title: Asn124 of Cel5A from Hypocrea jecorina not only provides the N-glycosylation site but is also essential in maintaining enzymatic activity.
abstract: To investigate the function of N-glycosylation of Cel5A (endoglucanase II) from Hypocrea jecorina, two N-glycosylation site deletion Cel5A mutants (rN124D and rN124H) were expressed in Saccharomyces cerevisiae. The weights of these recombinant mutants were 54 kDa, which were lower than that of rCel5A. This result was expected to be attributed to deglycosylation. The enzyme activity of rN124H was greatly reduced to 60.6% compared with rCel5A, whereas rN124D showed slightly lower activity (10%) than that of rCel5A. rN124D and rN124H showed different thermal stabilities compared with the glycosylated rCel5A, especially at lower pH value. Thermal stabilities were reduced and improved for rN124D and rN124H, respectively. Circular dichroism spectroscopy showed that the modification of secondary structure by mutation may be the reason for the change in enzymatic activity and thermal stability.
authors.affiliation: National Glycoengineering Research Center, and State Key Laboratory of Microbial Technology, Shandong University, 27, Shanda South Road, Jinan, Shandong 250100, China.
authors.forename: Yuqi
authors.initials: Y
authors.lastname: Qin
authors.affiliation: National Glycoengineering Research Center, and State Key Laboratory of Microbial Technology, Shandong University, 27, Shanda South Road, Jinan, Shandong 250100, China.
authors.forename: Yinbo
authors.initials: Y
authors.lastname: Qu
mesh_terms: D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry; D020134:Catalytic Domain / Q000235:genetics; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D035901:Hypocrea / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D001216:Asparagine; C407024:endoglucanase Cel5A; D002480:Cellulase