New structural features of the antigenic extracellular polysaccharides of Penicillium and Aspergillus species revealed with exo-beta-D-galactofuranosidase.

To study the structures of the epitopes of the extracellular polysaccharides from Penicillium and Aspergillus species, an exo-beta-D-galactofuranosidase was purified from a commercial crude enzyme preparation from Trichoderma harzianum. Analysis of ring size and linkage position of the galactose residues of the extracellular polysaccharide of Penicillium digitatum, before and after enzymatic treatment, was determined by the reductive-cleavage technique. In addition to terminal and beta (1-5)-linked galactofuranosides, beta (1-6)-linked and beta (1,5,6)-linked branched galactofuranose residues could be identified. After degradation with the purified exo-beta-D-galactofuranosidase, all initial linkages of the galactofuranose residues were still present, but the amount of beta (1-5)-linked galactofuranose residues had decreased considerably. Treatment of the extracellular polysaccharides of Penicillium and Aspergillus species with the purified exo-beta-D-galactofuranosidase resulted in complete disappearance of the enzyme-linked immunosorbent assay reactivity of these polysaccharides, using immunoglobulin G antibodies raised against P. digitatum. Therefore, with the use of this enzyme, it was proved that the beta (1-5)-linked galactofuranosyl residues only are responsible for the antigenicity of the extracellular polysaccharides of Penicillium and Aspergillus molds. A new structural model for the antigenic galactofuranose side chains of the galactomannan from P. digitatum is proposed.     

Microbial oxidases catalyzing conversion of glycolaldehyde into glyoxal

The present paper reviews oxidases catalyzing conversion of glycolaldehyde into glyoxal. The enzymatic oxidation of glycolaldehyde into glyoxal was first reported in alcohol oxidases (AODs) from methylotrophic yeasts such as Candida and Pichia, and glycerol oxidase (GLOD) from Aspergillus japonicus, although it had been reported that these enzymes are specific to short-chain linear aliphatic alcohols and glycerol, respectively. These enzymes continuously oxidized ethylene glycol into glyoxal via glycolaldehyde. The AODs produced by Aspergillus ochraceus and Penicillium purpurescens also oxidized glycolaldehyde. A new enzyme exhibiting oxidase activity for glycolaldehyde was reported from a newly isolated bacterium, Paenibacillus sp. AIU 311. The Paenibacillus enzyme exhibited high activity for aldehyde alcohols such as glycolaldehyde and glyceraldehyde, but not for methanol, ethanol, ethylene glycol or glycerol. The deduced amino acid sequence of the Paenibacillus AOD was similar to that of superoxide dismutases (SODs), but not to that of methylotrophic yeast AODs. Then, it was demonstrated that SODs had oxidase activity for aldehyde alcohols including glycolaldehyde. The present paper describes characteristics of glycolaldehyde oxidation by those enzymes produced by different microorganisms.     

Exploitation of a library of alpha-galactosidases for the synthesis of building blocks for glycopolymers.

After screening a library of fungal alpha-galactosidases for the synthesis of functionalized alkyl alpha-D-galactopyranosides, four enzymes (isolated from Aspergillus terreus CCM55, Aspergillus commune CCM 2969, Penicillium vinaceum CCM 2384, or Penicillium brasilianum 2155) proved to be suitable for these biotransformations. The effect of different concentrations of alcohol on activity and stability of these enzymes was investigated. After optimization of the reaction conditions, three galactose derivatives (allyl, 2-nitroethyl and 2-(2',2',2'-trifluoroacetamido)-ethyl alpha-D-galactopyranoside, 1a, 3a, and 4a, respectively), suitable for subsequent chemical polymerization, were synthesized using either the "reverse hydrolysis" or the "transglycosylation" protocols.     

Insights into the reaction mechanism of glycosyl hydrolase family 49. Site-directed mutagenesis and substrate preference of isopullulanase.

Aspergillus niger isopullulanase (IPU) is the only pullulan-hydrolase in glycosyl hydrolase (GH) family 49 and does not hydrolyse dextran at all, while all other GH family 49 enzymes are dextran-hydrolysing enzymes. To investigate the common catalytic mechanism of GH family 49 enzymes, nine mutants were prepared to replace residues conserved among GH family 49 (four Trp, three Asp and two Glu). Homology modelling of IPU was also carried out based on the structure of Penicillium minioluteum dextranase, and the result showed that Asp353, Glu356, Asp372, Asp373 and Trp402, whose substitutions resulted in the reduction of activity for both pullulan and panose, were predicted to be located in the negatively numbered subsites. Three Asp-mutated enzymes, D353N, D372N and D373N, lost their activities, indicating that these residues are candidates for the catalytic residues of IPU. The W402F enzyme significantly reduced IPU activity, and the Km value was sixfold higher and the k0 value was 500-fold lower than those for the wild-type enzyme, suggesting that Trp402 is a residue participating in subsite -1. Trp31 and Glu273, whose substitutions caused a decrease in the activity for pullulan but not for panose, were predicted to be located in the interface between N-terminal and beta-helical domains. The substrate preference of the negatively numbered subsites of IPU resembles that of GH family 49 dextranases. These findings suggest that IPU and the GH family 49 dextranases have a similar catalytic mechanism in their negatively numbered subsites in spite of the difference of their substrate specificities.     

