| Glycosylation is one of the most prevalent post-translational modifications and often has marked impact on the structure and function of protein.In our previous research,the gene ofα-L-rhamnosidase Rhal from Aspergillus niger was expressed and glycosylated in Pichia pastoris.However,the regulation mechanism of glycosylation towards the enzyme is still unclear.Therefore,in this study,the main type of glycosylated modification of α-L-rhamnosidase was determined.The enzymatic properties and structural characteristics of α-L-rhamnosidase with different N-glycosylation site mutation were investigated,and the effect of N-glycosylation on the conformation stability and structure of α-L-rhamnosidase.The main results are as follows:First,N-glycosylated modification was determined to be the main glycosylated modification type in α-L-rhamnosidase by using peptide-N-glycosidase(PNGase F)and beta-elimination reaction.Five sites of N-glycosylated modification were identified by LC-MS,and the structural analysis showed that N1325,N145,N220,N407 and N596 were all located on the Loop/Turn.The deglycosylated mutants were constructed and expressed.Then,the enzymatic properties and structural characteristics of α-L-rhamnosidase with different deglycosylated mutants were analyzed.The comparison of enzymatic properties of 5 deglycosylated mutants were carried out,and results showed the optimum pH,optimum temperature,secondary structure and tertiary structure of 5 deglycosylated mutants were similar.However,the enzyme activity,thermal stability and kinetic parameters of different deglycosylated mutants are different with each other.Then,7 potential glycosylated sites of α-L-rhamnosidase were identified using NetNGlyc.The potential deglycosylated mutants were constructed and expressed.Then comparison of enzymatic properties of 7 potential deglycosylated mutants were carried out,and results showed the effects of 7 mutations on the enzymatic properties and structural characteristics waer similar to the patterns of 5 deglycosylated mutants identified by LC-MS.Next,the enzymatic deglycosylation of α-L-rhamnosidase was investigated using Endoglycosidases F1(Endo F1),and the comparison study of enzymatic properties and structure between native and deglycosylated α-L-rhamnosidases were performed.Except for the same optimum temperature,the deglycosylated α-L-rhamnosidase presented many differences with native α-L-rhamnosidase.The relative enzymatic activity of deglycosylated α-L-rhamnosidase was 78%of native enzyme;the deglycosylated α-L-rhamnosidase showed a wider range of optimum pH,and a greater affinity to pNPR compared to glycosylated enzyme.DSC analysis exhibited that the deglycosylated a-L-rhamnosidase had lower Tm and higher ΔH to those of native α-L-rhamnosidase,indicating that the thermal stability of deglycosylatedα-L-rhamnosidase was decreased.Near-UV CD spectra and intrinsic fluorescence spectrum analyses confirmed some loss of tertiary conformation of deglycosylated a-L-rhamnosidase,but the secondary structure was nearly unaffected after the deglycosylation treatment.Finally,adding tunicamycin to prevent the assembly of lipid polysaccharide precursors,and affecting a-L-rhamnosidase glycosylatied during protein synthesis processing,and the comparison study of enzymatic properties and structures between a-L-rhamnosidase treated with Endo F1 and tunicamycin were carried out.The results indicated that N-glycosylation affects the enzymatic activity,catalytic activity and thermal stability of a-L-rhamnosidase,but did not affect the optimum pH and secondary structures.When using tunicamycin to affectingα-L-rhamnosidase glycosylatied during protein synthesis processings,the secondary and tertiary structures of obtained a-L-rhamnosidase were changed greatly.Compared to the Endo F1-treated-a-L-rhamnosidase,tunicamycin-treated-a-L-rhamnosidase showed lower catalytic activity,weaker thermal stability and conformational stability than Endo F1-treatedα-L-rhamnosidase. |
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