Different effects of N-glycosylation on the thermostability of highly homologous bacterial (1,3-1,4)-beta-glucanases secreted from yeast.
Genes encoding Bacillus amyloliquefaciens (1,3-1,4)-beta-glucanase (AMY), B. macerans (1,3-1,4)-beta-glucanase (MAC), and a series of hybrid enzymes containing N-terminal sequence segments of different length derived from AMY with the remaining C-terminal segment derived from MAC, were expressed in Saccharomyces cerevisiae. The cells secreted active enzyme into the medium. While the quantity of N-glycan linked to the different enzymes was similar, pronounced differences in thermotolerance were observed when the glycosylated enzymes were compared with the unglycosylated counterparts produced in Escherichia coli. Glycosylated AMY and hybrid enzyme H(A16-M), consisting of 16 N-terminal amino acids derived from AMY with the remaining C-terminal segment from MAC, exhibited a 7.5- and 1.6-fold increase in half-life at 70 degrees C, pH 6.0. N-terminal sequencing established that only two out of three sites for potential N-glycosylation of H(A16-M) secreted from yeast were actually glycosylated. Removal of N-glycans by endoglycosidase H and peptide:N-glycosidase F from H(A16-M) resulted in a 16- and 133-fold decrease of thermostability, demonstrating that N-glycans are a major determinant for the resistance of this enzyme to thermal inactivation. Glycosylated MAC and hybrid enzymes H(A36-M), H(A107-M) and H(A152-M) had increased thermostability but hybrid enzyme H(A78-M) was less thermostable. N-Glycosylation thus changes thermostability of (1,3-1,4)-beta-glucanases with similar primary structure in a variable, so far unpredictable way.
Meldgaard
M
M
Department of Physiology, Carlsberg Laboratory, Copenhagen, Denmark.
Svendsen
I
I
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014170:Transformation, Genetic
D006026:Glycoside Hydrolases; C028020:licheninase