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The glycosylation patterns of phytase (E C 3.1.3.8) from Aspergillus niger NRRL 3135 with recombinant phytase from A. niger van Tieghem were compared. The following characteristics were studied: size of the whole molecule, type of linkage (N-linked or O-linked oligosaccharide), profile (number of different type of oligosaccharides present), monosaccharide composition, their order, and configuration. The molecular weights of both glycoproteins, after deglycosylation, was approximately 55 kDa as determined by SDS-PAGE. Both glycoproteins were about 35.29% glycosylated (calculations were made on the basis of 85 kDa molecular weight before deglycosylation). Only N-linked oligosaccharides were present. When N-linked oligosaccharides were released and labeled, the same profile was obtained for both glycoproteins. Mannose residues were detected after digestion by combinations of various exoglycosidases. N-acetylneuraminic acid, galactose, and N-acetylglucosamine residues were not detected. Released mannose residues were (alpha 1-2,3,6) linked. The trisaccharide core structure was nonfucosylated for all the oligosaccharides released from both glycoproteins. The only major difference found between the two glycoproteins was the migration pattern of oligosaccharide bands on gel after digestion with alpha-mannosidases. The structure of N-linked oligosaccharides ranged from (Man)5(GlcNAc)2 to (Man)10 (GlcNAc)2.

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50,900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42 degrees C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 micrograms/ml) and not inhibited by antipain dihydrochloride (120 micrograms/ml), aprotinin (4 micrograms/ml), bestatin (80 micrograms/ml), chymostatin (50 micrograms/ml), E-64 (20 micrograms/ml), leupeptin (4 micrograms/ml), Pefabloc SC (2000 micrograms/ml), pepstatin (4 micrograms/ml), phosphoramidon (660 micrograms/ml), or phenylmethylsulfonyl fluoride (400 micrograms/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties.

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 crude extracellular extract of Aspergillus niger (syn A. ficuum) NRRL 3135 contains glucoamylase (exo-1,4-alpha-D-glucanohydrolase, EC 3.2.1.2). The enzyme, a glycoprotein, was purified 7-fold by ion-exchange chromatography, chromatofocusing, and conconavalin A affinity chromatography. The molecular weight of the enzyme was estimated to be 90 kDa by SDS-PAGE and gel permeation chromatography. The pI of the enzyme was 3.4. The temperature optimum of the enzyme was 60 degrees C and the pH optimum was 5.0. The Vmax values for soluble starch, maltose, maltotriose, maltotretraose, maltopentaose, and isomaltose were 55.2, 11.7, 32.3, 47.8, 59.2, 12.5 nKat glucose/sec, respectively and the Km values for the same substrates were 0.09%, 0.67 mM, 0.76 mM, 0.76 mM, 0.68 mM, and 122.01 mM, respectively.

The closely related soybean (Glycine max) cultivars Centennial and Pickett 71 were confirmed to be resistant and susceptible, respectively, to the root-knot nematode Meloidogryne incognita. Increases in superoxide dismutase (SOD) activity were detected in roots of both soybean cultivars 48 hours following inoculation. Superoxide dismutase activity increased in roots of the susceptible cultivar overall, but declined after 96 hours in roots of the resistant cultivar. The isoelectric points of SOD isolated from preparasitic and parasitic developmental stages of the nematode appeared to differ. The SOD activity increased dramatically as nematodes matured and enlarged. Plant and nematode SOD were present as ca. 40-kDa cuprozinc dimers. Initial increases in SOD activity in infected tissue appeared to involve nematode regulation of plant gene expression. However, as the nematode enlarged, SOD activity could be detected within the female body only.

Extracellular alpha-galactosidase, a glycoprotein from the extracellular culture fluid of Aspergillus ficuum grown on glucose and raffinose in a batch culture system, was purified to homogeneity in five steps by ion exchange and hydrophobic interaction chromatography. The molecular mass of the enzyme was 70.8 Kd by SDS polyacrylamide gel electrophoresis and 74.1 Kd by gel permeation HPLC. On the basis of a molecular mass of 70.7 Kd, the molar extinction coefficient of the enzyme at 279 nm was estimated to be 6.1 X10(4) M-1 cm-1. The purified enzyme was remarkably stable at 0 degrees C. It had a broad temperature optimum and maximum catalytic activity was at 60 degrees C. It retained 33% of its activity after 10 min. at 65 degrees C. It had a pH optimum of 6.0. It retained 62% of its activity after 12 hours at pH 2.3. The Kms for p-nitrophenyl-alpha-D-galactopyranoside, o-nitrophenyl-alpha-D-galactopyranoside and m-nitrophenyl-alpha-D-galactopyranoside are: 1462, 839 and 718 microM. The enzyme was competitively inhibited by mercury (19.8 microM), silver (21.5 microM), copper (0.48 mM), zinc (0.11 mM), galactose (64.0 mM) and fructose (60.3 mM). It was inhibited non-competitively by glucose (83.2 mM) and uncompetitively by mannose (6.7 mM).

The hemolytic plaque assay was adapted to the detection of antibodies to Newcastle disease virus (NDV) in an in vivo, an in vitro system, and a combined in vivo-in vitro system. Several conditions were tested for coupling of sheep erythrocytes to NDV and for the kinetics of plaque formation in the in vivo and in vitro systems. The one set of conditions which provided the best responses is presented. The effect of multiple injections of NDV into mice on plaque formation was optimized.

Two types of extracellular acid phosphatases are synthesized by Aspergillus ficuum NRRL 3135: a nonspecific orthophosphoric monoester phosphohydrolase (EC 3.1.3.2) with an optimum pH of 2.0, and an enzyme with restricted specificity, a mesoinositol-hexaphosphate phosphohydrolase (EC 3.1.3.8; phytase) with an optimum pH of 5.5. Although the pH 5.5 enzyme is termed a phytase, both enzymes hydrolyze phytin. Synthesis of the enzymes is repressed by high orthophosphate concentrations in the fermentation medium. The highest total level for each enzyme is synthesized in low orthophosphate medium. In high orthophosphate medium, more pH 5.5 enzyme is produced than pH 2.0 enzyme. In low orthophosphate medium, more pH 5.5 enzyme is produced than pH 2.0 enzyme during the early stages of growth, but the reverse occurs after 5 days. The enzymes are differentiated by heat denaturation at acid and alkaline pH levels. They are separated into two distinct fractions on Sephadex G-100 followed by carboxymethylcellulose column chromatography. This indicates that the two enzymes are structurally different. The K(m) for both enzymes is 1.25 mm when calcium phytate is the substrate. Orthophosphate competitively inhibits the pH 2.0 (K(i) = 1.1 x 10(-2)m) but not the pH 5.5 phosphatase. Neither enzyme is denatured by 50% (w/v) urea or inhibited by 0.01 m tartrate. Thus, they differ from human prostatic phosphatase.