Chemical and thermal stability of ferulic acid esterase-III from Aspergillus niger

The stability of ferulic acid esterase III (FAE-III) from Aspergillus niger was examined using chemical and thermal denaturation. Thermal denaturation was irreversible and the loss of activity was dependent on pH. At 60 degrees C and pH 6.0, the rate constant of unfolding was 0.76 10(-3)/s, and the change in free energy of irreversible inactivation, deltaG*, was 101.9 kJ/mol. Sinapic acid, a product of the reaction of methyl sinapate with FAE-III, reduced the rate of unfolding (0.66 10(-3)/s at 0.1 mM sinapic acid). Chemical denaturation was performed using guanidine hydrochloride. FAE-III was very sensitive to this denaturant, and the midpoint of unfolding was 1.38 M guanidine hydrochloride at 30 degrees C, pH 6.0. The stability of FAE-III is compared to other enzymes.     

The evolution of proteins from random amino acid sequences. I. Evidence from the lengthwise distribution of amino acids in modern protein sequences

We examine in this paper one of the expected consequences of the hypothesis that modern proteins evolved from random heteropeptide sequences. Specifically, we investigate the lengthwise distributions of amino acids in a set of 1,789 protein sequences with little sequence identify using the run test statistic (ro) of Mood (1940, Ann. Math. Stat. 11, 367-392). The probability density of ro for a collection of random sequences has mean = 0 and variance = 1 [the N(0,1) distribution] and can be used to measure the tendency of amino acids of a given type to cluster together in a sequence relative to that of a random sequence. We implement the run test using binary representations of protein sequences in which the amino acids of interest are assigned a value of 1 and all others a value of 0. We consider individual amino acids and sets of various combinations of them based upon hydrophobicity (4 sets), charge (3 sets), volume (4 sets), and secondary structure propensity (3 sets). We find that any sequence chosen randomly has a 90% or greater chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. We regard this as strong support for the random-origin hypothesis. However, we do observe significant deviations from the random expectation as might be expected after billions years of evolution. Two important global trends are found: (1) Amino acids with a strong alpha-helix propensity show a strong tendency to cluster whereas those with beta-sheet or reverse-turn propensity do not. (2) Clustered rather than evenly distributed patterns tend to be preferred by the individual amino acids and this is particularly so for methionine. Finally, we consider the problem of reconciling the random nature of protein sequences with structurally meaningful periodic "patterns" that can be detected by sliding-window, autocorrelation, and Fourier analyses. Two examples, rhodopsin and bacteriorhodopsin, show that such patterns are a natural feature of random sequences.     

Safety evaluation of amino peptidase enzyme preparation derived from Aspergillus niger

An amino peptidase enzyme preparation obtained from Aspergillus niger was subjected to a series of toxicological tests to document the safety for use as a processing aid for food. The enzyme preparation was examined for subacute and subchronic oral toxicity, and mutagenic potential. No evidence of oral toxicity or mutagenicity was found. Administration of the amino peptidase enzyme preparation at doses of 500, 1000 and 2000 mg/kg body weight/day for 90 days did not induce noticeable signs of toxicity. The no-observed-adverse-effect level (NOAEL) of the enzyme preparation in the subchronic toxicity study was 2000mg/kg body weight/day (equivalent to 1152 PHEA units/kg body weight/day). It can be concluded that no safety concerns were identified in the studies conducted with this amino peptidase enzyme preparation derived from Aspergillus niger and produced under controlled fermentation conditions.     

Degradation of feruloylated oligosaccharides from sugar-beet pulp and wheat bran by ferulic acid esterases from Aspergillus niger

The activity of two forms of ferulic acid esterase (FAE) from Aspergillus niger on a synthetic feruloylated substrate (methyl ferulate) and on 11 different feruloylated oligosaccharides from sugar-beet pulp and wheat bran was determined. The enzymes exhibited different specificities for the various feruloylated substrates and were more active on certain substrates of cell-wall origin than on methyl ferulate. Both enzymes preferred the arabinose residue to which ferulic acid is attached in the furanose form. FAE-I had no clear preference for the type of linkage involved between the ferulic acid units and the oligosaccharide chain. In contrast, FAE-III had a clear requirement for ferulic acid to be attached to O-5 of the Ara f ring while no catalysis was observed when ferulic acid was attached to O-2. Both enzymes showed maximum activity on feruloylated trisaccharides. An increase in the length of the oligosaccharide chain did not preclude catalysis, but feruloylated oligosaccharides of a dp > 3 were hydrolysed at a reduced rate. Our results support the hypothesis that different kinds of ferulic acid esterases exist with different specificities for the oligosaccharide chain of the feruloylated substrates.     

