Purification and properties of one component of acid phosphatase produced by Aspergillus niger.

One component, the i form, of acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) produced by Aspergillus niger was purified from the mycelial extract. The purified enzyme was homogenous on Sephadex G-200 gel filtration, disc electrophoresis and heat inactivation. The purified enzyme was studied and the following results were obtained: 1. The enzyme catalyzed the hydrolysis of a wide variety of phosphomonoesters, but not that of bis(p-nitrophenyl)phosphate, adenosine 3',5'-cyclic monophosphate, fructose 1,6-diphosphate, adenosine 5'-diphosphate or adenosine 5'-triphosphate. 2. Fluoride, orthophosphate, arsenate, borate, molybdate and (+)-tartrate acted as inhibitors. This enzyme was inactivated by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, and was not affected by p-chloromercuribenzoate, N-acetylimidazole, p-diazobenzenesulfonic acid and tetranitromethane. From these results, tryptophan was estimated to play an important role in the enzyme activity. 3. The apparent molecular weight was 310000 by Sephadex G-200 gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggested that the molecular weight of the subunit was approximately 89000. 4. The purified enzyme contained 29% carbohydrate consisting of glucosamine, mannose and galactose. The amino acid composition of this enzyme was not specific compared with other known acid phosphatases.     

Purification and some properties of NAD-degrading purine nucleosidase from Aspergillus niger.

An enzyme which degrades NAD at the adenine-ribose linkage has been purified from the mycelial extract of Aspergillus niger. NADP, deamido-NAD, and purine nucleosides and nucleotides were also susceptible to the hydrolytic cleavage. Pyrimidine- and nicotinamide-ribose linkages were not attacked. The substrate specificity showed that the enzyme may be classified as a N-ribosyl-purine ribohydrolase (EC 3.2.2.1). The enzyme had a maximum activity in the pH range of 4.0-4.5 toward NAD. The Km values for NAD, 5'-AMP, and inosine were 3.0, 2.9 and 1.6mM respectively.     

Purification and characterization of Aspergillus niger exo-1,4-glucosidase.

A specific exo-1,4-glucosidase (1,4-alpha-D-glucan glucohydrooase, EC 3.2.1.3) from Aspergillus niger has been partially purified and subsequently characterized by biochemical, physico-chemical and optical methods. Molecular sieve chromatography yields an enzyme with maximal activity at pH 4.2-4.5 close to its isoelectric point. Reduction and carboxymethylation leads to complete loss of activity and O-acetylation of 3 of the 13 tyrosine residues results in loss of 20 % of the activity. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicates that the native enzyme consists of two major components of molecular weights 63 000 and 57 500, respectively. Small amounts of dissociated material of molecular weight 28 000 and 16 000 as well as aggregates of the order of 100 000 are also present to the extent of 2-5% of the total potein. Following reduction and carboxymethylation under forcing conditions, the bands around 60 000 diminish and the 28 000-30 000, 16 000 and aggregate bands are dominant...     

Purification and properties of Aspergillus niger beta-glucosidase.

Beta-glucosidase was purified from a crude cellulase preparation from Aspergillus niger by affinity chromatography on a methacrylamide-N-methylene-bis-methacrylamide copolymer bearing cellobiamine. The purified enzyme was a dimer with an isoelectric point of 4.0. The molecular mass of the enzyme was estimated to be 240 kDa by gel-permeation chromatography. The enzyme hydrolyzed specifically beta-glucosidic bonds and catalyzed transglucosylation of the beta-glucosyl group of cellobiose to yield 4-O-beta-gentiobiosylglucose in the presence of organic solvents or under neutral conditions.     

Fractionation and purification of endo-1,4-beta-xylanases and exo-1,4-beta-xylosidases of Aspergillus niger]

Two endo-1,4-beta-zylanases (m. w. 24,000 and 41,000) and six exo-1,4-beta-xylosidases, differing in their molecular weights and isoelectric points, were found in a xylanase preparation from Aspergillus niger, using different methods of fractionation. An electrophoretically homogeneous exo-1,4-beta-xylosidase (m. w. 30,000) purified 120-fold, with pI 4.6, having optimal effect on methyl-beta-D-xyloside at pH 3.0 was obtained. Exo-1,4-beta-xylosidase splits off xylose from the ends of the xylan chains at xylotriose, xylobiose and methyl-beta-D-xyloside and is characterized by a high transglycosilase activity. An electrophoretically homogeneous endo-1,4-beta-xylanase (m. w. 24,000) purified 250-fold, with pI 4.2 and optimal effect on carboxymethylxylan at pH 4.2 was isolated. Endo-1,4-beta-xylanase splits arabinoglucuronoxylan to form xylooligosaccharides; however, it does not hydrolyze xylobiose.     

