Ficuum NRRL Research Articles

Optimization of phytase production by solid substrate fermentation.

The production of phytase by three feed-grade filamentous fungi ( Aspergillus ficuum NRRL 3135, Mucor racemosus NRRL 1994 and Rhizopus oligosporus NRRL 5905) on four commonly used natural feed ingredients (canola meal, cracked corn, soybean meal, wheat bran) was studied in solid substrate fermentation (SSF). A. ficuum NRRL 3135 had the highest yield [15 IU phytase activity/g dry matter (DM)] on wheat bran. By optimizing the supplementation of wheat bran with starch and (NH(4))(2)SO(4), phytase production increased to 25 IU/g DM. Optimization was carried out by Plackett-Burman and central composite experimental designs. Using optimized medium, phytase, phosphatase, alpha-amylase and xylanase production by A. ficuum NRRL 3135 was studied in Erlenmeyer flask and tray SSF. By scaling up SSF from flasks to stationary trays, activities of 20 IU phytase activity/g DM were reproducibly obtained.

Selection of phytase-producing moulds.

Organic phosphorous in the form of phytate salts are found in feed component originated from cereals which can not be digested by chicken unless after the addition of an enzyme, i.e. phytase. A research to investigate phytase-producing moulds had been carried out. A total of 60 isolates from various collection units and isolation from samples had been collected then screened in Czapek’s medium with Ca-phytate as the source of phosphorous. From mould growth measurements and clearing zones formation there were 4 mould isolates, i.e. Aspergillus ficuum NRRL 3135 and NRRL 320, Aspergillus niger M94/18/D2 (Balitvet isolate), and Aspergillus niger no. 58 (IPB isolate), which were further observed. Enzyme was produced from medium containing 8% of corn’s starch, 3% of glucose, 0.0005% of KCl and MgSO4.7H2O, 0.0002% of K2HPO4, and 0.00018% of FeSO4.7H2O. The enzyme activity, protein and biomass contents were observed at 16, 24, 32, 48 and 72 hours incubations for Aspergillus niger and at 1, 2, 3, 4 and 5 days incubations for Aspergillus ficuum . The highest protein content was found at 5 days incubation for Aspergillus ficuum NRRL 3135 and NRRL 320, (165 and 169 μg/ml respectively), while for Aspergillus niger (Balitvet and IPB isolates) at 16 hours incubation (102 and 112 μg/ml respectively). Meanwhile, the biomass contents were decreased in accordance with the duration of incubations. The maximum enzyme productions were reached at 24 hours incubation for Aspergillus niger and at 4 days incubation for Aspergillus ficuum . The highest phytase activities were reached at 2.808 U/ml for Aspergillus ficuum NRRL 3135, followed by 1.520 U/ml for Aspergillus ficuum NRRL 320. Aspergillus niger produced enzyme quicker than the other moulds, with lower activity level and decreases quicker at the following hours. Key words: Phytate, phytase, Aspergillus ficuum , Aspergillus niger

Culture conditions for a new phytase-producing fungus

Extracellular phytase produced by Aspergillus sp. 5990 showed a 5-fold higher activity in liquid culture when compared with cultures of Aspergillus ficuum NRRL 3135. The optimum fermentation conditions were determined to be 35 °C, neutral pH, and 4 days incubation. The phytase had a higher optimum temperature for its activity than the commercial enzyme, Natuphos, from Aspergillus ficuum NRRL 3135.

Comparison of Glycosylation Patterns of Phytase from Aspergillus Niger (A. Ficuum) NRRL 3135 and Recombinant Phytase

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 types 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 (α1-2,3,6) linked. The trisaccharide core structure was non-fucosylated 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 α-mannosidases. The structure of N-linked oligosaccharides ranged from (Man)5(GlcNAc)2 to (Man)10 (GlcNAc)2.

Identification, characterization, and partial purification of glucoamylase from Aspergillus niger (syn A. ficuum) NRRL 3135.

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.

