The Effect Of N-glycosylaton And CBM On Secretion, Activity And Stability Of AXE1Expressed In Pichia Pastoris

N-glycosylation, one of the most common and yet complex forms of post-translationalmodifcation,is intricately involved in a host of cellular processes including protein folding,protein secretion and intracellular traffcking. In recent research, N-glycosylation has been usedas an important strategy to modulate protein structure and function.In our previous research, we cloned a new AXE1from Volvariella volvacea. This AXE1iscomposed of a catalytic domain of CE1and a CBM of family1. It had been highlyN-glycosylated expressed in Pichia pastoris and presented two bounds located at66kDa and48kDa on SDS-PAGE separately. AXE1could only bind cellulose but not xylan. In thisresearch, we firstly vertify the importance of N-glycosylation of AXE1on its secretion, activityand stability. On basis of this, protein glycoengineering and CBM exchanging are used toimprove the characteristics of AXE1. The main contents and results of our research are asfollows.In this research tunicamycin is used to study the effect of N-glycosylation on AXE1secretion. As a result, secretion level of AXE1decreases as the tunicamycin concentrationincreases. About55%decrease has been observed when the tunicamycin concentration reach10μg/mL and then N-glycosylation of AXE1has been inhibited obviously. These data suggeststhat N-glycosylation played an essential role in the secretion of AXE1.In order to vertify the importance of N-glycans of AXE1on its activity and stability, wefurther analyse the catalytic properties and enzyme stability of three kinds of AXE1s (the wildtype, endo H-treated and tunicamycin-treated AXE1s).As a result, N-glycans have little effecton the catalytic activity of AXE1but do have some effect on its stability with respect to pH,temperature, urea and proteases.There are two N-glycosylation sites in AXE1, located at N31and N99respectively. We usesite-directed mutagenesis to get mutants (T33A,T33N,T33S) deglycosylated at N31andmutants (N99A,N99Q,T101A) deglycosylated at N99. We use these mutants to study the effectof individual N-glycans. As a result, the secretion level of mutants deglycosylated at N31decrease about26%-50%and mutants deglycosylated at N99decrease about18%-23%lessthan the wild type. All the mutants deglycosylated at N31or N99get less stable than the wildtype. When the two N-glycans have been both removed, the secretion level decreasesremarkably to20%of wild type AXE1and gets the worst stability. The two N-glycans play animportant role on AXE1secretion and stability, but both have little effect on its catalyticactivity.By increasing a N-glycosylation site positioned at Q24and N34of AXE1separately, we get mutants named Q24N and G36T. The secretion level of Q24N increases about5%thanAXE1, but it gets less stable. G36T gets better thermal stablily, but its secretion decreases36%less than AXE1. By increasing a N-glycosylation site at the two same positions of N31Aseparately,we get mutants named N31AG36T and Q24NN31A. The secretion level ofN31AG36T increases8times and its activity increased30%more than N31A. But Q24NN31Ahas no obvious change with N31A on the activity and secretion level.We synthesize two genes of xylan-binding domain named cbm2b-2and cbm6.Using thesetwo CBMs, we make two new AXE1s named AXE1CD-CBM2b-2and AXE1CD-CBM6. Thesetwo new AXE1s have better ability to bind Xylan. About20%increased activity towardacetylated xylan has been both observed in the two new AXE1s.