 truncated variants showed pululanase activity.Moreover,the extracellular enzyme secretion efficiency of the four truncated variants increased gradually with the decrease of protein molecular weight,and the proportion of extracellular enzyme activity in the total enzyme activity was 13.5%,18.2%,20.1% and 30.8%,respectively,which was greatly improved compared with the non-secretion ability of the Pul A strain.In addition,enzymatic analysis showed that the optimal p H of both the truncated variant and Pul A was about 6.0,but the p H range of the truncated variant became narrower.The optimal reaction temperature of Puld1,Puld2,Puld3 and natural pululanase was 35?,and that of Puld5 was 45?,which was 10? higher than that of natural pululanase,and its half-life reached 25 h,2.5 times of that of natural enzyme.However,the spatial structure change caused by domain excision leads to the reduction of the ability of truncating the binding of variants to substrates,and the catalytic efficiency is reduced accordingly.(2)Based on multiple sequence alignment and analysis of amino acid properties,we selected the conserved region and several amino acids around the conserved region as mutation sites,and constructed four site-specific variants and one superimposed variant.The results of enzymatic property analysis of the variants showed that the specific activity ratio of the variants G603 D and F654 Y decreased by 43.7% and 8.9%,and the specific activity ratio of the variants H611 Q and F751 L increased by 64% and 30.2%,respectively.Compared with the truncated variant Puld5,the superposition variants showed a 4% increase in extracellular secretion capacity.Except for the G603 D variant and Pul A,which both had an optimal interaction temperature of 35?,the optimal temperature of the other variants was increased.Additionally,different from variant G603 D,the others had the varying degree enhancement,the thermal stability of the Pul A only about 10 h,the half-life of pullulan enzyme variant F654 Y,H611Q,F751 L and Puld5 / H611 Q was 20 h,20 h,15 h and 50 h,which was 2 times,2 times,1.5 times and 5 times of pullulan natural enzyme,respectively.Thus,the superposition of truncated variants and point variants greatly improved the thermal stability.The results of kinetic parameter analysis showed that the Kcat/Km of truncated variant was 1.17 times of that of natural enzyme,which made up for the reduced catalytic efficiency caused by the removal of only structural domain.Through a series of molecular modifications,this study explored the influence of domain resection and site-specific mutation on the enzymatic properties of natural pululanase,and obtained Puld5/H611 Q,a superimposed variant with relatively good catalytic activity and enzymatic properties,which laid a good experimental foundation for the industrial production of pululanase.