Phytase activity in Aspergillus fumigatus isolates.

Extracellular phytase from Aspergillus fumigatus isolates was characterized and their genes were cloned and sequenced. Based on their banding pattern in SDS-PAGE all phytases were found to be glycosylated and have similar molecular mass. A correlation between lower optimum pH (4.0) and a higher optimum temperature (70 degrees C) was found in these enzymes. All enzymes characterized displayed a lower specific activity for phytic acid and were more susceptible to proteolytic degradation than the Aspergillus niger phytase that is now commercially available. DNA sequencing established almost no sequence variation in any of the genes and no correlation is evident between a specific amino acid sequence and any physicochemical and catalytic properties of the enzymes. Despite two of the isolates having identical deduced amino acid sequence, characterization of the enzymes encoded by these two identical genes revealed differences in both pH and temperature optimum. This suggests that differences in pH and temperature optimum in these four isolates of A. fumigatus may be due in part to subtle differences in posttranslational modification.

Biochemical characterization of cloned Aspergillus fumigatus phytase (phyA).

The gene for Aspergillus fumigatus phytase (phyA) was cloned and expressed in Pichia pastoris. The enzyme expressed was purified to near homogeneity using sequential ion-exchange chromatography and was characterized biochemically. Although A. fumigatus phytase shows 66.2% sequence homology with A. ficuum phytase, the most widely studied enzyme, the cloned phytase showed identical molecular weight and temperature optima profile to the benchmark phytase. The pH profile of activity and kinetic parameters, however, differed from A. ficuum phytase. The cloned enzyme contains the septapeptide RHGARYP motif, which is also identical to the active site motif of A. ficuum phytase. Chemical probing of the active site Arg residues using both cyclohexanedione and phenylglyoxal resulted in the inactivation of phytase. The cloned A. fumigatus phytase, however, was more resistant to phenylglyoxal-induced inactivation. Both cloned A. fumigatus and A. ficuum phytases were identically affected by cyclohexanedione. Both the thermal characterization data and kinetic parameters of cloned and expressed A. fumigatus phytase indicate that this biocatalyst is not superior to the benchmark enzyme. The sequence difference between A. fumigatus and A. ficuum phytase may explain why the former enzyme catalyzes poorly compared to the benchmark enzyme. In addition, differential sensitivity toward the Arg modifier, phenylglyoxal, indicates a different chemical environment at the active site for each of the phytases.

Conjugation and modeled structure/function analysis of lysozyme on glycine esterified cotton cellulose-fibers.

The antimicrobial activity of lysozyme covalently bound to glycine-derivatized cotton cellulose was assessed in a 96-well format. Lysozyme was immobilized on glycine-bound cotton through a carbodiimide reaction. The attachment to cotton fibers was made through both a single glycine and a glycine dipeptide esterified to cotton cellulose. Higher levels of lysozyme incorporation were evident in the diglycine-linked cotton cellulose samples. The antibacterial activity of the lysozyme-conjugated cotton cellulose against Bacillus subtilis was assessed as a suspension of pulverized cotton fibers in microtiter wells. Inhibition of B. subtilis growth was observed to be optimal within a range of 0.14-0.3 mM (equivalent to 4-20 mg of lysozyme-bound cotton/mL) of lysozyme. Enhancement of activity over soluble lysozyme may result from the solid-phase protection afforded by the cellulose linkage of the glycoprotein against proteolytic lysis. Computational models of lysozyme based on its crystal structure attached through aspartate, glutamate, and COOH-terminal residues to cellopentaose-(3) Gly-O-6-glycyl-glycine ester were constructed. The models demonstrate no steric constraints to the active-site cleft from the glycine-conjugated cellulose chain when lysozyme is bound at the carboxylates of Asp-87, Glu-7, Asp-119, Asp-18, and COOH-terminal Leu-129. The more robust antibacterial activity of the enzyme when bonded to cotton fibers suggests good potential for biologically active enzymes on cotton-based fabrics.

