| Glycosylation is one of the covalent modification which naturally occurring in eukaryotic proteins. It is estimated that more than half of the proteins found in nature have taken place in glycosylation and three quarters are N-glycosylation. N-Glycosylation, protein post-translational modification of the most complicated one, involved in various processes of the host cell, such as protein folding, protein secretion, intracellular transport intracellular communication and so on. N-glycosylated in regulating the protein structure and function has a significant advantage, the protein folding and the stability can be adjusted by a number of mechanisms. The glycosylation of cellulase have associated with different expression host and different growth conditions, therefore, its enzymatic activity and capacity of binding substrate will also be change. In recent years, many studies have shown that N-glycosylation also have a dramatic effect on enzyme activity and substrate specificity. Currently, more and more cellulase genes have been successfully expressed in Pichia pastoris, particularly for cellulase genes from Trichoderma reesei but there were less research reported on Rhizopus genes. Endoglucanase (EG) as a cellulase has a crucial role in the process of cellulose degradation.This article focuses on N-glycosylation of EG Ⅱ from R.stolonifer TP-02, analyzing its glycosylation induced changes of catalytic conformation. The main research findings are as follows:(1) Bioinformatics analysis found that Rhizopus stolonifer endoglucanase Ⅱ contains two potential glycosylation sites, namely, N68 and N134. The N-glycosylation site N68 was located on the linker close to carbohydrate binding modules (CBM), whereas the N-glycosylation site N134 was located at the entrance of the catalytic activity center of the catalytic domain (CD). We removed the glycosylation sites (N68, N134) by site-directed mutagenesis and combined the molecular dynamics simulation to study the N-glycosylation model of R.stolonifer endoglucanase Ⅱ gene. The results showed that, removing the N-glycan of N68 position does not involve changes in CD area, removing the N-glycan of N134 position make the total energy and the level of activity change of CD. This change led to the conformational rearrangements in N134.(2) Designing the experimental group, removing the glycosylation sites (Asn68, Asn134) by site-directed mutagenesis. The expression vector pPIC9K-N68D, pPIC9K-EG Ⅱ, pPIC9K-N134D were constructed, and the recombinant protein were expressed in Picha pastoris GS115. The maximum endoglucanase activity of the N68D, EG Ⅱ and N134D were 0.789 IU/ml,1.013 IU/ml,1.383 IU/ml, respectively.(3) 10L fermenter was used for amplification experiments, to vary different initial wet cell weight and induction temperature, the results showed that when the initial cell wet weight of 220g/L, induction temperature was adjusted to 28℃, after inducing 108h, N68D, EG Ⅱ, N134D enzyme activity reached 12.09 IU/ml,15.89 IU/ml,20.14 IU/ ml, respectively. Using Ni column to isolated and purified expression product, of three specific activities were measured, compared to native protein, N68D protein specific activity decreased 29.3%, N134D protein specific activity increased nearly 57.6%. N68D exhibited the highest kinetic constant (Km), followed by EG Ⅱ and N134D. Km can be approximated showed the affinity of enzymes and substrate, Km smaller, indicating that the greater affinity of the protein.(4) The enzyme characterization of three recombinant proteins showed that N-glycosylation influence on them mainly in the thermal stability, even thermal insulation 70 min at 70℃, the relative activity of EG Ⅱ still maintain 40%, but there were no relative activity of EG Ⅱ mutant proteins. Some other enzymatic properties in the three recombinant proteins were very close except subtle differences:EG Ⅱ has a optimum catalytic activity at 55℃ and pH 5.0, N68D has a optimum catalytic activity at 50℃ and pH 4.8 and N134D has a optimum catalytic activity at 55℃ and pH 5.0; From the metal ions, The catalytic activity of three recombinant proteins could be enhanced by metal ion Mn2+, Zn2+ and Fe2+, but strong inhibited by Cu2+ at a high concentration.Based on the former three theoretical analysis and experimental results, proposes a hypothesis. In the whole process, N-glycans play a role in stabilizing the conformation. The N-glycans of N68 help CBM firmly grasp the cellulose chains, playing a supporting role; The N-glycans of N134 also to stabilize the conformation of the CD, however, this stablity just formed a sterically hindered, impeded the full binding between CD and cellulose chain and the catalytic activity can’t contact with the cellulose chains in a effective plane and the catalytic efficiency is greatly reduced. |
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