Purification and properties of a cellulase from Aspergillus niger.

A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.     

Purification and characterization of pectinesterase produced by a strain ofAspergillus niger

After an 88-fold purification, pectinesterase produced by a strain ofAspergillus niger, isolated from rotten lemons, showed the following main characteristics: maximum activity at 45°C, pH 5; Km, with pectin as substrate, 1.01 mg/L; G^*, 4750 Cal/mol. Polygalacturonic acid and methanol acted as competitive and non-competitive inhibitors, respectively. The activity of the enzyme was impaired by MgCl₂ and stimulated by NaCl.     

The active site of an acidic endo-1,4-beta-xylanase of Aspergillus niger

The substrate binding site of an acidic endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) of Aspergillus niger was investigated using 1,4-beta-xylooligosaccharides (1-3H)-labelled at the reducing end. Bond cleavage frequencies and V/Km parameters of the oligosaccharides were determined under conditions of unimolecular hydrolysis and, according to the method of Suganuma et al. (J. Biochem. (Tokyo) (1978) 84, 293-316), used for evaluation of subsite affinities. The substrate binding site of the enzyme was found to consist of seven subsites, numbered -IV, -III, -II, -I, I, II and III, towards the subsite binding the reducing end unit of xyloheptaose. The catalytic groups were localized between subsites -I and I, the affinities of which have not been determined. All other subsites showed positive values of affinities for binding xylosyl residues. The values decrease from subsites -II and II, similarly in both directions. As a consequence of such an almost symmetric distribution of affinities around the catalytic groups, the enzyme cleaves preferentially the bonds in the oligosaccharides which are most distant from both terminals. Thus, the acidic A. niger beta-xylanase appears to be an endo-1,4-beta-xylanase attacking polymeric substrates in a random fashion. This conclusion was supported by viscosimetric measurements with carboxymethylxylan as a substrate.     

Purification and properties of a 4-nitrophenylphosphatase from Aspergillus niger.

A 4-nitrophenylphosphatase (EC 3.1.3.41) was identified in extracts of Aspergillus niger. The production of this activity was decreased by growth on a phosphate-limiting medium and was greatest in a medium supplemented with corn steep liquor. The phosphatase activity was purified by hydrophobic, ion-exchange, and molecular sieve chromatography. The purified enzyme has a native size of approximately 80,000, polypeptide subunits with sizes of 37,000 upon denaturation, and a pI of 4.6. The activity was optimal at pH 8.0 and was stimulated by Mg2+ and to a lesser extent by Mn2+ but was inhibited by Zn2+ and Ca2+. The enzyme was highly specific for 4-nitrophenyl phosphate as substrate, having a Km of 0.77 mM and a turnover number of 108 s-1. The purified enzyme did not hydrolyze any of 22 sugar phosphates, mononucleotides, or other phosphocompounds tested. A small, but reproducible, amount of activity was measured using 5'-DNA phosphate as a substrate. Although some similarities exist to three previously characterized 4-nitrophenylphosphatases from Saccharomyces cerevisiae, the enzyme from A. niger is distinctly different from at least two of these activities.     

Purification and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger

Five endo-polygalacturonases (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) and one exo-polygalacturonase (poly(1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67) were isolated from a commercial pectinase preparation derived from Aspergillus niger. All five endo-enzymes could be purified to homogeneity by affinity chromatography on cross-linked alginate, ion-exchange chromatography, chromatofocusing, and gel permeation chromatography. The exo-polygalacturonase was only partially purified but free from endo-polygalacturonase activity. The two most abundant endo-polygalacturonases (endo-I and endo-II), with molecular masses of 55 and 38 kDa, respectively, are quite different with respect to their isoelectric point, specific activity, mode of action on oligomeric substrates, and amino acid composition. The physicochemical properties of the other three endo-polygalacturonases (endo-IIIA, endo-IIIB, and endo-IV), present in low amounts, are quite similar to those of the endo-I type. The pH optima of all these endo-polygalacturonases are in the range of 4.3-4.9.     