Advances in phytase research.

Since its discovery in 1907, a complex of technological developments has created a potential $500 million market for phytase as an animal feed additive. During the last 30 years, research has led to increased use of soybean meal and other plant material as protein sources in animal feed. One problem that had to be overcome was the presence of antinutritional factors, including phytate, in plant meal. Phytate phosphorus is not digested by monogastric animals (e.g., hogs and poultry), and in order to supply enough of this nutrient, additional phosphate was required in the feed ration. Rock phosphate soon proved to be a cost-effective means of supplying this additional phosphorus, and the excess phytin phosphorus could be disposed of easily with the animals' manure. However, this additional phosphorus creates a massive environmental problem when the land's ability to bind it is exceeded. Over the last decade, numerous feed studies have established the efficacy of a fungal phytase, A. niger NRRL 3135, to hydrolyze phytin phosphorus in an animal's digestive tract, which benefits the animal while reducing total phosphorus levels in manure. The gene for phytase has now been cloned and overexpressed to provide a commercial source of phytase. This monomeric enzyme, a type of histidine acid phophatase (HAP), has been characterized and extensively studied. HAPs are also found in other fungi, plants, and animals. Several microbial and plant HAPs are known to have significant phytase activity. A second A. niger phytase (phyB), a tetramer, is known and, like phyA, has had its X-ray crystal structure determined. The model provided by this crystal structure research has provided an enhanced understanding of how these molecules function. In addition to the HAP phytase, several other phytases that lack the unique HAP active site motif RHGXRXP have been studied. The best known group of the non-HAPs is phytase C (phyC) from the genus Bacillus. While a preliminary X-ray crystallographic analysis has been initiated, no enzymatic mechanism has been proposed. Perhaps the pivotal event in the last century that created the need for phytase was the development of modern fertilizers after the Second World War. This fostered a transformation in agriculture and a tremendous increase in feed-grain production. These large quantities of cereals and meal in turn led to the transition of one segment of agriculture into "animal agriculture," with their its animal production capability. The huge volumes of manure spawned by these production units in time exceeded both the capacity of their crops and crop lands to utilize or bind the increased amount of phosphorus. Nutrient runoff from this land has now been linked to a number of blooms of toxin-producing microbes. Fish kills associated with these blooms have attracted public and governmental concern, as well as greater interest in phytase as a means to reduce this phosphorus pollution. Phytase research efforts now are focused on the engineering of an improved enzyme. Improved heat tolerance to allow the enzyme to survive the brief period of elevated temperature during the pelletization process is seen as an essential step to lower its cost in animal feed. Information from the X-ray crystal structure of phytase is also relevant to improving the pH optimum, substrate specificity, and enzyme stability. Several studies on new strategies that involve synergistic interactions between phytase and other hydrolytic enzymes have shown positive results. Further reduction in the production cost of phytase is also being pursued. Several studies have already investigated the use of various yeast expression systems as an alternative to the current production method for phytase using overexpression in filamentous fungi. Expression in plants is underway as a means to commercially produce phytase, as in biofarming in which plants such as alfalfa are used as "bioreactors," and also by developing plant cultivars that would produce enough transgenic phytase so that additional supplementation of their grain or meals is not necessary. Ultimately, transgenic poultry and hogs may produce their own digestive phytase. Another active area of current phytase research is expanding its usage. One area that offers tremendous opportunity is increasing the use of phytase in aquaculture. Research is currently centered on utilizing phytase to allow producers in this industry to switch to lower-cost plant protein in their feed formulations. Development of a phytase for this application could significantly lower production costs. Other areas for expanded use range from the use of phytase as a soil amendment, to its use in a bioreactor to generate specific myo-inositol phosphate species. The transformation of phytase into a peroxidase may lead to another novel use for this enzyme. As attempts are made to widen the use of phytase, it is also important that extended exposure and breathing its dust be avoided as prudent safety measures to avoid possible allergic responses. In expanding the use of phytase, another important consideration has been achieved. Conservation of the world's deposits of rock phosphate is recognized as important for future generations. Phosphorus is a basic component of life like nitrogen, but, unlike nitrogen, phosphorus does not have a cycle to constantly replenish its supply. It is very likely that the use of phytase will expand as the need to conserve the world's phosphate reserves increases.

