PubMed Health⌕ Search

Biomedical subjects

A H Ullah

Publications and source records attributed to A H Ullah.

At least 19 recordsLinked to original sources
PubMed1998

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.

6-Phytase↗
PubMed1998

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.

6-Phytase↗
PubMed1996

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.

6-Phytase↗
PubMed1996

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.

6-Phytase↗
PubMed1995

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.

Acid Phosphatase↗
PubMed1994

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.

Chromatography↗
PubMed1994

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.

Acid Phosphatase↗
PubMed1994

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.

Acid Phosphatase↗
PubMed1993

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.

6-Phytase↗
PubMed1993

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.

6-Phytase↗
PubMed1993

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.

Acid Phosphatase↗
PubMed1993

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).

Aflatoxins↗
PubMed1991

Cyclohexanedione modification of arginine at the active site of Aspergillus ficuum phytase.

Reaction of Aspergillus ficuum phytase with the arginine specific modifier 1,2-cyclohexanedione causes a rapid loss of activity. The inactivation can be partially reversed by 0.2 M hydroxylamine and exhibits pseudo-first order kinetics. The reaction order and second order rate constant of inactivation were 0.87 and 6.72 M-1 Min-1, respectively. Amino acid analysis of modified phytase indicates that about 7 arginine of the total 19 were modified. While the chymotryptic maps of treated and untreated phytase wer virtually identical, the tryptic maps had 4 peaks of altered mobility. An Arg containing tripeptide was identified in the phytase which is also present in other phosphohydrolases and may represent one of the labile Arg involved in the formation of the active site.

6-Phytase↗
PubMed1991

Positive identification of a lambda gt11 clone containing a region of fungal phytase gene by immunoprobe and sequence verification.

As the initial step in a project to provide a more cost-effective source of the phytase enzyme, this paper reports on the use of a polyclonal antibody raised to phytase purified from an isolate of Aspergillus niger (A. ficuum) to screen an A. niger lambda gt11 expression library and the use of amino acid sequencing to identify a clone containing part of the fungal phytase gene. The described use of amino acid sequence fragments to verify the cloning of a gene has potential applications in other cloning projects.

6-Phytase↗
PubMed1990

Extracellular alpha galactosidase (E.C. 3.2.1.22) from Aspergillus ficuum NRRL 3135 purification and characterization.

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).

Aspergillus↗