Production of Aspergillus niger β-mannosidase in Pichia pastoris

β-Mannosidase (EC 3.2.1.25) is an exoglycosidase specific for the hydrolysis of terminal β-linked mannoside in various sugar chains. cDNA corresponding to the β-mannosidase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Pichia pastoris. The β-mannosidase gene contains an open reading frame which encodes the protein with 933 amino acid residues. The wild type and recombinant proteins were purified to apparent homogeneity and biochemically characterized (K(M) 0.28 and 0.44mmol/l for p-nitrophenyl β-d-mannopyranoside, pI 4.2 and 4.0, and their pH optima were at pH 4.5 and 5.5 and 65°C, respectively).     

Optimization of β-glucosidase production from Aspergillus niger

本研究对Aspergillus niger Glu05生产β-葡萄糖苷酶的培养基组分及培养条件进行了优化.优化后的培养基组成和培养条件分别为:麸皮4％,tryptone 4％,1μmol MnSO4,1μmol NaCl,KH2PO40.2％,oH自然,摇床转速250 r/min,培养温度30℃,培养周期5d.优化后发酵液中酶活力达到44.11 IU/mL,与初始的产酶水平32.87 IU/mL相比,提高了36％.     

Fed-batch biotransformation of β-ionone by Aspergillus niger

Aspergillus niger IFO 8541 was found to be an efficient biocatalyst for the biotransformation of -ionone into hydroxy and oxo derivatives. The reaction had to be carried out with an inoculum made of about 4 × 10⁷ fresh spores/l and with a preliminary growth period giving at least 3 g/l biomass. The fungus developed in the form of pellets when cultivated as free mycelium; entrapment of the microorganism in calcium alginate beads was an efficient way to mimic this feature in an aerated, stirred bioreactor. The biotransformation was carried out using a fed-batch mode of operation involving sequential precursor addition. -Ionone stopped the fungal growth and was converted into metabolites only when the carbon source remained present in the medium; it was fully oxidized after sucrose exhaustion. These conditions allowed recovery of about 2.5 g/l aroma compounds after 230 h cultivation with a molar yield close to 100%.     

Formation of β-Fructosyl Compounds of Pyridoxine in Growing Culture of Aspergillus niger

Two pyridoxine compounds were found to be formed in a culture filtrate of Aspergillus niger and A. sydowi, when grown in a medium containing sucrose and pyridoxine. Each of the two compounds I and II was obtained as a white powdered preparation by preparative paper chromatography, gel filtration on Toyopearl HW-40S and Sephadex G-10 columns, DEAE-cellulose column chromatography, and lyophilization. Compounds I and II were identified as 5?-O-(β-D-fructofuranosyl)-pyridoxine and 5?-O-(β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl]-pyridoxine, on the basis of the various experimental results, viz., elementary analyses, UV, ¹H-, and ¹³C-NMR spectra, products by hydrolysis with acid and yeast β-D-fructofuranosidase, migration on paper electrophoresis, and Gibbs reaction in the presence and absence of boric acid. Levansucrase from Microbacterium laevaniformans and yeast β-D-fructofuranosidase did not catalyze the β-D-fructofuranosyl transfer from sucrose to pyridoxine to give rise to β-D-fructofuranosyl-pyridoxine.     

Simple inheritance of variations in male mouse kidney β-glucuronidase activity

Kidney -glucuronidase activity in C57BL/K1 and DBA/2/K1 male mice differs about ten times, C57 giving low and DBA high values. F₁ males have intermediate activities. Male liver as well as female kidney and liver invariably give low values. The most likely interpretation for this difference between the two strains is a genetic variation at a single locus.This work was supported by the Nilsson-Ehle fund, the Marcus Borgström fund, and the Hierta memorial fund.     

