Molecular Modification Of Cellobiose Phosphorylase From Clostridium Thermocellum Towards Broader Substrate Specificity

Cellobiose phosphorylase(CBP)can phosphorolyze cellobiose into glucose and glucose-1-phosphate.As an important member of the cellulose degrading enzyme system,CBP plays an irreplaceable role and has very high application value.However,CBP can only phosphorolyze cellobiose with high-efficiency,and has no activity on oligosaccharides with higher polymerization degrees such as cellotriose and cellotetraose,which limits the further application of CBP.In order to break through the limitations of CBP application,this study modified the broadness of CBP substrates through three aspects:the replacement of the catalytic loop of CBP,the mutation of the key site of the catalytic ring,and the superimposed mutation of the N-terminal site.The main findings of this research are:(1)Deleting the partial section of the CBP catalytic loop,insert the partial section of the CDP catalytic loop at the same position,and gradually narrow the replacement range on the premise of introducing the helical structure of the CDP catalytic loop.A total of 7 modified enzymes were obtained at this stage,which were named CBP-?1,CBP-?2,CBP-?3,CBP-?4,etc.to CBP-?7 according to the order of modification.Among them,CBP-?1 and CBP-?2 are not active.Starting from CBP-?3,the engineered enzyme has weak activity.CBP-?4 is the first modified enzyme obtained in the study to have a higher activity on cellobiose than wild-type CBP,which is 1.5 times the latter,but its activity on the natural substrate xylose is only 4.8%that of wild-type CBP.CBP-?5 is the best modified enzyme in the experiment.Its activity on xylose is increased to 6.8%of that wild-type CBP,and its activity on cellobiose is 2.1 times that of wild-type.However,the activities of CBP-?6 and CBP-?7,which were modified on the basis of CBP-?5,decreased significantly.(2)Mutations were made to key positions 496-499 on the catalytic loop of CBP to eliminate side chains of residues and open the substrate channel.Three modified enzymes were obtained at this stage.The first is CBP-GGGG,where all 4 sites are mutated to glycine and CBP-AAAA where all the pictures are changed to alanine,but the activity of both is very low.After that,only the 497 position was mutated,and serine was mutated to glycine to obtain S497G.The activity of this modified enzyme is significantly better than that of wild-type CBP.Its activity on natural substrate xylose is 1.3 times that of wild-type,and its activity on cellobiose is 2.3 times.(3)On the basis of S497G,the N-terminal positions 165 and 166 were superimposed and mutated to obtain modified enzymes S497G-Q165G/R166G and S497G-Q165A/R166A.After testing,it was found that the two modified enzymes basically lost their activity.(4)Determine the enzyme kinetic parameters of modified enzyme S497G.Its optimum pH is 7.0,and its optimum temperature is 50?,which is not much different from wild-type CBP,and it can maintain more than 80%of its activity after a water bath at the optimum temperature for 50 minutes,indicating that it has good thermal stability.Analyzing the enzymatic kinetic parameters,it is found that the catalytic ability of S497G on the natural substrate xylose in the synthesis reaction is 5.5 times that of the wild-type CBP,and 3.1 times for cellobiose;in the phosphorolation rection,S497G has 1.5 times the catalytic ability of the natural substrate cellobiose,and 3.5 times that of cellotriose.The activity of S497G is significantly improved.In this study,the broadness of the CBP substrate was modified.The modified enzyme S497G not only improved the activity of its natural substrate,but also significantly improved the catalytic ability of cellotriose with a higher degree of polymerization.This breaks through the limitations of CBP application to a certain extent.It is believed that S497G will have higher value in future production applications.