Purification and characterization of invertase from Aspergillus niger

Invertase produced by a strain of Aspergillus niger showed the following main characteristics: maximum activity at 60°C, pH 5.0; K _m with sucrose as substrate, 0.0625mm; V _max 0.013 mol/min; and free energy 9132 cal/mol. The metal ions and p-chloromercuribenzoate (PCMB) acted as inhibitors respectively.     

Purification and characterization of a protease-resistant cellulase from Aspergillus niger

An endo-β-1,4-glucanase (EC 3.2.1.4) was purified from a culture filtrate of Aspergillus niger IFO31125 by column chromatography through TSK-gel DEAE-3SW and TSK-gel DEAE-5PW, and by gel filtration through TSK-gel G2000SW by high performance liquid chromatography. The enzyme was estimated to have a molecular weight of about 40 kDa by both gel filtration and SDS-polyacrylamide gel electrophoresis, and appeared to consist of a monomeric protein. It contained 8.9% carbohydrate. The optimal pH for activity was 6.0–7.0, and the stable pH range was 5.0–10.0. The optimum temperature at pH 6.0 was around 70°C. The enzyme was very thermally stable and no loss of original activity was found on incubation at 60°C for 2 h. The enzyme efficiently hydrolyzed carboxymethylcellulose and lichenan, but crystalline forms of cellulose, curdlan, laminarin, cellobiose, p-nitrophenyl-β-d-glucopyranoside and p-nitrophenyl-β-d-cellobioside were barely hydrolyzed. The activity of the enzyme was inhibited by Hg²⁺ and Cu²⁺ but was not affected by other inhibitors of thiol enzymes such as p-chloromercuribenzoate and N-ethylmaleimide. N-Bromosuccinimide showed a strong inhibitory effect, suggesting that a tryptophan residue is essential for the activity of the enzyme. The N-terminal amino acid sequence of the enzyme showed considerable homology to those of endo-β-1,4-glucanases from some other microorganisms, including Sclerotinia sclerotiorum and Schizophyllum commune. The enzyme had very strong protease-resistance, and showed no loss of activity when incubated with proteases such as Savinase at 40°C, even for 2 weeks.     

Purification and characterization of a nitrilase from Aspergillus niger K10

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg^-1) at 45°C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of d-sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.     

Purification of lipase from Aspergillus niger and its characterization analysis

从黑曲霉发酵液中经硫酸铵分级沉淀,Phenyl-Sepharose疏水柱层析,DEAE-Sepharose 4B阴离子交换柱得到电泳纯的脂肪酶,纯化倍数达10倍,回收率50％.对脂肪酶的性质分析表明:该酶分子质量约为35 kDa,最适温度和最适pH分别为37℃和9.5,50℃以下和pH6.0～11.0之间保持稳定,属于碱性脂肪酶.Mg2+、Ca2+、Cu2+、Zn2+、Co2+、Mn2+对该酶有激活作用,而Al3+、Fe2+、Fe3+对酶有严重抑制作用.变性剂盐酸胍和脲对其未见显著影响,而SDS强烈抑制其酶活.用不同氨基酸修饰剂对酶进行修饰,其中NBS和PMSF强烈抑制该酶活性,NBSF和DTT在低浓度下对酶活影响不大,2,3-丁二酮在高浓度下影响其活性.外加稳定剂如NaCl、PEG、甘油、山梨醇、海藻酸钠,均可不同程度的延长脂肪酶的半衰期.在一定质量比条件下,该酶有良好的抗蛋白酶性质.     

黑曲霉(Aspergillus niger)脂肪酶的纯化及性质分析

从黑曲霉发酵液中经硫酸铵分级沉淀,Phenyl-Sepharose疏水柱层析,DEAE-Sepharose 4B阴离子交换柱得到电泳纯的脂肪酶,纯化倍数达10倍,回收率50％。对脂肪酶的性质分析表明：该酶分子质量约为35kDa,最适温度和最适pH分别为37℃和9．5,50℃以下和pH6．0～11．0之间保持稳定,属于碱性脂肪酶。Mg^2＋、Ca^2＋、Cu^2＋、Zn^2＋、Co^2＋、Mn2^＋对该酶有激活作用,而Al^3＋、Fe^2＋、Fe^3＋对酶有严重抑制作用。变性剂盐酸胍和脲对其未见显著影响,而SDS强烈抑制其酶活。用不同氨基酸修饰剂对酶进行修饰,其中NBS和PMSF强烈抑制该酶活性,NBSF和DTT在低浓度下对酶活影响不大,2,3-丁二酮在高浓度下影响其活性。外加稳定剂如NaCl、PEG、甘油、山梨醇、海藻酸钠,均可不同程度的延长脂肪酶的半衰期。在一定质量比条件下,该酶有良好的抗蛋白酶性质。     