Production of phytase during solid state fermentation using Aspergillus ficuum NRRL 3135 in canola meal

Effects of moisture content of media, inoculum age and homogenization on production of phytase and reduction of phytic acid content in canola meal by Aspergillus ficuum NRRL 3135 during static solid-state fermentation (SSF) have been considered. Optimum mostirue content of media for these processes was 64%. Rate of phytase production increased with an increase in inoculum age between 2 and 5 days. Both the level and the rate of phytase production were lower in initial stages of SSF when inoculum was homogenized for longer periods of time, while after 36 h of the process an increase in phytase production was noticed with an increase in homogenization time. Mathematical correlations of phytase activity and phytic acid content reduction with moisture content and with time of homogenization of inoculum are proposed in this work.

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 pl 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.

Phytic acid content reduction in canola meal by various microorganisms in a solid state fermentation process

AbstractSolid state fermentation of canola meal has been carried out for the reduction of its phytic acid content using the following microorganisms: Rhizopus oligosporus NRRL 2990, Aspergillus niger NRC 5765 and NRC 401 121, Aspergillus ficuum NRRL 3135 and a wild Saccharomyces cerevisiae strain.The results showed that all these microorganisms can be used for the reduction of the phytic acid content in the tested material. A. ficuum which completely hydrolyzed the phytic acid in 48 hours was the most efficient.Buffered systems, aeration and an increase in inoculum concentration caused faster and higher reduction of phytic acid content in canola meal.

Reduction of phytic acid content in canola meal by Aspergillus ficuum in solid state fermentation process

Solid state fermentation (SSF) of canola meal has been carried out to reduce its phytic acid content using Aspergillus ficuum NRRL 3135. In certain batches, a complete reduction of phytic acid content in canola meal was achieved in 48 h. A larger amount of biomass in the inoculum and older inoculum increased the rate of phytic acid hydrolysis. The optimum moisture content of the medium was found to be 67% for phytic acid hydrolysis in an SSF process. The substitution of water in the semi-solid medium with acetate buffer resulted in faster reduction of the phytic acid content. A 15% increase in the amount of protein after 120 h of incubation was observed in the treated meal. The crude phytase preparation extracted from the canola meal after it was treated in an SSF process was also used for reduction of the phytic acid content in new batches of canola meal both in semi-solid medium and in liquid medium. In the semi-solid medium, 58% of the phytic acid was hydrolysed at 45°C in 20 h, while 100% hydrolysis was recorded at 50°C in 12 h in the liquid medium. The SSF process seems to be beneficial for the upgrading of canola meal by reducing both its phytic acid content and increasing the amount of protein.

DNA mediated transformation of Aspergillus ficuum

Two DNA-mediated transformation systems were successfully adapted to Aspergillus ficuum. Both the Escherichia coli hygromycin B resistance gene and the A. nidulans amdS gene transformation systems produced stable A. ficuum NRRL 3135 transformants. Cotransformation with the E. coli lacZ gene was also achieved with the hygromycin B system. In cotransformation a second unselected gene, in this case the lacZ gene which codes for β-galactosidase, was also integrated and expressed in hygromycin B transformants. Since both of these transformation systems utilized dominant selection markers, they are potentially useful in other genetically uncharacterized filamentous fungi.

Production of phytate-hydrolysing enzyme by some fungi

Eighty-four fungi from twenty five species have been examined for the production of extracellular enzymes capable of hydrolysing phytate (3-phytase, myo-inositol hexakisphosphate 3-phosphohydrolase, EC 3.1.3.8, and 6-phytase, myo-inositol hexakisphosphate 6-phosphohydrolase, EC 3.1.3.26) when grown in: (1) rapeseed meal (RSM); (2) a semisynthetic medium containing phytate as the sole phosphorus source (PSM); (3) potato dextrose broth (PDB). Although 58 active strains showed substantial activity, results in either of the media were of no value in indicating activity in RSM. There was no relationship between the ability of a fungus to hydrolyse phytate and its taxonomic position. Aspergillus ficuum NRRL 3135 had the greatest activity in the synthetic medium, and was relatively active in RSM. The extracellular enzyme had maximum activity after 10 days growth in PSM and had a temperature optimum of 55°C. Two pH optima were noted at pH 2.0 and 5.5. Inorganic phosphate inhibited enzyme production; ammonia ions were a better nitrogen source than nitrate or urea.

