Glucoamylase of Aspergillus niger

Aspergillus niger produces two extracellular glucoamylases (GAI of Mr 85 300 and GAII of Mr 77 600) separable on DEAE-cellulose. The enzymes differes in electrophoretic mobility, thermostability and substrate specificity. The GAI/GAII ratio depends on the concentration and form of nitrogen (nitrate or ammonium) in the culture medium. Proteinase VIII from Bacillus subtilis converts GAI to a form showing properties similar to those of GAII. Possible proteolytic degradation of GAI to GAII by Asp. niger endogenous proteinase(s) is suggested.     

Aspergillus niger sulfhydryl oxidase

A procedure for the isolation of a sulfhydryl oxidase from an Aspergillus niger cell suspension involved three major steps and yielded enzyme preparations exhibiting a single but diffuse protein-containing zone when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight estimated to be 53,000. Sedimentation equilibrium experiments indicated a native molecular weight of 106,000. Analyses for sugar residues showed that the enzyme is a glycoprotein, containing 20.3% neutral hexose and 1.9% aminohexose by weight. This enzyme catalyzed the conversion of reduced glutathione (GSH) to its disulfide form, with concomitant consumption of O2 and release of H2O2. The ratio of GSH consumed to H2O2 produced was determined to be 2:1. At 25 degrees C, the optimum pH for the oxidation of GSH was 5.5. Under these conditions, the enzyme had a Michaelis constant of 0.3 mM for GSH. Other low molecular weight thiol compounds (cysteine, dithiothreitol, and 2-mercaptoethanol) were also oxidized, but the Michaelis constants for these substrates were substantially higher than that for GSH under identical conditions of temperature and pH. The rate of reactivation of reductively denatured ribonuclease A was enhanced by the presence of sulfhydryl oxidase, indicating that the latter is capable of oxidizing protein-associated thiol groups. The UV-visible spectrum of sulfhydryl oxidase solution had absorbance maxima at 274, 364.5, and 442.5 nm and was otherwise characteristic of the spectra of known flavoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biotransformation of dopamine to norlaudanosoline by Aspergillus niger

Norlaundanosoline is a key intermediate in the synthesis of the benzylisoquinoline alkaloids providing the upper isoquinoline portion of the morphinan skeleton. This study evaluates the feasibility of using Aspergillus niger as an in situ biotransformation system to produce norlaudanosoline from dopamine. A. niger was chosen because monoamine oxidase can be readily induced in this organism. Monoamine oxidase catalyzes the conversion of dopamine to 3,4-dihydroxyphenylacetaldehyde. In the presence of dopamine, this aldehyde will then undergo a spontaneous Picket-Spengler condensation to form norlaudanosoline. Fermentation condition to form norlaudanosoline. Fermentation conditions were optimized for the production monoamine oxidase by using a two-stage process consisting of a growth stage and an induction stage. pH control was found to be important, and at pH 4.5 dopamine accumulation in the cells was high as was the level of monoamine oxidase. With pH control at 4.5, up to 21% of the cellular dopamine was converted to norlaudanosoline. It is proposed that with further protein engineering improvements, this system may prove suitable for the in situ bio-transformation of dopamine to norlaudanosoline.     

Rassenumwandlung bei Aspergillus niger

Zusammenfassung Es wurde versucht, zwei physiologisch durch die Oxalsäurebildung und die dadurch beeinflußte Richtung der Autolyse verschiedene Rassen von Aspergillus niger ineinander überzuführen. Dies gelang durch Einwirkung von Trocknen bzw. Erhitzen der Sporen und durch Passage über Tannin- und Oxalsäurelösung.Der Übergang der Rasse Göttingen neu (viel Oxalsäure, saure, helle Autolyse) in die Rasse Göttingen alt (wenig Oxalsäure, neutrale bzw. alkalische, dunkle Autolyse) ging bedeutend leichter als umgekehrt.Durch Oxalsäurepassage wird die Rasse Göttingen alt stärker in der Richtung der sauren, die Rasse Göttingen neu stärker in der Richtung der neutralen Autolyse beeinflußt.Die jeweiligen Veränderungen erwiesen sich drei bis acht Generationen, soweit untersucht, konstant.Mit Hilfe ähnlicher Methoden dürften die Veränderungen bei diesem Pilz der experimentellen Analyse zugänglich werden.Kurzer Auszug aus der Dissertation von Paul Hugo Kreutzmann, Untersuchungen über die Variabilität von Aspergillus niger. Göttingen 1935.     

Yellow Pigments of Aspergillus niger and Aspergillus awamori

Alkaline degradation of Aurasperone A, C₃₂H₂₆O₁₀, gave a binaphthyl (IIa), m.p. 255°C and acetone. (IIa) afforded a tetraacetate (IIb), C₃₂H₃₀O₁₂ m.p. 219°C and a tetramethyl ether (IId), C₂₈H₃₀O₈, m.p. 188°C. These facts along with the NMR spectra of aurasperone A and (IIb) confirm that aurasperone A is a dimeric 2-methyl-5-hydroxy-6,8-dimethoxy-4H-naphtho[2,3-b]pyran-4-one with asymmetric C-C linkage (7-10′ or 9-10′). The ether (IId) is not identical with 1,1′ ,3,3′ ?6,6′ ,8,8′-octamethoxy-4,4′-binaphthyl. Thus, it follows that (IId) is a 2,4′-binaphthyl and hence aurasperone A is 2,2′-dimethyl-5,5′- dihydroxy-6,6′,8,8′-tetrahydroxy-7,10′-bi[4H-naphtho[2,3-b]pyran-4-one] (I).     

