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Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens) and screened for phytase activity in yeast. One full-length phytaseencoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed in Aspergillus oryzae, purified, and characterized. The phytases revealed temperature optima between 40 and 60°C and pH optima at 5.0 to 6.0, except for the P. lycii phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U ⅐ mg, of protein ؊1 and were capable of hydrolyzing phytate down to myo-inositol monophosphate. Surprisingly, 1 H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC 3.1.3.26).Phytases (myo-inositol hexakisphosphate phosphohydrolases) belong to the family of histidine acid phosphatases sharing the sequence consensus pattern(27; http://www.expasy.ch /cgi-bin/get-prodoc-entry?PDOC00538). They are capable of catalyzing the hydrolysis of phosphomonoester bonds of phytate (salts of myo-inositol hexakisphosphate or myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate (22,24). Phytases are grouped according to the specific position of the phosphate ester group on the phytate molecule at which hydrolysis is initiated, i.e., as 3-phytases (EC 3.1.3.8) or as 6-phytases (EC 3.1.3.26).Phytate is the primary source of inositol and the primary storage form of phosphate in plant seeds (23). Seeds, cereal grains, and legumes are important components of food and, in particular, of animal feed preparations. However, monogastric animals such as poultry and swine are incapable of utilizing the phosphorus bound in phytate due to low levels of phytase activity in the digestive tract. Furthermore, phytate acts as an antinutrient by chelating divalent cations and preventing the uptake of minerals, e.g., Zn (9). Thus, phytases are used as a cereal feed additive that enhances the phosphorus and mineral uptake in monogastric animals and reduces the level of phosphate output in their manure. Recently, there have been several reports on the cloning of fungal PhyA phytases from Aspergillus niger (16,20, 28), Aspergillus fumigatus (19), Aspergillus terreus, Myceliophthora thermophila (14), Emericella nidulans, Talaromyces thermophilus (18), and Thermomyces lanuginosus (2). Based on their characteristics and on their s...

A Microdochium nivale carbohydrate:acceptor oxidoreductase was purified, cloned, heterologously expressed, and characterized. The gene encoding the protein showed one intron, and the ORF showed a sequence with low homology (# 25% identity or 65% similarity) to other known flavin-containing carbohydrate oxidases. The maturation of the protein required the cleavage of a tetrameric propeptide in addition to an 18 amino-acid signal peptide. The enzyme was found to have a relative molecular mass of 55 000 Da, an isoelectric point of 9, and one FAD per protein. It could oxidize mono-, oligo-, or polymeric saccharides, and transfer their electrons to O 2 or other acceptors. When d-glucose served as electrondonating substrate, an activity of 2 s 21 was observed at pH 5.5 and 23 8C. Among various oligosaccharides, the enzyme preferred tetrameric dextrins, indicating a favorable interaction of four linked glucose units with the substrate pocket. The unique structure and ability of oxidizing oligo/ polymeric saccharides suggest a promising prospect of this enzyme for various industrial/medicinal applications. Keywords:oxidoreductase; flavin; carbohydrate; Microdochium.Carbohydrate oxidases are a family of flavin-or Cu-containing enzymes that catalyze the oxidation of the primary or secondary alcohol in various saccharides with the concomitant reduction of O 2 to H 2 O 2 . These enzymes are widely distributed and play important roles in various metabolism steps. Extensive physical and chemical characterizations, including X-ray crystallography and sitedirected mutagenesis, have been carried out to elucidate the fundamental aspects of the structure-function relationship of these enzymes [1±3].Recently, carbohydrate oxidases are receiving increased attention as potential diagnostic reagents or industrial biocatalysts [3±8]. Attractive applications include (a) biotransformation of glycopolymers (glycolipids, glycoproteins, polysaccharides) into desirable materials such as sweeteners, flavourants, or paper strength additives; (b) in situ generation of oxidant H 2 O 2 for uses in food manufacturing, dyeing or bleaching of lignocellulolytic/ keratin materials, detergent, or waste-water treatment; (c) utilization of economic and stable carbohydrates, rather than expensive and unstable NAD(P)H, as electron-donor for various biocatalyses; (d) construction of biosensors for blood sugar, O 2 , or other substances; and (e) biosynthesis of functional/chiral pharmaceutical molecules. The high substrate specificity, mild operation conditions, and environmental-friendliness of bio-oxidative systems hold advantages over conventional chemical systems that are often hard to control, nonspecific, costly, or hazardous.We report here the identification, cloning, expression, and characterization of a novel carbohydrate:acceptor oxidoreductase from Microdochium nivale (MnCO). We found that this flavin-containing enzyme differs significantly from other functional analogs [such as glucose oxidase (GO; EC 1.1.3.4)] as demonstrated by its low sequence...

In the filamentous fungus Aspergillus fumigatus, the vast majority of the cellwall-associated proteins are secreted proteins that are in transit in the cell wall. These proteins can be solubilized by detergents and reducing agents. Incubation of a SDS/β-mercaptoethanol-treated cell-wall extract with various recombinant enzymes that hydrolyse cell-wall polysaccharides resulted in the release of a unique protein in minute amounts only after incubation of the cell wall in the presence of 1,3-β-glucanase. Sequence analysis and biochemical studies showed that this glycoprotein, with an apparent molecular mass of 80 kDa, was an acid phosphatase (PhoAp) that was active on both phosphate monoesters and phosphate diesters. PhoAp is a glycosylphosphatidylinositolanchored protein that was recovered in the culture filtrate and cell-wall fraction of A. fumigatus after cleavage of its anchor. It is also a phosphaterepressible acid phosphatase. The absence of PhoAp from a phosphate-rich medium was not associated with a reduction in fungal growth, indicating that this cell-wall-associated protein does not play a role in the morphogenesis of A. fumigatus.

The kinetics of thermally induced aggregation of the glycoprotein Peniophora lycii phytase (Phy) and a deglycosylated form (dgPhy) was studied by dynamic (DLS) and static (SLS) light scattering. This provided a detailed insight into the time course of the formation of small aggregates ( approximately 10-100 molecules) of the enzyme. The thermodynamic stability of the two forms was also investigated using scanning calorimetry (DSC). It was found that the glycans strongly promoted kinetic stability (i.e., reduced the rate of irreversible denaturation) while leaving the equilibrium denaturation temperature, T(d), defined by DSC, largely unaltered. At pH 4.5-5.0, for example, dgPhy aggregated approximately 200 times faster than Phy, even though the difference in T(d) was only 1-3 degrees C. To elucidate the mechanism by which the glycans promote kinetic stability, we measured the effect of ionic strength and temperature on the aggregation rate. Also, the second virial coefficients (B(22)) for the two forms were measured by SLS. These results showed that the aggregation rate of Phy scaled with the concentration of thermally denatured protein. This suggested first-order kinetics with respect to the concentration of the thermally denatured state. A similar but less pronounced correlation was found for dgPhy, and it was suggested that while the aggregation process for the deglycosylated form is dominated by denatured protein, it also involves a smaller contribution from associating molecules in the native state. The measurements of B(22) revealed that dgPhy had slightly higher values than Phy. This suggests that dgPhy interacts more favorably with the buffer than Phy and hence rules out strong hydration of the glycans as the origin of their effect on the kinetic stability. On the basis of this and the effects of pH and ionic strength, we suggest that the inhibition of aggregation is more likely to depend on steric hindrance of the glycans in the aggregated form of the protein.