Characterization of recombinant fungal phytase (phyA) expressed in tobacco leaves.

The phyA gene from Aspergillus ficuum coding for a 441-amino-acid full-length phytase was expressed in Nicotiana tabacum (tobacco) leaves. The expressed phytase was purified to homogeneity using ion-exchange column chromatography. The purified phytase was characterized biochemically and its kinetic parameters were determined. When the recombinant phytase was compared with its counterpart from Aspergillus ficuum for physical and enzymatic properties, it was found that catalytically the recombinant protein was indistinguishable from the native phytase. Except for a decrease in molecular mass, the overexpressed recombinant phytase was virtually the same as the native fungal phytase. While the temperature optima of the recombinant protein remain unchanged, the pH optima shifted from pH 5 to 4. The results are encouraging enough to open the possibility of overexpressing phyA gene from Aspergillus ficuum in other crop plants as an alternative means of commercial production of this important enzyme.

Identification of a histidine acid phosphatase (phyA)-like gene in Arabidopsis thaliana.

A close examination of the protein sequence encoded by the Arabidopsis thaliana gene F21M12.26 reveals the gene product to be a phosphomonoesterase, acid optimum (EC 3.1.3.2). A subclass of this broad acid phosphatase is also known as 'histidine acid phosphatase. ' This is the first sequence-based evidence for a 'histidine acid phosphatase' in a dicotyledon. One important member of this class of enzymes is Aspergillus niger (ficuum) phytase, which came into prominence for its commercial application as a feed additive. The putative protein from A. thaliana gene F21M12.26 shares many important features of Aspergillus phytase, namely, size, active-site sequence, catalytic dipeptide and ten cysteine residues located in the key areas of the molecule, but lacks all nine N-glycosylation sites.

Myo-inositol hexasulfate is a potent inhibitor of Aspergillus ficuum phytase.

Myo-inositol hexasulfate (MIHS), a structural analog of the substrate myo-inositol hexaphosphate, is a potent competitive inhibitor of both phyA and phyB enzymes. The Ki of inhibition for the phyA and phyB proteins were estimated to be 4.6 and 0.2 microM, respectively. Thus, the phyB protein is 23-fold more sensitive to MIHS inhibition than the phyA protein. The active-site geometry of phyB protein is presumed to be very different from the phyA protein as deduced by chemical probing of the enzymes by Arg-specific modifiers, i.e., 1,2-cyclohexanedione and phenylglyoxal. Probing the catalytic site of the same proteins by this newly developed specific inhibitor also gives a similar conclusion.

Differences in the active site environment of Aspergillus ficuum phytases.

While Aspergillus ficuum phytaseA (phyA) was rapidly inactivated by 1,2-cyclohexanedione and phenylglyoxal, both specific modifiers of arginine, phytaseB (phyB) showed a markedly different behavior. First, phyB was totally insensitive to 1,2-cyclohexanedione even in the presence of 0.2 M guanidinium hydrochloride; second, the enzyme showed a great deal of resistance to inactivation by phenylglyoxal. Taken together, these results indicate that the chemical environment of the active site of phyB is very different from that of the active site of phyA. Despite sequence similarities of the active site region in these two proteins, their differential behavior to arginine modifiers indicates that other parts of the protein play a role in the active site formation. We expected some differences in the structure since the proteins have dissimilar kinetic parameters and pH optima.

Conservation of the active site motif in Aspergillus niger (ficuum) pH 6.0 optimum acid phosphatase and kidney bean purple acid phosphatase.