Inositol Phosphate Phosphatases of Microbiological Origin. Some Properties of the Partially Purified Phosphatases of Aspergillus ficuum NRRL 3135

Partially purified samples of the acid phosphatase (EC 3.1.3.2) and of the phytase (EC 3.1.3.8) from A. ficuum were prepared by gel-filtration and ion-exchange dextran chromatography.

Effect of Supplemental Phytase on the Utilization of Phytate Phosphorus by Chicks

Chicks were used to determine whether the addition of a mold phytase to a diet containing natural phytate phosphorus causA§dthe phosphorus in the phytate to be hydrolyzed in vivo to a form which could be utilized by the chick. Phytase, produced by Aspergillus ficuum NRRL 3135, was fed as an ace tone-dried preparation. The diets contained from 0.18 to 0.24% natural phytate phosphorus supplied primarily by corn and soybean meal. Phytase supplement was added at levels ranging from 1 to 8 g/kg of diet. One gram of the supple ment hydrolyzed 950 mg of inorganic phosphate from calcium phytate in vitro in 1 hour under standard conditions. For comparative purposes, graded levels of either monobasic sodium orthophosphate, or beta-tricalcium phosphate were added at levels to supply from 0.04 to 0.21% phosphorus. The percentage of bone ash was used to evaluate in vivo phytase activity. The addition of phytase to the diet produced an increase in percentage bone ash indicating hydrolysis of phytate by the enzyme. Total hydrolysis of phytate occurred when 3 g phytase supplement was used per kilogram diet. Chicks utilized the hydrolyzed phytate phosphorus as well as supplemental inorganic phosphorus. Phytase activity oc curred in the alimentary tract of the chick and not in the feed prior to ingestion. J. Nutr. 101: 1289-1294, 1971. The ability of poultry to utilize phytate phosphorus is a controversial subject (1, 2). The probability that the differences re ported for the availability of phytate phos phorus to the chick are caused by dietary variables was discussed in a recent review (3). The presence of phytase, an enzyme which hydrolyzes phytate to phosphoric acid and inosinic acid (4-6) in some feed ingredients (7, 8) may have enhanced the utilization of phytate phosphorus. Phytase of plant origin appears to retain its activity when added to animal diets (7-10). The reaction conditions of in vitro studies with some phytases suggest that the enzyme is active in the digestive tract of animals (11, 12) since the in vitro conditions are similar to those found in the chick's ali mentary tract. A purified extract of phytase from wheat bran was inactive in the rat (13). Phytase obtained from Escherichia coli was active when fed to chicks (14). The present report describes quantitative studies of the effect of a mold phytase on J. NUTRITION, 101: 1289-1294. the utilization of phytate phosphorus by chicks. EXPERIMENTAL PROCEDURE The breed, sex and number of chicks used are listed in the design of each ex periment. The replication of treatments varied and was dependent on the amount of phytase preparation available. Day-old chicks were allotted to the treatments and were housed in electrically heated brooders with raised wire mesh floors. Feed and water were supplied ad libitum. Body weights and feed consumption were re corded weekly. At the end of week 3 the Received for publication February 9, 1971. 1Presented in part at the 51st annual meeting of the Federation of American Societies for Experimental Biology, Chicago, Illinois, 1967. Federation Proc. 26: 416 (abstr.). 2Present address: Department of Animal Sciences, University of Arkansas, Fayetteville, Arkansas 72701. 3Reprint requests should be addressed to this au thor. 4Present address: Department of Biological Sci ences, Florida Technological University, Orlando, Flor ida 32816. 5Present address: 2921 Glenwood Lane, Billings, Montana 59102.

Regulation of the formation of acid phosphatases by inorganic phosphate in Aspergillus ficuum.

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.

Survey of Microorganisms for the Production of Extracellular Phytase

A culture enrichment technique was used to isolate phytase-producing microorganisms. Also, microorganisms from various culture collections were tested for their phytase-producing ability. A number of the Aspergillus niger group produced extracellular phytase which dephosphorylated calcium phytate in acidic solution. A soil isolate, A. ficuum NRRL 3135, produced the most active phytase in a cornstarch-based medium. Production of phytase was strongly repressed by inorganic phosphates and required a high carbon to phosphorus ratio in the medium.