Plant endogenous β-glucuronidase activity: how to avoid interference with the use of theE. coli β-glucuronidase as a reporter gene in transgenic plants

We have detected a plant β-glucuronidase activity, present in several tissues and organs of plant species belonging to different families. The fluorimetric β-glucuronidase assay was used to partially characterize this activity in post-ribosomal supernatants of tobacco leaves. The tobacco activity is very stable at low temperatures, but quickly inactivated above 45°C. It is relatively resistant to proteases and insensitive to-SH group reagents and to ionic conditions. It does not require, nor is it inhibited by, divalent cations. Although these properties are shared by theEscherichia coli β-glucuronidase, the two activities can be distinguished by: (i) their different sensitivity to the specific inhibitor saccharic acid-1,4-lactone; (ii) their different thermal stability (iii) their different pH optima (5.0 for the plant activity and close to neutral for the bacterial enzyme). Therefore, under appropriate experimental conditions, it should be possible to assay theE. coli β-glucuronidase in transgenic plants without interference from the endogenous plant activity.     

Non-destructive assay systems for detection of β-glucuronidase activity in higher plants

β-glucuronidase (GUS) can be qualitatively assayed in seedlings and fully grown plants without injury or irreversible damage by short term incubations in X-gluc or by spraying 4-MUG.     

Synthesis of alkyl β-glucosides from cellobiose with Aspergillus niger β-glucosidase II

An extracellular -glucosidase II of Aspergillus niger catalyzed the synthesis of methyl -glucoside and ethyl -glucoside with 5.0% (v/v) cellobiose as glucosyl donor in a biphasic media containing 20% (v/v) methanol and 30% (v/v) ethanol, respectively. The maximum yield of methyl -glucoside and ethyl -glucoside was 83% (mol/mol; 12 mg/ ml) and 53% (mol/mol; 5.5 mg/ml), based on cellobiose consumed. © Rapid Science Ltd. 1998     

Over-production of β-glucosidase by Aspergillus niger mutant from lignocellulosic biomass

The maximum yield of -glucosidase by A. niger KK2 mutant, grown on the basal medium for 7 days, was 514 I U g^–1 ground rice straw, and was about twice those obtained from wheat straw or bran by previous researchers. Optimal activity of -glucosidase was at 60–70 °C and pH 4.8.     

Fractionation of β-Glucosidases and Related Extracellular Enzymes from Aspergillus niger

Industrial concentrates from Aspergillus niger culture filtrates were fractionated by ion-exchange and adsorption chromatography. Several other types of hydrolases were completely removed. Eight partially purified components were obtained. Using specific activity as an estimate of purification, one aryl-β-glucosidase was purified 35-fold. Another component showed 147-fold purification using a viscosimetric assay with carboxymethylcellulose as substrate. The aryl-β-glucosidase was distinctly more thermolabile than the carboxymethylcellulase.     

Enhancing the activity and thermostability of thermostable β-glucosidase from a marine Aspergillus niger at high salinity

To evaluate the effect of salinity on the catalyzing ability of β-glucosidase in the marine fungus Aspergillus niger, the thermodynamic parameters of the β-glucosidase were investigated at different salinities. At the optimum salinity of 6% NaCl (w/v) solution, the optimum temperature and pH of the β-glucosidase activity was 66 °C and 5.0, respectively. Under these conditions, the β-glucosidase activity increased 1.46 fold. The half-life of denaturation in 6% NaCl (w/v) solution was approximately twice as long as that in NaCl free solution. The Gibb's free energy for denaturation, ΔG, was 2 kJ/mol higher in 6% NaCl (w/v) solution than in NaCl free solution. The melting point (68.51 °C) in 6% NaCl (w/v) solution was 1.71 °C higher than that (66.80 °C) in NaCl free solution. Similarly, the activity and thermostability of the pure β-glucosidase increased remarkably at high salinity. The thermostable β-glucosidase, of which the activity and the thermostability are remarkably enhanced at high salinity, is valuable for industrial hydrolyzation of cellulose in high salinity environments.     