Host specificity of Eupenicillium ochrosalmoneum, E. cinnamopurpureum and two Penicillium species associated with the conidial heads of Aspergillus

The genus Penicillium comprises species that mostly colonize plant matter. However early reports suggest that several species are capable of parasitizing Aspergillus and sporulating on the conidial heads of the host. More recently Eupenicillium ochrosalmoneum and E. cinnamopurpureum, both with Penicillium anamorphs, have been observed sporulating on the heads of Aspergillus species belonging to section Flavi during the colonization of peanut seeds. Little is known about the host specificity underlying these Aspergillus-Penicillium associations. In this study Aspergillus species representing nine taxonomic sections were paired in culture with E. ochrosalmoneum, E. cinnamopurpureum and two unnamed Penicillium species. Eupenicillium ochrosalmoneum, E. cinnamopurpureum and Penicillium sp. 1 sporulated predominantly on the heads of section Flavi species. In contrast Penicillium sp. 2 was restricted to the heads of section Nigri species. All species spread across Aspergillus colonies by means of aerial hyphae that grew from head to head. Additional studies are required to clarify whether Eupenicillium and Penicillium species are parasitic or simply epibiotic on their hosts.     

Hydrolysis of beta-galactosyl ester linkage by beta-galactosidases

p-Hydroxybenzoyl beta-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of beta-galactosyl ester linkage by beta-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl beta-galactoside (pNP-Gal), a usual substrate with a beta-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that beta-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H218O to liberate galactose containing 18O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.     

Cloning, sequence analysis and heterologous expression of the Myrothecium gramineum orotidine-5'-monophosphate decarboxylase gene

A 2918 bp sequence coding for the orotidine-5'-monophosphate decarboxylase enzyme (OMPD) was isolated from the genome of Myrothecium gramineum. This sequence was analysed and, remarkably, it is the first OMPD gene of a Sordariomycete that has an intron. The gene codes for an enzyme of 282 amino acids. The nucleotide sequence and the amino acid sequence were compared with fungal OMPD sequences. They show the highest similarity to OMPD genes and enzymes of Aspergillus sp., Penicillium sp. and Cladosporium fulvum. The functionality of the gene as a selection marker was proven by complementation of the uracil auxotrophy of Aspergillus nidulans FGSC A722.     

Identity of Escherichia coli D-1-amino-2-propanol:NAD+ oxidoreductase with E. coli glycerol dehydrogenase but not with Neisseria gonorrhoeae 1,2-propanediol:NAD+ oxidoreductase.

The properties of D-1-amino-2-propanol oxidoreductase from wild-type Escherichia coli have been compared with those of a glycerol dehydrogenase from mutant E. coli 424 and of a 1,2-propanediol oxidoreductase from Neisseria gonorrhoeae. Several independent lines of evidence indicate that the former two enzymes are identical. (i) Both enzymatic activities purified to virtual homogeneity in an identical manner, and the ratio of specific activities (glycerol/aminopropanol) remained constant at all stages. (ii) When electrophoresed, both purified enzymes showed a major as well as a minor band of protein coincident with activity, and these two bands from each enzyme had the same mobility. (iii) The subunit molecular weights and isoelectric points were identical for each enzyme, and (iv) kinetic constants (Km and Vmax values) determined with three different substrates were the same. The somewhat greater stability of the glycerol dehydrogenase to controlled heat denaturation at 74 degrees C was the only difference observed between these two enzymes. In contrast, D-1-amino-2-propanol oxidoreductase was found to be immunochemically and kinetically distinct from the 1,2-propanediol oxidoreductase from N. gonorrhoeae.     