The citric acid fermentation by Aspergillus niger: regulation by zinc of growth and acidogenesis

The citric acid fermentation by Aspergillus niger is divided into two consecutive phases, a growth phase when the cells proliferate but do not accumulate citrate, followed by an accumulating phase when they produce citrate but do not proliferate, or else do so at a much reduced rate. When studied in a low sucrose (0.4-0.8%) mpinimal salts medium the growth-accumulation alternative was controlled by the concentration of zinc: high zinc (about 1-2 muM) maintained growth phase, while at low zinc (below 1 muM) growth became limited by zinc deficiency and the cultures passed into accumulating phase. Addition of zinc to accumulating cultures resulted in their reversion to growth phase. Iron, manganese, calcium at concentrations as high as 5-10 muM had no influence on either growth or citrate accumulation. These results suggest that zinc plays a role in the regulation of growth and citric acid accumulation.     

Phylogenetic analysis of Acinetobacter strains based on the nucleotide sequences of gyrB genes and on the amino acid sequences of their products

Partial nucleotide sequences of the gyrB genes (DNA gyrase B subunit genes) of 15 Acinetobacter strains, including the type and reference strains of genomic species 1 to 12 (A. calcoaceticus [genomic species 1], A. baumannii [genomic species 2], Acinetobacter genomic species 3, A. haemolyticus [genomic species 4], A. junii [genomic species 5], Acinetobacter genomic species 6, A. johnsonii [genomic species 7], A. lwoffii [genomic species 8], Acinetobacter genomic species 9, Acinetobacter genomic species 10, Acinetobacter genomic species 11, and A. radioresistens [genomic species 12]), were determined by sequencing the PCR-amplified fragments of gyrB. The gyrB sequence homology among these Acinetobacter strains ranged from 69.6 to 99.7%. A phylogenetic analysis, using the gyrB sequences, indicates that genomic species 1, 2, and 3 formed one cluster (87.3 to 90.3% identity), while genomic species 8 and 9 formed another cluster (99.7% identity). These results are consistent with those of DNA-DNA hybridization and of biochemical systematics. On the other hand, the topology of the published phylogenetic tree based on the 16S rRNA sequences of the Acinetobacter strains was quite different from that of the gyrB-based tree. The numbers of substitution in the 16S rRNA gene sequences were not high enough to construct a reliable phylogenetic tree. The gyrB-based analysis indicates that the genus Acinetobacter is highly diverse and that a reclassification of this genus would be required.     

Horsetail (Equisetum telmateia) ferredoxins I and II. Amino acid sequences

Two ferredoxins were isolated from horsetail (Equisetum telmateia) and their amino acid sequences were determined by use of a sequence analyzer in combination with carboxypeptidase digestion and manual Edman degradation of tryptic peptides of carboxymethyl-ferredoxins. Ferredoxins I and II each had only four cysteine residues in a total of 95 and 93 residues, respectively. The amino-terminal residues of both ferredoxins were heterogeneous, but alanine was concluded to be their genuine terminal residue. The comparison of these isozymelike molecules showed 29 differences in amino acid residues with three inverted replacements. One gap was inserted in ferredoxin II at position 32 to align the ferredoxins with greatest homology. Despite the many differences in amino acid residues there was no difference in net charges of the two ferredoxins.     

The amino acid sequences of two acylphosphatase isoforms from fish muscle (Lamna nasus)

Two acylphosphatase isoenzymes have been purified from Lamna nasus muscle, and their complete amino acid sequences have been determined. The former (E1) consists of 99 amino acid residues, while the latter (E2) consists of 102 residues. Both are acetylated at their N termini. E1 has the FFRK active site motif characteristic of all common-type acylphosphatase isoenzymes, whereas E2 contains the CFRM active site motif characteristic of all muscle-type acylphosphatase isoenzymes. They have quite similar kinetic properties. The comparison of sequences of fish E1 and E2 isoenzymes with other known mammalian and bird acylphosphatases reveals that the E2 isoenzyme has an N terminus tail, four residues long, similar to those previously found in all known bird species muscle-type isoenzymes. Among organ-common-type acylphosphatases about 50% of residues are completely conserved, whereas about 60% of muscle-type acylphosphatase residues are completely conserved, indicating that the latter type of isoenzyme has a slower evolutionary rate than the former. The sequences of E1 and E2 acylphosphatases from L. nasus represent the first primary structures of this kind of enzyme determined among fish species.     

Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger

A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme. In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expression of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently induced after 3 h of growth on apple pectin. The rhamnogalacturonan hydrolase B gene was not induced by apple pectin, but the rhgB gene was derepressed after 18 h of growth on either apple pectin or sucrose. Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain high-producing A. awamori transformants which were then used for the purification of the two A. niger rhamnogalacturonan hydrolases. High-performance anion-exchange chromatography of oligomeric degradation products showed that optimal degradation of an isolated highly branched pectin fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogalacturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeatus rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5). Compared to the A enzymes, the A. niger B enzyme appears to have a different substrate specificity, since additional oligomers are formed.     

A tobacco soluble protoporphyrinogen-oxidizing enzyme similar to plant peroxidases in their amino acid sequences and immunochemical reactivity

Partial amino acid sequences of a soluble protoporphyrinogen-oxidizing enzyme (PPO) purified from tobacco cultured cells were identified. The sequences of two regions of the soluble PPO corresponded to the acid/base catalysis and heme binding regions of plant peroxidases. Anti-soluble PPO IgG cross-reacted with these plant peroxidases. Thus, the soluble PPO seems to be a kind of peroxidase.     

The specificity of peptide bond cleavage of acid proteinase A from Aspergillus niger var. macrosporus toward oxidized ribonuclease A

In order to investigate the specificity of peptide bond cleavage by acid proteinase A from Aspergillus niger var. macrosporus (Aspergillopepsin II), performic acid-oxidized bovine pancreatic ribonuclease A was digested by the enzyme at pH 1.8 or 5.5, and the resulting peptides were separated by HPLC and analyzed. Among the total 123 peptide bonds approximately thirty and thirteen bonds were cleaved at pH 1.8 for 2 h and at pH 5.5 for 20 h, respectively. Cleavages occurred fairly specifically at Tyr-X, Phe-X, His-X, Asn-X, Asp-X, Gln-X, and Glu-X bonds.     

Extraction of Ce and Th from Monazite Using REE Tolerant Aspergillus niger

In the present study, bioleaching of monazite, one of oldest and most water-resistant mineral to microbial attack, was tested for its amenability toward extraction of Ce, Th, and U using heterotrophic fungal species, Aspergillus niger. Shake flask experiments were carried out using Bromfield, sucrose, synthetic, and standard media containing 2%(w/v) of monazite for 60 days. Several parameters such as media pH, redox potential, siderophore production, biomass, and metal oxidation were analyzed at different intervals during the process of bioleaching. The maximum concentration of Ce obtained was 0.701, 0.229, 0.132, and 0.052 mg/L in Bromfield, synthetic, sucrose, and standard media(s), respectively. Thorium was identified only in synthetic (0.026 mg/L) and sucrose (0.0175 mg/L) media, whereas uranium was not observed in any of the media attempted. The results were corroborated with analysis by SEM characterization of the head and residues.     

Complete amino acid sequences of chitinase-1 and -2 from bulbs of genus Tulipa

The complete amino acid sequences of tulip bulb chitinase-1 and -2 (TBC-1 and -2) were determined. The sequences of the TBC-1 and TBC-2 were solved by analysis of peptides derived by enzymatic digestions as well as by chemical cleavages with cyanogen bromide (CNBr), o-iodosobenzoic acid, and hydroxylamine. TBC-1 and TBC-2 both consisted of 275 amino acid residues and had molecular masses of 30,825 and 30,863, respectively. They shared 247 identical residues, that is 90% identity. Comparison of their sequences with that of gladiolus bulb class IIIb chitinase-a (GBC-a) showed that 63% of the residues of both TBC-1 and TBC-2 are identical to that of GBC-a. From these results, it was seen that TBC-1 and -2 are class IIIb chitinases. The characteristic difference in specific activity between TBC-1 and -2 was also discussed on the basis of their amino acid sequences.     

