Effect of N -Linked Glycosylation on Secretion, Activity, and Stability of α-Amylase from Aspergillus oryzae

Abstract

The effect of N-linked glycosylation on secretion, activity, and stability of alpha-amylase from Aspergillus oryzae grown as dispersed filaments was studied. In the presence of tunicamycin the fungus grew either as dispersed filaments or as one large pellet, whereas growth was as dispersed filaments in all control cultures. The presence of tunicamycin affected neither biomass, level of secreted alpha-amylase, nor total amount of secreted protein in cultures growing as dispersed filaments. In these cultures both glycosylated and nonglycosylated alpha-amylase appeared in the culture medium as well as in the cells, whereas in control cultures only the glycosylated form of alpha-amylase was found in the medium and in the cells. The presence of nonglycosylated alpha-amylase in the medium seemed to result from active secretion rather than from autolysis of the mycelium or extracellular deglycosylation. Deglycosylation with Endo H of crude alpha-amylase in culture filtrate did not affect its stability towards heat, acid pH, or proteolytic degradation.

Identification, Molecular Cloning, and Recombinant Gene Expression of an Extracellular A-Amylase fromDactylis GlomerataL. Embryogenic Suspension Cultures

“…This result suggests that lack of posttranslational modifications (in particular glycosylation, as predicted for DgAmy1) is not necessary either for the enzymatic function of DgAmy1, or for the stability of the protein and its insensitivity towards protease attack from the cytosol of E. coli. In agreement with this result, the carbohydrate moiety was not essential for the catalytic activity and stability of extracellular amylases secreted by fungi (16). The homogeneity of the purified α-amylase was additionally confirmed by 2-DE that revealed a single 46 kDa spot with pI 5.4 (Fig.…”

Isolation of nine Phytophthora capsici pectin methylesterase genes which are differentially expressed in various plant species

“…2 -4). We note that the Pcpme1, Pcpme5, Pcpme7, Pcpme8 and Pcpme9 products have more potential Nlinked glycosylation sites on the amino acid sequences than the other Pcpme genes in this study, which may affect enzymatic stability, secretion, solubility, or activity [42,43]. It is possible that these factors may be related to the five genes' higher expression level relative to other Pcpme genes in cucumber fruits [37].…”

The influence of N-glycosylation and C-terminal sequence on secretion of HBV large surface antigen fromS. cerevisiae

“…Reportedly, the N-glycosylation of heterologous proteins significantly affects intracellular proteolytic processing, secretion efficiency, and post-translational stability of proteins secreted from eukaryotic host cells (Doyon et al, 2002;Eriksen et al, 1998;Gahring et al, 2001;Livi et al, 1991;Pratap et al, 2000;Rothwell et al, 1993;Zhu et al, 1998). For instance, the N-glycosylation significantly increased the Results of Western blot analyses with the samples from culture broth (100 mL) of recombinant S. cerevisiae producing pre-S::S (A), pre-S88::S (B), pre-S88::S8 (C), and pre-S88::S88 (D, E), using the mouse anti-HBV pre-S2 (A to D) and anti-HBV S (E) monoclonal IgG1 as primary antibody (Lanes 1, 2, and 3 in Fig.…”