Mannitol Biosynthesis in Pyrenochaeta terrestris I. D‐Maunitol‐1‐Phosphate: NAD Oxidoreductase

Abstract

Cell‐free extracts of mycelial mats of Pgrenochaeta terrestris grown in stationary culture on synthetic glucose or sucrose ‐ salts liquid media contained D‐mannitol‐1‐Phosphate:NAD oxidoreductase (EC 1.1.1.17) activity. Greatest activity occurred early in the growth period. The optimum pH for the reduction of NAD+ in the presence of Fru‐6‐P was 7.4–7.5 while the optimum pH for the oxidation of NADH in the presence of Mtl‐1‐P was 8.1–8.2. The enzyme was stabilized to some extent in Tris‐maleate buffer, pH 7.5, and by the addition of 10% (NH4)2SO4, to this buffer. A 10‐ to 16‐fold purification was attained by a combination of (NH4)2SO4 fractionation and gel filtration on Sephadex G‐100. The enzyme was relatively specific in its substrate and coenzyme requirements. The Km values were determined as: Fru‐6‐P ‐ 3 × 10−4 M, Mtl‐1‐P ‐ 1 × 10−4 M, and NAD+ and NADH ‐ 3 × 10−5 M.

Mannitol-1-Phosphate Dehydrogenase in Pseudevernia Furfuracea

“…3, insets) allow direction determination of K m values from the X-axis intercept of the regression lines and gave the same K m values as for the direct non-linear method. The apparent K m value obtained for NADH is comparable to those reported for other fungi, whereas the apparent K m value for fructose-6phosphate appears higher than other reported data (30 uM NADH, 0-3 mM F-6-P, Aitken et al 1969a; 5 uM NADH, 0-54 mM F-6-P, Kiser & Niehaus 1981). The use of crude extracts from stored (frozen) material and chemical impurities may have led to an overestimate on the K m for fructose-6-phosphate.…”

“…2. There is a narrow pH optimum at about pH 71 for the reductive reaction, which is similar to the pH optima reported for fungi (pH 7-5, Aitken et al 1969a, Kiser & Niehaus 1981, a green alga (pH 7-5, Richter & Kirst 1987) and a brown alga (pH 6-5, Ikawa et al 1972). In addition, the requirement for Ca + + and Mg + + ions was tested.…”