Biochemical and catalytic properties of two intracellular β-glucosidases from the fungus Penicillium decumbens active on flavonoid glucosides

In the presence of rutin as sole carbon source, Penicillium decumbens produces two intracellular β-glucosidases named G_I and G_II, with molecular masses of 56,000 and 460,000 Da, respectively. The two proteins have been purified to homogeneity. G_I and G_II composed of two and four equal sub-units, respectively and displayed optimal activity at pH 7.0 and temperature 65–75 °C. Both β-glucosidases were competitively inhibited by glucose and glucono-δ-lactone. G_I and G_II exhibited broad substrate specificity, since they hydrolyzed a range of (1,3)-, (1,4)- and (1,6)-β-glucosides as well as aryl β-glucosides. Determination of k_cat/K_m revealed that G_II hydrolyzed 3–8 times more efficiently the above-mentioned substrates. The ability of G_I and G_II to deglycosylate various flavonoid glycosides was also investigated. Both enzymes were active against flavonoids glycosylated at the 7 position but G_II hydrolyzed them 5 times more efficiently than G_I. Of the flavanols tested, both enzymes were incapable of hydrolyzing quercetrin and kaempferol-3-glucoside. The main difference between G_I and G_II as far as the hydrolysis of flavanols is concerned, was the ability of G_II to hydrolyze the quercetin-3-glucoside.     

Septum formation in Aspergillus nidulans

Filamentous fungi form multicellular hyphae that are partitioned by septa. In A. nidulans, septum formation requires the assembly of a septal band following the completion of mitosis. Recent observations show that this band is a dynamic structure composed of actin, a septin and a formin. In addition, assembly is dependent upon a conserved protein kinase cascade that regulates mitotic exit and septation in yeast. Hyphal differentiation may reflect the regulation of this cascade by cyclin-dependent kinase activity. In this review, the dynamics and regulation underlying the assembly of the septal band are discussed.     

At least four regulatory genes control sulphur metabolite repression in Aspergillus nidulans

The Staphylococcus aureus chromosomal gene pcrA, identified by mutations, such as pcrA3, that affect plasmid pT181 replication, has been cloned and sequenced. The pcrA gene encodes a protein with significant similarity (40% identity) to two Escherichia coli helicases: the helicase II encoded by the uvrD gene and the Rep helicase. The pcrA3 mutation was found to be a C to T transition leading to a threonine to isoleucine substitution at amino acid residue 61 of the protein. The pcrA gene seems to belong to an operon containing at least one other gene, tentatively named pcrB, upstream from pcrA. The PcrA protein was shown to be essential for cell viability and overproduction has deleterious effects on the host and plasmid replication.     

Thermostability of β-xylosidase from Aspergillus sydowii MG49

Heating of Aspergillus β-xylosidase at 85°C ± 1°C and pH 5.5–6.0 (optimum for activity), causes irreversible, covalent thermoinactivation of the enzyme, involving oxidation of the thiol groups that are required for catalysis. Exogenous addition of cysteine, DTT, GSH and mercaptoethanol stabilizes the enzyme by extending its half-life. A similar effect is also exhibited by bivalent cations like Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺and Zn²⁺ while, on the other hand Cu²⁺ accelerates thermoinactivation. Chemical modification of crude β-xylosidase with cross-linking agents like glutaraldehyde or covalent immobilization to a nonspecific protein like gelatin and BSA also enhances enzyme thermostability. These results suggest that addition of thiols and bivalent metal ions to a crude β-xylosidase preparation or immobilization/chemical modification enhances its thermal stability, thus preventing loss of catalytic activity at elevated temperatures.     

The Aspergillus niger carbon catabolite repressor encoding gene, creA

In order to undertake a comparative analysis of carbon catabolite repression in two Aspergillus species, the creA gene has been isolated from A. niger by cross hybridization, using the cloned A. nidulans gene. The A. niger gene has been shown to be functional in A. nidulans by heterologous complementation of the creA204 mutation of A. nidulans. Overall, the genes show 90% sequence similarity (82% identity) at the amino acid (aa) level. There were some striking similarities between the aa sequences encoded by the two fungal creA genes and two genes involved in carbon catabolite repression in Saccharomyces cerevisiae. The zinc-finger regions showed 96% similarity (84% identity) with the zinc-finger region of the MIG1 gene of S. cerevisiae. The CREA protein contains a stretch of 42 aa that is identical in A. niger and A. nidulans, and these show 81% similarity (33% identity) with a region of the S. cerevisiae RGR1 gene.     

Complementation among developmental mutants in Aspergillus nidulans

Summary In heterokaryons between pairs of aconidial mutants of Aspergillus nidulans one of the component strains usually shows a striking prevalance in the contribution to the conidial crop. By assuming that the prevailing strain is blocked earlier and the succumbent one later in the process of differentiation, a series of mutations can be arranged in a consistent order.Some mutant strains do not fit the scheme exactly but show a general tendency to be succumbent to early mutants and prevalent over the late ones. A criterion for arraying genes involved in differentiation according to the order of their physiological action is proposed.     

