Rouwenhorst R J, Hensing M, Verbakel J, Scheffers W A, van Duken J P
Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
Appl Environ Microbiol. 1990 Nov;56(11):3337-45. doi: 10.1128/aem.56.11.3337-3345.1990.
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.
在马克斯克鲁维酵母中存在两种形式的菊粉酶，即分泌到培养液中的菊粉酶和保留在细胞壁中的菊粉酶。两种形式的菊粉酶均经纯化，并通过变性和非变性聚丙烯酰胺凝胶电泳进行分析。使用内切β-N-乙酰葡糖胺酶H确定，保留在细胞壁中的酶和分泌到培养液中的酶具有相似的亚基，由一个64 kDa的多肽组成，碳水化合物含量不同（占分子质量的26%至37%）。两种形式的菊粉酶大小不同，因为它们的亚基聚集情况不同。培养液中存在的酶是二聚体，而保留在细胞壁中的酶是四聚体。寡聚化的差异不影响对底物蔗糖和棉子糖的表观Km值。这些发现支持了这样一种假说，即酵母细胞壁中糖蛋白的保留可能是由对较大糖蛋白的通透性屏障引起的。测定了菊粉酶的氨基末端，并与密切相关的转化酶的氨基末端进行了比较。动力学和结构证据表明，在酵母中存在两种不同的β-果糖苷酶，即转化酶和菊粉酶。