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 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 characterization of a novel carbaryl hydrolase from Aspergillus niger PY168

A fungus capable of using carbaryl as the sole source of carbon and energy was isolated from a soil enrichment, and characterized as Aspergillus niger and designated strain PY168. A novel carbaryl hydrolase from cell extract was purified 262-fold to apparent homogeneity with 13.6% overall recovery. It had a monomeric structure with a molecular mass of 50,000 Da and a pI of 4.6, and the enzyme activity was optimal at 45 degrees C and pH 7.5, The activities were strongly inhibited by Hg(2+), Ag+, rho-chloromercuribenzoate, iodoacetic acid, diisofluorophosphate and phenylmethylsulfonyl fluoride but not EDTA and phenanthroline. The purified enzyme hydrolyzed various N-methylcarbamate insecticides. Carbaryl is the preferred substrate.     

Purification and characterization of extracellular phytase from Aspergillus niger ATCC 9142

Extracellular phytase produced by Aspergillus niger ATCC 9142 was purified to homogeneity by employing an initial ultrafiltration step, followed by chromatography using ion exchange, gel filtration and chromatofocusing steps. The purified enzyme was an 84 kDa, monomeric protein. It possessed a temperature optimum of 65 degrees C, and a pH optimum of 5.0. Km and Vmax values of 100 microM and 7 nmol/s, respectively, were recorded and these values fall well within the range of those previously reported for microbial phytases. Substrate specificity studies indicated that, while the enzyme could hydrolyse a range of non-phytate-based phosphorylated substrates, its preferred substrate was phytate. Phytase activity was moderately stimulated in the presence of Mg2+, Mn2+, Cu2+, Cd2+, Hg2+, Zn2+ and F- ions. Activity was not significantly affected by Fe2- or Fe3- and was moderately inhibited by Ca2+. The enzyme displayed higher thermostability at 80 degrees C than did two commercial phytase products. Initial characterisation of the purified enzyme suggested that it could be a potential candidate for use as an animal feed supplement.     

Purification and characterization of acidic lipase from Aspergillus niger NCIM 1207

An extracellular lipase from Aspergillus niger NCIM 1207 has been purified to homogeneity using ammonium sulfate precipitation followed by phenyl sepharose and Sephacryl-100 gel chromatography. This protocol resulted in 149 fold purification with 54% final recovery. The purified enzyme showed a prominent single band on SDS-PAGE. The purified enzyme is a monomeric protein of 32.2 kDa molecular weight and exhibits optimal activity at 50 degrees C. One interesting feature of this enzyme is its highly acidic pH optimum. The isoelectric point (pI) of lipase was 8.5. The purified lipase appears to be unique since it cleaved triolein at only 3-position releasing 1,2-diolein. Chemical modification studies revealed that His, Ser, Carboxylate and Trp are involved in catalysis.     

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 an extracellular exo-D-galacturonanase of Aspergillus niger.

A D-galacturonanase (EC 3.2.1.67) catalyzing the degradation of D-galacturonans by terminal action pattern was purified from a culture filtrate of Aspergillus niger by a procedure including the salting-out with ammonium sulfate, precipitation by ethanol, chromatography on DEAE-cellulose, and gel chromatography on Sephadex G-100. The obtained preparation was slightly contaminated by an enzymically inactive protein fraction. Maximum activity and stability of the enzyme was observed at pH 5.2. The enzyme degrades digalacturonic acid, p-nitrophenyl-alpha-D-galactopyranuronide, as well as oligogalacturonides containing at the nonreducing end 4-deoxy-L-threo-hexa-4-enopyranosyluronate. It differs from all A. niger enzymes so far described which degrade D-galaturonans by the terminal action pattern, in not clearly preferring low-molecular substrates. It is therefore classified as an exo-D-galacturonanase.     

Purification and characterization of endo-xylanases from Aspergillus niger. II. An enzyme of pl 4.5

A homogeneous endo-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) was obtained from a crude Aspergillus niger pentosanase by chromatography with Ultrogel AcA 54, SP-Sephadex C-25 at pH 4.5, DEAE-Sephadex A-25 at pH 5.4, Sephadex G-50, and SP-Sephadex C-25 with a gradient from pH 2.8 to pH 4.6. It was much more active on soluble than on insoluble xylan, yielding large amounts of unreacted xylan and a mixture of oligosaccharides with chain lengths from two to six. No xylose or L-arabinose was produced. There was high activity on a xylopentaose through xylononaose mixture, but not on xylobiose, xylotriose, or xylotetraose. The enzyme had slight activity on untreated cellulose, carboxymethylcellulose, and pectin. Molecular weight was ca. 1.4 x 10(4), with an isoelectric point of 4.5 and an amino acid profile high in acidic but low in sulfur-containing residues. In a 25-min assay at pH 4.7, this endo-xylanase was most active at 45 degrees C, with an activation energy from 5 to 35 degrees C of 33.3 kJ/mol. The optimum pH for activity was 4.9. Decay in buffer was first order, with an activation energy at pH 4.7 from 48 to 53 degrees C of 460 kJ/mol. Optimum pH for stability was about 5.6, where the half-life at 48 degrees C in buffer was ca. 40 h.     

