Phytases catalyse the hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. In this study genes encoding novel phytases from two different filamentous fungi, Aspergillus terreus strain 9A-1 and Myceliophthora thermophila were isolated. The encoded PhyA phytase proteins show 60% (A. terreus) and 489'0 (M. thermophila) identity, respectively, to the PhyA of Aspergillus niger and have 21-29% identity compared to other histidine acid phosphatases. All three PhyA proteins, in contrast to the A. niger pH 25-optimum acid phosphatase, prefer phytic acid as substrate and show enzyme activity at a broad range of acidic pH values. Based on their enzyme characteristics and protein sequence homology, the phytases form a novel subclass of the histidine acid phosphatase family.

The repressible acid phosphatase gene PHOS of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHOS gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHOS promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PH04 binding. Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASpl) and the other located at -239 to -262 (UASp2). Exonuclease m footprint experiments revealed stops at -349 and -368 (UASpl) as well as at -245 and -260 (UASp2). Gel retardation assays with the PH02 protein revealed a binding region that lay between the two PH04-binding sites. DNase I footprint analysis suggested a PH02-binding site covering the region between -277 and -296.The promoter region of the strongly regulated acid phosphatase gene PH05 (4) has been recently analyzed by deletion mapping (6,19,24). In addition to the TATA box, two regions were found (24) to be essential for activation of the PHOS promoter. Sequence comparisons defined four elements from which a 19-base-pair dyad consensus sequence was deduced. Three of the elements, called UASp, were located within deletions showing reduced transcription of PHOS (24), at -367, -245, and -185 relative to the translational start.Not much is known about the physical interaction of regulatory proteins with the PHOS promoter. Since mutations defective in PH02 or PH04 are epistatic to all other regulatory mutations and show a negative phenotype, it is likely that the PHO2 and PHO4 proteins are positive transcription factors that interact with the PHOS promoter DNA. The finding that a pho2 mutation can be complemented by overexpression of the PHO4 protein (20) is a further indication that the PHO2 and PHO4 proteins exert their functions at the same late hierarchical level of PH05 activation.Extensive studies of the chromatin fine structure of the PH05 gene (1, 2, 5, 7) have been done. Interestingly, the element at -367, which is essential for gene induction (24), resides in the center of a hypersensitive region under conditions of PH05 repression (1, 2).In press). Interestingly, the pho2 mutation showed no influence on the basal level of TRP4 expression but rather modulated the general control response of TRP4.MATERIALS AND METHODS Escherichia coli vector for PH04 and PH02 expression. The PH04 gene (13, 15) was reisolated from a S288C background by screening an ordered centromere library (25). An NcoI site at the ATG of PH04 was introduced by site-specific mutagenesis (14). The PH04 gene (NcoI-BamHI fragment) was placed under control of the inducible PL promoter by using the vector pPLmu, a derivative of PLc24 (23) containing the phage Mu Ner gene ribosome-binding site with an NcoI site at its ATG start codon (obtained from Biogen).The PH02 gene (25) was fused to the same vector but carrying a differ...

Switzerland. 514%, respectively, and also decreased whole-body lipid contents signi¢cantly from 120 to 123 g kg À1 in ¢sh fed the basal diet and phytase-supplemented diet to 106 g kg À1 in ¢sh fed with lysine-forti¢ed diet.

Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, andTalaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli. All of the phytases examined are monomeric proteins. WhileE. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in A. niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of A. fumigatus andE. nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.

Yeast cells produce a set of enzymes which are involved in the metabolism of phosphate, and include acid and alkaline phosphatases as well as permeases. Most of these enzymes are synthesized in response to the presence or absence of inorganic phosphate. In the past few years a considerable amount of genetic and molecular evidence has accumulated and a rather precise overall picture emerges which describes the mechanism of phosphate control at the level of gene activation. This mini-review summarizes these data. The main focus lies on the regulatory features associated with the control of transcription of PHO5, a gene coding for most of the regulated acid phosphatase activity produced by yeast cells.

Two regulatory proteins, PHO2 and the general control regulator GCN4, bind in vitro to the promoter of the tryptophan biosynthetic TRP4 gene; the TRP4 gene product catalyses the phosphoribosylation of anthranilate. PHO2 binds specifically to the TRP4 promoter, but does not bind to any other TRP promoter. PHO2 and GCN4 proteins bind in a mutually exclusive manner to the same sequence, UAS1, one of two GCN4 binding sites in the TRP4 promoter. UAS1 is the major site for GCN4‐dependent TRP4 activation. The second GCN4 binding site, UAS2, interacts with GCN4 alone. PHO2 binding interferes with the general control response of TRP4 under low phosphate conditions and simultaneous amino acid starvation and thus the PHO2 regulatory protein connects phosphate metabolism and amino acid biosynthesis in yeast. The GCN4 protein mediates the response of the transcriptional apparatus to the environmental signal ‘amino acid limitation’, while PHO2 seems to be the phosphate sensor that adjusts the response to the availability of phosphate precursors.

A simple, robust and highly reproducible method for the determination of xylanase, beta-glucanase, and cellulase in commercial feed enzyme preparations is described. The method is based on measurement of reducing moieties released by the enzymes from arabinoxylan, beta-glucan, or carboxymethylcellulose (CMC) and is independent of enzyme standards.

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