Purification and properties of two β-glucosidases isolated from Aspergillus niger

The cellulase complex of the fungus Aspergillus niger (strain CBS 554.65 = ATCC 16 888) was fractionated by gel filtration yielding six pronounced peaks. Only proteins from the fraction corresponding to the first peak (96 kDa) showed β-glucosidase activity vs. the substrate 4-nitrophenyl-β-D-glucopyranoside (pNPG). These proteins have been fractionated by chromatofocusing, yielding two β-glucosidases (I and II) which are shown to be homogeneous in isoelectric focusing experiments (pI = 4.6 and 3.8, respectively). Kinetic experiments with pNPG, MU-glucopyranoside and cellobiose revealed that both types of β-glucosidases behave like aryl-β-glucosidases. β-Glucosidase-I acting on pNPG exhibits a split kinetics characterized by high and low substrateconcentration kinetics which are differentiated by different values of V and of K_m. In addition, β-glucosidase-II is shown to be an exo-glucohydrolase as deduced from experiments with MU-cellobiopyranoside. Experimental features should be emphasized; usual soft-gel ion-exchange materials did not work in the chromatofocusing separation of the two β-glucosidases, in contrast to the 10μ-Si 500 = DEAE exchange material (Serva) typically used in HPLC-experiments. Furthermore, protein content determinations based on different procedures yielded widely differing values.     

Effect of carbon source on the biochemical properties of β-xylosidases produced by Aspergillus versicolor

The filamentous fungus Aspergillus versicolor produced large amounts of mycelial β-xylosidase activity when grown on xylan or xylose as the only carbon source. The presence of glucose drastically decreased the level of β-xylosidase activity, while cycloheximide prevented the induction of the enzymes by xylan or xylose. The β-xylosidases induced by xylose or xylan were purified by a simple protocol involving DEAE-cellulose chromatography and ammonium sulphate precipitation. The purified enzymes were acidic proteins, with carbohydrate contents of 21% for that induced by xylose, and 47% for that induced by xylan. Their apparent molecular masses, estimated by gel filtration, and optimal temperatures for β-xylosidase activities, were about 60 and 100 kDa, and 40 and 45 °C, respectively, for the enzymes induced by xylose and xylan. Xylose-induced β-xylosidase exhibited an optimum pH of 6.0, while that of the xylan-induced enzyme was 5.5. Both purified β-xylosidases exhibited also β-galactosidase, β-glucosidase and -arabinosidase activities. In addition to synthetic substrates, the enzymes hydrolysed xylobiose and xylotriose, suggesting a physiological role. K_M values for p-nitrophenyl β- -xylopyranoside were 0.32 mM, for the xylose-induced β-xylosidase, and 0.19 mM for the xylan-induced one. Xylose competitively inhibited both β-xylosidases, with K_I values of 5.3 and 2.0 mM, for the enzymes induced by xylose or xylan, respectively.     

sodC Is an α-COP-Related Gene Which Is Essential for Establishing and Maintaining Polarized Growth in Aspergillus nidulans

Strains of Aspergillus nidulans carrying the conditional-lethal mutation sodVIC1 (stabilization of disomy) are defective in nuclear division and hyphal extension. The mutation affects both the establishment and maintenance of polar growth, since mutant spores do not germinate at restrictive temperature and preexisting hyphae stop growing upon upshift. The defect is reversible within the first 3-4 h at restrictive temperature but longer periods of incubation are lethal due to cell lysis and morphological abnormalities. There is no evidence for a specific cell cycle lesion, suggesting the existence of a feedback mechanism whereby hyphal extension is coordinated with nuclear partitioning. The sodVIC gene has been cloned from a chromosome VI-specific cosmid library and its product exhibits strong homology to the alpha-COP subunit of the coatomer complex involved in the secretory pathway in yeast and higher organisms. Molecular disruption of the gene is lethal, indicating that SodVIC is essential for growth in A. nidulans.     

Growth-mediated metabolic activation of promutagens in Aspergillus nidulans

7 procarcinogens belonging to different chemical classes (nitrosamines, hydrazoalkanes, oxazaphosphorines and aromatic amines) were tested in A. nidulans for the induction of point mutations with two genetic systems (8-AG resistance and induction of methionine suppressors).

Dimethylnitrosamine, diethylnitrosamine, nitrosomorpoline, dimethyl-hydrazine, procarbazine and cyclophosphamide gave positive results with a good dose—effect relationship in the growth-mediated assay, whereas they gave negative or borderline positive results in the plate incorporation assay. 2-Aminoanthracene was completely negative with both experimental procedures.

DMN, DEN and NM were also tested for their ability to induce somatic segregation: all were positive when assayed in the growth-mediated assay.     

Isolation and some properties of two extracellular β-glucosidases from Trichosporon adeninovorans

The yeast Trichosporon adeninovorans secretes two multiple forms of β-glucosidase at a high rate if grown in a medium containing cellobiose. Following mutagenesis a mutant strain resistant to 2-deoxy-D-glucose was selected. This strain produced more β-glucosidase activity and had acquired a strong resistance against repression by glucose. The β-glucosidases were separated one from each other by chromatography on hydroxylapatite and by gel filtration. Both enzymes have similar properties. The optimal temperature for their activity was 60 to 63°C and the enzymes displayed highest activity at pH of 4.5. The molecular weight of β-glucosidase I was found to be 570,000 and that for β-glucosidase II was 525,000. The K_m value for cellobiose was determined to be 4.1 mM for β-glucosidase I and 3.0 mM for β-glucosidase II.     

Regulation of homocysteine metabolizing enzymes in Aspergillus nidulans

Summary Mutants of Aspergillus nidulans blocked in the main pathway of cysteine synthesis show an elevated level of the enzymes involved in the synthesis of cysteine from homocysteine i.e. cystathionine -synthase and -cystathionase and a depressed level of homocysteine methyltransferase. This results in a considerable change in the sulfur amino acids pool as compared to the wild type. Upon addition of cysteine to the growth medium the first two enzymes are repressed while the level of the third one increases. These data indicate that the two diverging pathways of homocysteine metabolism are anti-coordinately regulated.