Facile production of Aspergillus niger α-N-acetylgalactosaminidase in yeast

α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC.3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the α-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae. The α-GalNAc-ase gene contains an open reading frame which encodes a protein of 487 amino acid residues. The molecular mass of the mature protein deduced from the amino acid sequence of this reading frame is 54 kDa. The recombinant protein was purified to apparent homogeneity and biochemically characterized (pI4.4, K(M) 0.56 mmol/l for 2-nitrophenyl 2-acetamido-2-deoxy-α-d-galactopyranoside, and optimum enzyme activity was achieved at pH2.0-2.4 and 50-55°C). Its molecular weight was determined by analytical ultracentrifuge measurement and dynamic light scattering. Our experiments confirmed that the recombinant α-GalNAc-ase exists as two distinct species (70 and 130 kDa) compared to its native form, which is purely monomeric. N-Glycosylation was confirmed at six of the eight potential N-glycosylation sites in both wild type and recombinant α-GalNAc-ase.     

Mycelia-associated β-galactosidase activity in microbial pellets of Aspergillus and Penicillium strains

Pellet formation and production of mycelia-associated -galactosidase were investigated in 15 Aspergillus and Penicillium strains. Mycelia-associated enzyme activity was measured in sonicated homogenates. The properties of the mycelia-associated -galactosidase of A. phoenicis QM 329 was investigated. The pH optimum of the mycelia-associated enzyme was 4.0. The optimum temperature under assay conditions was 70°C and the optimum temperature for repeated lactose hydrolysis was 60°C. Repeated batch hydrolysis of lactose was made with pellets from five Aspergillus strains. A. phoenicis QM 329 showed the least enzyme leakage from the pellets during hydrolysis. From repeated lactose hydrolysis experiments it was estimated that 50% of the mycelia-associated -galactosidase activity remained after 1300 h. Correspondence to: F. Tjerneld     

Influence of Environmental Conditions on Hyphal Morphology in Pellets of Aspergillus niger: Role of β-N-Acetyl-D-Glucosaminidase

The influence of modifications of the environmental conditions of growth on β-N-acetyl-D-glucosaminidase (EC 3.2.1.30) activity and on hyphal morphological patterns in pellets of Aspergillus niger was studied. It was found that changes in the degree of branching and, to a lesser extent, in the number of bulbous cells were directly related to the activity of the enzyme. Nevertheless, since β-N-acetyl-D-glucosaminidase is not the only enzyme involved in the lytic potential of the fungus, these findings do not exclude the possibility that other enzymes may be involved. Received: 30 December 1998 / Accepted: 9 March 1999     

Synthesis of cello-oligosaccharides from cellobiose with β-glucosidase II from Aspergillus niger

An extracellular -glucosidase II of Aspergillus niger catalysed the synthesis of cello-oligosaccharides from cellobiose (15%, w/v). The enzyme was stable at and below 4°C for at least 230 days and also stable at 30°C with the presence of 2.0% (w/v) cellobiose. The maximum yield of cello-oligosaccharides was about 30% (mol/mol), based on cellobiose (130 mg/mL) consumed. © Rapid Science Ltd. 1998     

The locations of cathepsin activity and β-glucuronidase in the Guerin T8 tumour

Tumour homogenate fractions, isolated by differential centrifugation, were subfractionated by density-gradient centrifugation. Biochemical and electron microscopic analyses revealed that beta-glucuronidase and cathepsin activity were associated with a class (possibly two) of lysosomal particles of density greater than those of mitochondria and the endoplasmic reticulum. Lysosomes sedimented by low g forces were vacuolar, electron-dense, delineated by a unit membrane and about 0.2mum in diameter. beta-Glucuronidase was also apparently associated with ribosomes whereas cathepsin was bound in part to the endoplasmic reticulum. Catalase and glucose 6-phosphatase possessed slightly different density-gradient sedimentation profiles.