Purification and properties of an endo-1,4-beta-glucanase from Clostridium josui.

An enzyme active against carboxymethyl cellulose (CMC) was purified from the stationary-phase-culture supernatant of Clostridium josui grown in a medium containing ball-milled cellulose. The purification in the presence of 6 M urea yielded homogeneous enzyme after an approximately 50-fold increase in specific activity and a 13% yield. The enzyme had a molecular mass of 45 kilodaltons. The optimal temperature and pH of the enzyme against CMC were 60 degrees C and 6.8, respectively. The enzyme hydrolyzed cellotetraose, cellopentaose, and cellohexaose to cellobiose and cellotriose but did not hydrolyze cellobiose or cellotriose. A microcrystalline cellulose, Avicel, was also hydrolyzed significantly, but the extent of hydrolysis was remarkably less than that of CMC. On the basis of these results, the enzyme purified here is one of the endo-1,4-beta-glucanases. The N-terminal amino acid sequence of the enzyme is Tyr-Asp-Ala-Ser-Leu-Lys-Pro-Asn-Leu-Gln-Ile-Pro-Gln-Lys-Asn-Ile-Pro-Asn- Asn-Asp-Ala-Val-Asn-Ile-Lys.     

Purification and characterization of glucoamylase produced by Aspergillus niger in solid state fermentation

Glucoamylases produced by Aspergillus niger grown on wheat bran in solid cultures were purified. Four different forms, GA I, GA I', GA II and GA III, were found having apparent molecular weights of 112 000, 104 000, 74 000 and 61 000 Da respectively. The enzymes are glycoproteins with a carbohydrate content of 16%, and optimal activity at 60C and pH 4.4. Activity was strongly inhibited by Hg²⁺ while Mn²⁺ and Fe²⁺ were stimulatory. The K_m values for the degradation of starch and maltose were 3.5 and 7.8 mg ml^-1, respectively.     

Purification and properties of an alpha-D-xylosidase from Aspergillus niger

Two components of alpha-D-xylosidase (alpha-D-xylosidase I and II) were detected in the culture filtrate of Aspergillus nigher grown in a medium containing Sanzyme 1000-treated Glyloid 2A. The major component (alpha-D-xylosidase I) was purified to an electrophoretically pure state. The purified enzyme showed approximately 540-fold increase in specific activity over the original culture filtrate. The purified enzyme was shown to be an oligomeric protein consisting of four subunits, each of which had a molecular weight of 123,000. The enzyme showed the highest activity at pH 2.5-3.0 and 45 degrees C, and was stable in the pH range from 3.0 to 7.0 and at the temperatures up to 60 degrees C. The isoelectric point of this enzyme was pH 5.6. The purified enzyme was highly specific for p-nitrophenyl alpha-D-xylopyranoside and isoprimeverose (6-O-alpha-D-xylopyranosyl-D-glucopyranose). The apparent Km and Vmax values of the enzyme for p-nitrophenyl alpha-D-xylopyranoside and isoprimeverose were 10.5 mM and 40.8 mumol/min/mg protein, and 2.2 mM and 30 mumol/min/mg protein, respectively. The purified enzyme could also split off the alpha-D-xylopyranosyl residue on the non-reducing terminal of the backbone of oligoxyloglucans such as alpha-D-xylopyranosyl-(1----6)-beta-D-glucopyranosyl- (1----4)-[(alpha-D-xylopyranosyl-(1----6)-]-beta-D-glucopyranosyl- (1----4)-] 2-D-glucopyranose.     

黑曲霉纤维素酶的纯化及酶学性质研究

黑曲霉(Aspergillusniger)固态发酵后粗酶液经硫酸铵盐析,2次SephadexG-200柱层析后可提纯8倍左右.CMC酶最适作用温度为60℃,最适作用pH为3.5,30℃～70℃区间酶活力较稳定,在pH3.0～5.0范围内,50℃保温30min能保持80%的酶活力.CMC酶的Km、Vmax值分别为7.69%CMCg/ml、0.33mg/ml·