Structure and properties of the extracellular inulinase of Kluyveromyces marxianus CBS 6556.
In the yeast Kluyveromyces marxianus two forms of inulinase were present, namely, an inulinase secreted into the culture fluid and an inulinase retained in the cell wall. Both forms were purified and analyzed by denaturing and nondenaturing polyacrylamide gel electrophoresis. With the use of endo-beta-N-acetyl-glucosaminidase H, it was established that the enzyme retained in the cell wall and the enzyme secreted into the culture fluid have similar subunits consisting of a 64-kDa polypeptide with varying amounts of carbohydrate (26 to 37% of the molecular mass). The two forms of inulinase differed in size because of their differences in subunit aggregation. The enzyme present in the culture fluid was a dimer, and the enzyme retained in the cell wall was a tetramer. The differences in oligomerization did not affect the apparent Km values towards the substrates sucrose and raffinose. These findings support the hypothesis that the retention of glycoproteins in the yeast cell wall may be caused by a permeability barrier towards larger glycoproteins. The amino-terminal end of inulinase was determined and compared with the amino terminus of the closely related invertase. The kinetic and structural evidence indicates that in yeasts two distinct beta-fructosidases exist, namely, invertase and inulinase.
Rouwenhorst
R J
RJ
Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
Hensing
M
M
Verbakel
J
J
Scheffers
W A
WA
van Duken
J P
JP
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000201:enzymology; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007444:Inulin / Q000378:metabolism; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011887:Raffinose / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism
D013395:Sucrose; D007444:Inulin; D006026:Glycoside Hydrolases; C023086:inulinase; D011887:Raffinose