Aspergillus niger (ficuum) and the kidney bean purple acid phosphatases retained all the essential amino acids in the active site despite a low degree of total sequence homology. This high degree of homology in the sequence motif of A. niger fungal acid phosphatase (Apase6) active site with Kidney bean metallo phosphoesterase (KBPAP) and the absence of the RHG-XRXP sequence motif indicates Apase6 to be a metallophosphoesterase rather than a histidine acid phosphatase.

Disulfide bonds are necessary for structure and activity in Aspergillus ficuum phytase.

The function of disulfide bonds in Aspergillus ficuum phytase was elucidated by unfolding studies, using guanidinium hydrochloride (Gu.HCl) as denaturant. Although the enzyme is totally inactivated by 0.8 M Gu.HCl, at pH 5.0, the active conformation is instantaneously restored by 0.6 M Gu.HCl, at pH 5.0. Conditions which would permit refolding of phytase are completely negated by 10 mM beta-mercaptoethanol and causes its catalytic demise at pH 7.5. Assay of free thiols using Ellman's reagent indicates that none of the thiols in the ten cysteines in phytase are free; five disulfide bonds were predicted for the enzyme. Sequence comparison of mold phytases and yeast acid phosphatases indicates four conserved cysteines. Thus, disulfide bonds play an important role in the folding of fungal phytase; any perturbation of the process of its formation causes an altered three-dimensional structure that is inconsistent with catalytic activity.

Phytase.

Of all the sources of phytase that have been studied (plant, animal, and microorganisms), the highest yields are produced by a wild-type strain A. niger NRRL 3135 (12.7 mg P/hr/ml = 6.8 microns P/ml/min = 113.9 nKat/ml) in a mineral salt medium in which total phosphate (4 mg %) is limiting for growth and cornstarch and glucose are the carbon sources. Synthesis of the enzyme is repressed by phosphate in the wild-type strain. Aspergillus niger NRRL 3135 produces two phytases one with pH optima at 2.5 and 5.5 (phyA) and one with an optimum at pH 2.0 (phyB). It also produces a pH 6.0 optimum phosphatase that has no phytase activity. These three glycoproteins have been purified to homogeneity, characterized, sequenced, and cloned. The sequences have been compared to each other, other phytases, and to known phosphatases. Their homology has been determined. The active sites of phytases show remarkable homology to the active site residues of the members of a particular class of acid phosphatase (histidine phosphatase). The most conserved sequence is RHGXRXP. Phytase has been covalently immobilized on Fractogel TSK HW-75 F and glutaraldehyde-activated silicate. It has been immobilized on agarose. Losses of activity have been noted on immobilization but these may be minimized by future research. It should be possible to commercially produce and recover penta-, tetra-, tri-, di-, and monoinositol phosphates using immobilized phytase if markets develop for those products. Phytase (phyA) from A. niger NRRL 3135 has been cloned into an A. niger glucoamylase producing strain CBS 513.88 using a construct that has a glucoamylae promoter and an A. niger NRRL 3135 leader sequence, and that is devoid of phosphate repression. The yield of the secreted enzyme was increased 52-fold above that of wild-type A. niger NRRL 3135. The bioengineered organism produces 270 microns P/ml/min (4500 nKat/ml) which is approximately 7.9 g/liter in the medium. The yield of the secreted enzyme was increased 1440-fold above that of wild type CBS 513.88. Commercial preparations of the cloned enzyme are available. Phytase (phyA) has been cloned into tobacco and canola. The enzyme is localized in the seed and expressed at high levels. Feeding of the seed to animals has made the phytin-P in the commercial diets available to the animals. The efficacy of feeding phytase to monogastric animals (poultry and swine) has been established. The amount of enzyme that is necessary to be added to commercial diets has been titred for broilers, layers, turkeys, ducks, and swine. The units of enzyme required are related to the phytin-P content in the diet. The use of the enzyme as a feed additive has been cleared in 22 countries. If phytase were used in the diets of all of the monogastric animals reared in the U.S., it would release phosphorus that has a value of $1.68 x 10(8) per year. The FDA has approved the enzyme preparation as GRAS. The effect of feeding phytase to animals enables assimilation of the P found in feed ingredients and diminishes the amount of phosphate in the manure and subsequently entering the environment. The effect of feeding phytase to animals on pollution has been quantitatively determined. If phytase were used in the diets of all of the monogastric animals reared in the United States, it would preclude 8.23 x 10(7) kg P from entering the environment.