Unraveling terminal C-domain-mediated condensation in fungal biosynthesis of imidazoindolone metabolites

The fungal peptidyl alkaloids of the tryptoquialanine and fumiquinazoline families are nonribosomally assembled by annulation of the indole side chain of fumiquinazoline F (FQF) with an alaninyl or aminoisobutyryl unit by monomodular NRPS enzymes containing adenylation, thiolation, and condensation (A-T-C) domains. The Af12060 and Af12050 enzyme pair from Aspergillus fumigatus thereby converts FQF to FQA, while the homologous TqaH and TqaB enzyme pair from Penicillium aethiopicum makes the 2'-epi diastereomer of FQA, differing only in the stereochemistry of one of the C-N bonds formed in the annulation with l-Ala. To evaluate the basis for this stereochemical control, we have mixed and matched the flavoprotein oxygenases Af12060 and TqaH with the A-T-C modular enzymes Af12050 and TqaB to show that the NRPS enzymes control the stereochemical outcome. The terminal 50 kDa condensation domains of Af12050 and TqaB are solely responsible for the stereochemical control as shown both by making chimeric (e.g., A-T-C* and A*-T*-C) forms of these monomodular NRPS enzymes and by expression, purification, and assay of the excised C-domains. The Af12050 and TqaB condensation domains are thus a paired set of diastereospecific annulation catalysts that act on the fumiquinazoline F scaffold.     

Isolation and characterization of the acetyl-CoA synthetase from Penicillium chrysogenum. Involvement of this enzyme in the biosynthesis of penicillins.

Acetyl-CoA synthetase (ACS) of Penicillium chrysogenum was purified to homogeneity (745-fold) from fungal cultures grown in a chemically defined medium containing acetate as the main carbon source. The enzyme showed maximal rate of catalysis when incubated in 50 mM HCl-Tris buffer, pH 8.0, at 37 degrees C. Under these conditions, ACS showed hyperbolic behavior against acetate, CoA, and ATP; the Km values calculated for these substrates were 6.8, 0.18, and 17 mM, respectively. ACS recognized as substrates not only acetate but also several fatty acids ranging between C2 and C8 and some aromatic molecules (phenylacetic, 2-thiopheneacetic, and 3-thiopheneacetic acids). ATP can be replaced by ADP although, in this case, a lower activity was observed (37%). ACS in inhibited by some thiol reagents (5,5'-dithiobis(nitrobenzoic acid), N-ethylmaleimide, p-chloromercuribenzoate) and divalent cations (Zn2+, Cu2+, and Hg2+), whereas it was stimulated when the reaction mixtures contained 1 mM dithiothreitol, reduced glutathione, or 2-mercaptoethanol. The calculated molecular mass of ACS was 139 +/- 1 kDa, and the native enzyme is composed of two apparent identical subunits (70 kDa) in an alpha 2 oligomeric structure. ACS activity was regulated "in vivo" by carbon catabolite inactivation when glucose was taken up by cells in which the enzyme had been previously induced. This enzyme can be coupled "in vitro" to acyl-CoA:6-aminopenicillanic acid acyltransferase from P. chrysogenum, thus allowing the reconstitution of the functional enzymatic system which catalyzes the two latter reactions responsible for the biosynthesis of different penicillins. The ACS from Aspergillus nidulans can also be coupled to 6-aminopenicillanic acid acyltransferase to synthesize penicillins. These results strongly indicate that this enzyme can catalyze the activation (to their CoA thioesters) of some of the side-chain precursors required in these two fungi for the production of several penicillins. All these data are reported here for the first time.     

A comparative study of hydrolysis and transglycosylation activities of fungal β-glucosidases

β-glucosidases (BGs) from Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and investigated for their (simultaneous) hydrolytic and transglycosylation activity in samples with high concentrations of either cellobiose or glucose. The rate of the hydrolytic process (which converts one cellobiose to two glucose molecules) shows a maximum around 10–15 mM cellobiose and decreases with further increase in the concentration of substrate. At the highest investigated concentration (100 mM cellobiose), the hydrolytic activity for the different enzymes ranged from 10% to 55% of the maximum value. This decline in hydrolysis was essentially compensated by increased transglycosylation (which converts two cellobiose to one glucose and one trisaccharide). Hence, it was concluded that the hydrolytic slowdown at high substrate concentrations solely relies on an increased flow through the transglycosylation pathway and not an inhibition that delays the catalytic cycle. Transglycosylation was also detected at high product (glucose) concentrations, but in this case, it was not a major cause for the slowdown in hydrolysis. The experimental data was modeled to obtain kinetic parameters for both hydrolysis and transglycosylation. These parameters were subsequently used in calculations that quantified the negative effects on BG activity of respectively transglycosylation and product inhibition. The kinetic parameters and the mathematical method presented here allow estimation of these effects, and we suggest that this may be useful for the evaluation of BGs for industrial use.     

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.     

The structure and function of acid proteases. V. Comparative studies on the specific inhibition of acid proteases by diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(p-nitrophenoxy) propane and pepstatin.