Prediction of secondary structural content of proteins from their amino acid composition alone. II. The paradox with secondary structural class

The success rates reported for secondary structural class prediction with different methods are contradictory. On one side, the problem of recognizing the secondary structural class of a protein knowing only its amino acid composition appears completely solved by simply applying jury decision with an elliptically scaled distance function. Chou and coworkers repeatedly (see Crit. Rev. Biochem. Mol. Biol. 30:275-349, 1995) published prediction accuracies near 100%. On the other hand, traditional secondary structure prediction techniques achieve success rates of about 70% for the secondary structural state per residue and about 75% for structural class only with extensive input information (full sequence of the query protein, its amino acid composition and length, multiple alignments with homologous sequences). In this article, we resolve the paradox and consider (1) the question of the secondary structural class definition, (2) the role of the representativity of the test set of protein tertiary structure for the current state of the Protein Data Bank (PDB); and (3) we estimate the real impact of amino acid composition on secondary structural class. We formulate three objective criteria for a reasonable definition of secondary structural classes and show that only the criterion of Nakashima et al. (J. Biochem. 99:153-162, 1986) complies with all of them. Only this definition matches the distribution of secondary structural content in representative PDB subsets, whereas other criteria leave many proteins (up to 65% of all PDB entries) simply unassigned. We review critically specialized secondary-structural class prediction methods, especially those of Chou and coworkers, which claim almost 100% accuracy using only amino acid composition, and resolve the paradox that these prediction accuracies are better than those from secondary structure predictions from multiple alignments. We show (i) that these techniques rely on a preselection of test sets which removes irregular proteins and other proteins without any class assignment (about 35% of all PDB entries); and (ii) that even for preselected representative test sets, the success rate drops to 60% and lower for a 4-type classification (alpha, beta, alpha + beta, alpha/beta). The prediction accuracies fall to about 50% if the secondary structural class definition of Nakashima et al. is applied and only few irregular proteins are preselected and removed from automatically generated, representative subsets of the PDB. We have applied two new vector decomposition methods for secondary structural content prediction from amino acid composition alone, with and without consideration of amino acid compositional coupling in the learning set of tertiary structures respectively, to the problem of class prediction and achieve about 60% correct assignment among four classes (alpha, beta, mixed, irregular) as well as single sequence-based secondary structure prediction methods like GORIII and COMBI. Our results demonstrate that 60% correctness is the upper limit for a 4-type class prediction from amino acid composition alone for an unknown query protein and that consideration of compositional coupling does not improve the prediction success. The prediction program SSCP offering secondary structural class assignment for query compositions and sequences has been made available as a World Wide Web and E-mail service.     

A new crystal form of proteinase A, a non-pepsin-type acid proteinase from Aspergillus niger var. macrosporus

Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase, whose catalytic residues and mechanism remain to be elucidated. A new form of proteinase A crystals more suitable for crystallography than that obtained previously was prepared from an ammonium sulfate solution at pH 3.5 by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1), with unit cell dimensions of a = 69.75 +/- 0.06 A, b = 87.55 +/- 0.05 A, and c = 60.83 +/- 0.04 A. On the assumption of two enzyme molecules per asymmetric unit, the calculated volume to unit protein mass ratio (Vm) was 2.08 A3/Da. By assuming the specific volume to be 0.74 cm3/g, the solvent content (Vso1) was estimated to be 41%, i.e., much larger than that of the crystal form obtained previously at pH 2.0 (Vso1 = 26%). Diffraction data were collected up to a resolution higher than 1.6 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation.     

Nucleotide and amino acid sequences of the metallo-beta-lactamase, ImiS, from Aeromonas veronii bv. sobria

The Aeromonas veronii bv. sobria metallo-beta-lactamase gene, imiS, was cloned. The imiS open reading frame extends for 762 bp and encodes a protein of 254 amino acids with a secreted modified protein of 227 amino acids and a predicted pI of 8.1. To confirm the predicted sequence, purified ImiS was digested and the resulting peptides were identified, yielding an identical sequence for ImiS, with 98% identity to CphA. Both possessed the putative active-site sequence Asn-Tyr-His-Thr-Asp at positions 88 to 92, which is unique to the Aeromonas metallo-beta-lactamases.     

Identification and cloning of a mobile transposon from Aspergillus niger var. awamori

Aspergillus niger var. awamori contains multiple copies of a transposable element, Vader. This element was detected as a 437-bp insertion in four independently isolated spontaneous mutants of the niaD (nitrate reductase) gene. The Vader element is present in approximately 15 copies in both A. niger var. awamori and A. niger. A single copy of Vader was detected from only one of the two laboratory strains of A. nidulans which were also examined. Insertion of the Vader element into the niaD gene of A. niger var. awamori caused a 2-bp duplication (TA) of the target sequence. The Vader element is flanked by a 44-bp inverted repeat. The genetic stabilities of the inserted Vader elements at niaD were examined by studying reversion frequencies resulting in colonies able to grow on nitrate as a sole nitrogen source. Mutants niaD392 and niaD436 reverted at a frequency of 9x10(-3) and 4x10(-2), respectively. Two of the mutants, niaD587 and niaD410, reverted at a lower frequency of 6x10(-4).