The Aspergillus niger (ficuum) aphA gene encodes a pH 6.0-optimum acid phosphatase.

We have used the Aspergillus niger (An) aphA gene as a probe and cloned the A. ficuum (Af) SRRC 265 gene encoding an extracellular pH 6.0-optimum acid phosphatase (APase6) from a genomic library. The identity of the Af aphA gene was confirmed and its nucleotide (nt) sequence verified by comparing its deduced amino acid (aa) sequence to that of purified Af APase6. A comparison of the nt sequences of the An and Af genes suggested that errors were made in the previously reported An aphA sequence. Several regions of the An aphA were resequenced and the mistakes corrected. With its nt sequence corrected, the An aphA is nearly identical to the cloned Af gene encoding APase6, and in 90.4% agreement in the coding regions. Both genes have three conserved introns and when translated, both nt sequences code for a polypeptide of 614 aa. There is now evidence that the two cloned genes are homologous and code for acid phosphatases that are 96% identical.

Purification and some properties of inositol 1,3,4,5,6-Pentakisphosphate 2-kinase from immature soybean seeds.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase was purified from immature soybean seeds harvested approximately 5 weeks post-anthesis. A crude extract was clarified using polyethyleneimine and purified by chromatography on DEAE-cellulose, Cibacron Blue 3GA-agarose, Toyopearl DEAE 650M, and Toyopearl phenyl 650M columns. The enzyme had a relative molecular mass, M(r), of 52,000 as determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and retained 50% of its activity after 6 weeks at 0 degrees C. The Km values for inositol 1,3,4,5,6-pentakisphosphate and MgATP, respectively, were 2.3 microM and 8.4 microM, and the Vmax was 243 nmol/min/mg. The pH and temperature optima, respectively, were 6.8 and 42 degrees C. Maximum activity was obtained when the magnesium ion concentration was 4 mM. The kinase specifically phosphorylated the 2-position on the inositol ring and could also utilize D-inositol 1,4,5,6-tetrakisphosphate as a substrate. The K for the reaction was 14, indicating that the enzyme may be involved in both inositol hexakisphosphate formation in maturing seeds and ATP resynthesis in germinating seeds. Substrate concentrations in mature seeds were favorable for ATP formation, whereas additional factors appeared to drive the accumulation of inositol hexakisphosphate in maturing seeds.

An acid phosphatase from Aspergillus ficuum has homology to Penicillium chrysogenum PhoA.

Three secreted acid phosphatases had previously been characterized from Aspergillus ficuum grown under conditions of limited phosphate. One of these could not be readily separated from AFPhyB, a pH 2.5 optimum acid phosphatase with phytase activity. From extensive protein sequence analysis and subsequent cloning of the gene, we have shown that the AFPhyB protein fraction contains a fourth secreted acid phosphatase (AFPhoA) that has 64% homology to a phosphate-repressible acid phosphatase from Penicillium chrysogenum. Garnier plot analysis revealed that the putative phosphate catalytic domain of AFPhoA at His215Asp216 is similar to those of other acid phosphatases, but that AFPhoA lacks the phosphate-binding motif RHGXRXP of known histidine phosphatases.

The complete primary structure elucidation of Aspergillus ficuum (niger), pH 6.0, optimum acid phosphatase by Edman degradation.