Zum Ertragsgesetz bei Aspergillus niger

Zusammenfassung A. Einleitend wird kritisch zu den Grundsätzen der bisherigen Untersuchungsverfahren des Ertragsgesetzes Stellung genommen; insbesondere geht daraus hervor, daß das Ausprobieren von analytischen Funktionen für eine mathematisch-physiologische Ertragsgesetztheorie ohne Bedeutung ist und nicht als mathematische Behandlung des Ertragsgesetzes angesehen werden darf.B. I. Es wird an Hand experimenteller Ergebnisse gezeigt daß eine für die Ertragskurve charakteristische Größe, nämlich die Anstiegstangente, bei Aspergillus niger für Stickstoff weitgehend unabhängig von der Phosphorsäuregabe, ist, und daß auch die reziproke Beziehung zu bestehen scheint. Die Versuche zeigen ferner, daß die Stickstoffertragskurve bei hohen Phosphorsäuregaben einen Wendepunkt ganz zu Beginn des aufsteigenden Astes hat.B. II. Es wird für den gleichen Organismus gezeigt, daß die Abszissenlage des Maximums der Stickstoffertragskuve mit der Phosphorsäuregabe veränderlich ist in der Weise, daß diese Abszisse zunächst mit steigender Phosphorsäuregabe wächst (Rechtläufigkeit der Maximum-verschliebung), später wieder abnimmt (Rückläufigkeit). Hieran anschließende theoretische Erörterungen legen es nahe, dieses Verhalten als Wettstreit zwischen der Nährstoffkonzentration und der sogenannten Minimumfaktor-Eigenschaft der veränderlichen Nährstoffgabe zu deuten. Das Verhalten der Anstiegstangente ist bei diesen Erörterungen mit verwertet.B. III. Es werden Häufigkeitsverteilungskurven der Einzelgewichte von je 100 unter gleichen Bedingungen gewachsener Aspergillus-Mycelien nach verschiedener Wachstumszeit vorgeführt, nach denen es sich empfiehlt, die Fehlerausgleichsrechnung auf biologische Fragen nur formal und mit äußerster Vorsicht anzuwenden.     

产糖化酶黑曲霉的固定化研究

采用多孔聚酯材料作为固定化载体,考察并比较了载体吸附固定化黑曲霉菌丝细胞的条件,当菌丝体细胞与载体预培养的条件为pH值5.0、孢子浓度为105个/ml、固液比为1/75时,有利于菌丝体的生长、吸附固定及发酵产酶.在产糖化酶的发酵过程中,与游离菌丝体细胞相比,发酵过程持续产酶时间有一定程度的延长,产糖化酶活力始终高于游离菌丝体.     

Directed evolution of Aspergillus niger glucoamylase to increase thermostability

Using directed evolution and site‐directed mutagenesis, we have isolated a highly thermostable variant of Aspergillus niger glucoamylase (GA), designated CR2‐1 . CR2‐1 includes the previously described mutations Asn20Cys and Ala27Cys (forming a new disulfide bond), Ser30Pro, Thr62Ala, Ser119Pro, Gly137Ala, Thr290Ala, His391Tyr and Ser436Pro. In addition, CR2‐1 includes several new putative thermostable mutations, Val59Ala, Val88Ile, Ser211Pro, Asp293Ala, Thr390Ser, Tyr402Phe and Glu408Lys, identified by directed evolution. CR2‐1 GA has a catalytic efficiency (k_cat/K_m) at 35°C and a specific activity at 50°C similar to that of wild‐type GA. Irreversible inactivation tests indicated that CR2‐1 increases the free energy of thermoinactivation at 80°C by 10 kJ mol^?1 compared with that of wild‐type GA. Thus, CR2‐1 is more thermostable (by 5 kJ mol^?1 at 80°C) than the most thermostable A. niger GA variant previously described, THS8 . In addition, Val59Ala and Glu408Lys were shown to individually increase the thermostability in GA variants by 1 and 2 kJ mol^?1, respectively, at 80°C.     

Cellulase Enzyme from Aspergillus niger

Cellulase enzyme was produced by a selected strain of Aspergillus niger isolated from deteriorated wood and grown on different carbon sources. Filter paper gave the highest yield, followed by carboxymethyl cellulose (CMC). Cellobiose as well as glucose gave a low yield, while the yield from lactose was negligible. The concentration of filter paper cellulose that induced the maximum yield of the enzyme was 1%. Both soluble cellulose (CMC) and cotton cellulose treated with phosphoric acid (swollen) were easily hydrolyzed by cellulase; an increase in cellulase concentration lead to more hydrolysis of CMC and gave linearity in the reaction velocity. At certain concentrations of the enzyme, increase in CMC concentration, (up to 1%) resulted in more reducing sugar. Beyond this point no more hydrolysis occur.