Functional analysis of O-linked oligosaccharides in threonine/serine-rich region of Aspergillus glucoamylase by expression in mannosyltransferase-disruptants of yeast.
The glaA gene encoding glucoamylase I (GAI) of Aspergillus awamori var. kawachi was heterologously expressed in mannosyltransferase mutants of Saccharomyces cerevisiae, in which the pmt1 gene and the kre2 gene were disrupted. The GAI enzymes expressed in these yeast mutant cells exhibited a lesser extent of O-glycosylation. Secretion of GAI expressed in the pmt1-disruptant and in the kre2-disruptant, respectively, was almost the same as that of GAI expressed in wild type (wt) strains. The number of O-linked mannose in GAI from wt yeast strain ranged in size from one (Man1) to five (Man5). On the other hand, the O-linked oligosaccharides of GAI from the pmt1-disruptant ranged in size from Man1 to Man4. Man5 was not detected and Man2-Man4 were reduced in proportion to the reduction of Man1. The O-linked oligosaccharides of GAI from the kre2-disruptant ranged from Man1 to Man4, and the molar amount of Man4 was reduced to 27.3%, compared to that of the wt strain. The hydrolyzing abilities for soluble starch and the adsorbing abilities on raw starch were comparable between both disruptants and wt strains. However, the digesting abilities for raw starch of the disruptants were decreased to 70% of those of the wt strains. Stabilities of GAI of the disruptants were reduced toward extreme pH and high temperature, compared to those of the wt strains. These results demonstrate that the O-linked oligosaccharides of GAI are responsible for the enzyme stability and activity toward insoluble substrates but not for secretion.
Goto
M
M
Laboratory of Applied Microbiology, Departemnt of Agricultural Chemistry, Kyushu University, Hakozaki, Fukuoka, Japan. m_goto@agr.kyushu-u.ac.jp
Tsukamoto
M
M
Kwon
I
I
Ekino
K
K
Furukawa
K
K
D001230:Aspergillus / Q000201:enzymology* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D008364:Mannosyltransferases / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014162:Transfection
D009844:Oligosaccharides; D013912:Threonine; D012694:Serine; D008364:Mannosyltransferases; D005087:Glucan 1,4-alpha-Glucosidase