The primary structure of the Aspergillus ficuum (niger) NRRL 3135 extracellular, pH 6.0, optimum acid phosphatase (E.C.3.1.3.2) was elucidated by gas phase sequencing. It was deduced by sequence overlap of peptides obtained from trypsin, chymotrypsin, clostripain, and cyanogen bromide digests of the pyridylethylated protein. The mature, active protein is composed of 583 amino acids, including 13 glycosylated Asn residues. The unglycosylated protein has a MW of 64,245-KDa and a pI of 4.97. Two putative metal binding sites were identified in the molecule. This enzyme may represent a special class of high molecular weight acid phosphatase, since it lacks the active site sequence RHGXRXP and shows no significant homology with known acid phosphatases containing this active site. Homology to human type 5 and A.niger APases was detected, however.

Identification and cloning of a second phytase gene (phyB) from Aspergillus niger (ficuum).

An Aspergillus niger (ficuum) genomic DNA lambda EMBL3 library was probed with a 354-bp DNA fragment obtained by polymerase chain reaction of A. niger DNA with oligonucleotides based on partial amino acid sequence of a pH 2.5 optimum acid phosphatase. A clone containing a 1605 bp segment (phyB) encoding the 479 amino acid enzyme was isolated and found to contain four exons. Global alignment revealed 23.5% homology to Aspergillus niger phytase (PhyA); four regions of extensive homology were identified. Some of these regions may contain catalytic sites for phosphatase function.

Identification of active-site residues in Aspergillus ficuum extracellular pH 2.5 optimum acid phosphatase.

Primary structure elucidation of peptides generated by cyanogen bromide, endoproteinase Glu-C, and clostripain cleavage of an Aspergillus ficuum extracellular pH optimum 2.5 acid phosphatase identified a region which contains the active site of the enzyme. The 23-residue segment contains the fragment RHGXRXP, which is homologous to acid phosphatase from Saccharomyces spp., Aspergillus ficuum, mammals, and bacteria. Homologous or conservative substitutions are observed in the 10-amino acid fragment preceding this region.

Aspergillus ficuum phytase: complete primary structure elucidation by chemical sequencing.

The primary structure of Aspergillus ficuum phytase was deduced from overlaps in peptide sequences. The unglycosylated enzyme is a 441 residue protein with a molecular mass of 48.5-KDa, as calculated from the total covalent structure. The estimated pl of the protein is about 4.76. Of the 19 Asn residues, 9 were found to be glycosylated. The phytase consists of 37% non-polar, 42% polar, 11.5% acidic, and 9.5% basic amino acids. The putative active site of the enzyme containing the sequence RHG is located at the N-terminal region of the molecule and shows homology to the active site of both microbial and mammalian acid phosphatases, and phosphoglycerate mutase.

Purification of a 40-kilodalton methyltransferase active in the aflatoxin biosynthetic pathway.

The penultimate step in the aflatoxin biosynthetic pathway of the filamentous fungi Aspergillus flavus and A. parasiticus involves conversion of sterigmatocystin to O-methylsterigmatocystin. An S-adenosylmethionine-dependent methyltransferase that catalyzes this reaction was purified to homogeneity (> 90%) from 78-h-old mycelia of A. parasiticus SRRC 163. Purification of this soluble enzyme was carried out by five soft-gel chromatographic steps: cell debris remover treatment, QMA ACELL chromatography, hydroxylapatite-Ultrogel chromatography, DEAE-Spherodex chromatography, and Octyl Avidgel chromatography, followed by MA7Q high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein peak from this step on silver staining identified a single band of approximately 40 kDa. This purified protein was distinct from the dimeric 168-kDa methyltransferase purified from the same fungal strain under identical growth conditions (D. Bhatnagar, A. H. J. Ullah, and T. E. Cleveland, Prep. Biochem. 18:321-349, 1988). The chromatographic behavior and N-terminal sequence of the 40-kDa enzyme were also distinct from those of the 168-kDa methyltransferase. The molar extinction coefficient of the 40-kDa enzyme at 278 nm was estimated to be 4.7 x 10(4) M-1 cm-1 in 50 mM potassium phosphate buffer (pH 7.5).