Comparative studies have been made on the effects of diazoacetyl-DL-norleucine methyl ester (DAN), 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and pepstatin on acid proteases, including those from Acrocylindrium sp., Aspergillus niger, Aspergillus saitoi, Mucor pusillus, Paecilomyces varioti, Rhizopus chinensis, and Trametes sanguinea, and also porcine pepsin [EC 3.4.23.1] and calf rennin [EC 3.4.23.4] for comparative purposes. These enzymes were rapidly inactivated at similar rates and in 1:1 stiochiometry by reaction with DAN in the presence of cupric ions. The pH profiles of inactivation of these enzymes were similar and had optima at pH 5.5 to 6. They were also inactivated at similar rates by reaction with EPNP, with concomitant incorporation of nearly 2 EPNP molecules per molecule of enzyme. The pH profiles of inactivation were again similar and maximal inactivation was observed at around pH 3 to 4. Some of the EPNP-inactivated enzymes were treated with DAN and shown still to retain reactivity toward DAN. All these enzymes were inhibited strongly by pepstatin, and the reactions of DAN and EPNP with them were also markedly inhibited by prior treatment with pepstatin. These results indicate that the active sites of these enzymes are quite similar and that they presumably have at least two essential carboxyl groups at the active site in common, one reactive with DAN in the presence of cupric ions and the other reactive with EPNP, as has already been demonstrated for porcine pepsin and calf rennin. Pepstatin appears to bind at least part of the active site of each enzyme in a simmilar manner.     

Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.     

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.     

Kinetically controlled drug resistance: how Penicillium brevicompactum survives mycophenolic acid

The filamentous fungus Penicillium brevicompactum produces the immunosuppressive drug mycophenolic acid (MPA), which is a potent inhibitor of eukaryotic IMP dehydrogenases (IMPDHs). IMPDH catalyzes the conversion of IMP to XMP via a covalent enzyme intermediate, E-XMP*; MPA inhibits by trapping E-XMP*. P. brevicompactum (Pb) contains two MPA-resistant IMPDHs, PbIMPDH-A and PbIMPDH-B, which are 17- and 10(3)-fold more resistant to MPA than typically observed. Surprisingly, the active sites of these resistant enzymes are essentially identical to those of MPA-sensitive enzymes, so the mechanistic basis of resistance is not apparent. Here, we show that, unlike MPA-sensitive IMPDHs, formation of E-XMP* is rate-limiting for both PbIMPDH-A and PbIMPDH-B. Therefore, MPA resistance derives from the failure to accumulate the drug-sensitive intermediate.     

Novel Aspergillus hemicellulases enhance performance of commercial cellulases in lignocellulose hydrolysis

A novel hemicellulase-producing fungal strain was isolated from a local soil sample. The organism is identified as Aspergillus fumigatus based on ribosomal RNA analyses. The Aspergillus strain, designated as 2NB, produces both enzymes acting on xylan backbone (xylanase and β-xylosidase), and those acting on side chains (or accessory enzymes) notably α-arabinofuranosidase and acetyl-xylan esterase. The Asperigillus hemicellulases are characterized as having relatively low xylanase and β-xylosidase activities but high side chain removal activities. The activity ratio of side-chain acting enzymes to xylanase is higher than that of the Multifect enzyme, a commercial hemicellulase product. The potential of the novel hemicellulases in lignocelluloses bioprocessing was demonstrated with alkaline-pretreated switchgrass as lignocellulose substrate with hemicellulase supplemented with a ratio of xylanase activity to filter paper unit of 2:1. Supplement of Aspergillus hemicellulases to commercial cellulases significantly enhanced the hydrolysis of lignocellulose, achieving a 94% hydrolysis yield based on reducing sugar measurement, compared to 60% when no hemicellulase or 75% when Multifect enzyme was used under otherwise identical conditions. The significant improvement resulting from supplementing a hemicellulase mix with high side-chain removal activities suggests the importance of accessory hemicellulases in lignocellulose processing.     

Activation parameters for the cleavage reaction of cytidine-2',3'-monophosphate catalyzed by Penicillium brevicompactum and Aspergillus clavatus RNAses]

On the basis of coincidence of all activation parameters (E, deltaH*, deltaF* and deltaS*) for the reaction of cleavage of cytidine-2';3'-monophosphate catalyzed by "acid" (pH-optimum 4.7) nonspecific RNAses from Aspergillus clavatus and Penicillium brevicompactum (EC 3. 1. 4. 23) it has been proposed that the mechanism of action on this reaction stage (hydrolysis) for both enzymes is equal.