Purification and characterization of endo-xylanases from Aspergillus niger. III. An enzyme of pl 3.65

An endo-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) from Aspergillus niger was purified to homogeneity by chromatography with Ultrogel AcA 54, SP-Sephadex C-25 at pH 4.5, DEAE-Sephadex A-25 at pH 5.4, Sephadex G-50, and DEAE-Sephadex A-25 at pH 5.15. The enzyme was active on soluble xylan, on insoluble xylan only after arabinosyl-initiated branch points were removed, and on xylooligosaccharides longer than xylotetraose. There was slight activity on carboxymethyl-cellulose, arabinogalactan, glucomannan, and p-nitrophenyl-beta-D-glucopyranoside. The main products of the hydrolysis of soluble and insoluble xylan were oligosaccharides of intermediate length, especially the tri- and pentasaccharides. The isoelectric point of the enzyme was 3.65. It had a molecular weight of 2.8 x 10(4) by SDS-gel electrophoresis, and was high in acidic amino acids but low in those containing sulfur. Highest activity in a 20-min assay at pH 5 was between 40 and 45 degrees C, with an activation energy up to 40 degrees C of 11.1 kJ/mol. The optimum pH for activity was at 5.0. The enzyme was strongly activated by Ca(2+).     

Purification and thermodynamic characterization of glucose oxidase from a newly isolated strain of Aspergillus niger

An intracellular glucose oxidase (GOD) was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger NFCCP. The enzyme was partially purified to a yield of 28.43% and specific activity of 135 U mg(-1) through ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The enzyme showed high specificity for D-glucose, with a K(m) value of 25 mmol L(-1). The enzyme exhibited optimum catalytic activity at pH 5.5. Optimum temperature for GOD-catalyzed D-glucose oxidation was 40 degrees C. The enzyme displayed a high thermostability having a half-life (t(1/2)) of 30 min, enthalpy of denaturation (H*) of 99.66 kJ mol(-1), and free energy of denaturation (G*) of 103.63 kJ mol(-1). These characteristics suggest that GOD from A. niger NFCCP can be used as an analytical reagent and in the design of biosensors for clinical, biochemical, and diagnostic assays.     

Purification and partial characterization of fructosyltransferase and invertase from Aspergillus niger AS0023

Fructosyltransferase (EC.2.4.1.9) and invertase (EC.3.2.1.26) have been purified from the crude extract of Aspergillus niger AS0023 by successive chromatographies on DEAE-sephadex A-25, sepharose 6B, sephacryl S-200, and concanavalin A-Sepharose 4B columns. On acrylamide electrophoresis the two enzymes, in native and denatured forms, gave diffused glycoprotein bands with different electrophoretic mobility. On native-PAGE and SDS-PAGE, both enzymes migrated as polydisperse aggregates yielding broad and diffused bands. This result is typical of heterogeneous glycoproteins and the two enzymes have proved their glycoprotein nature by their adsorption on concanavalin A lectin. Fructosyltransferase (FTS) on native PAGE migrated as two enzymatically active bands with different electrophoretic mobility, one around 600 kDa and the other from 193 to 425 kDa. On SDS-PAGE, these two fractions yielded one band corresponding to a molecular weight range from 81 to 168 kDa. FTS seems to undergo association-dissociation of its glycoprotein subunits to form oligomers with different degrees of polymerization. Invertase (INV) showed higher mobility corresponding to a molecular range from 82 to 251 kDa, on native PAGE, and from 71 to 111 kDa on SDS-PAGE. The two enzymes exhibited distinctly different pH and temperature profiles. The optimum pH and temperature for FTS were found to be 5.8 and 50 degrees C, respectively, while INV showed optimum activity at pH 4.4 and 55 degrees C. Metal ions and other inhibitors had different effects on the two enzyme activities. FTS was completely abolished with 1 mM Hg(2+) and Ag(2+), while INV maintained 72 and 66% of its original activity, respectively. Furthermore, the two enzymes exhibited distinctly different kinetic constants confirming their different nature. The K(m) and V(m) values for each enzyme were calculated to be 44.38 mM and 1030 micromol ml(-1)min(-1) for FTS and 35.67 mM and 398 micromol ml(-1) min(-1) for INV, respectively. FTS and INV catalytic activity was dependent on sucrose concentration. FTS activity increased with increasing sucrose concentrations, while INV activity decreased markedly with increasing sucrose concentration. Furthermore, INV exhibited only hydrolytic activity producing exclusively fructose and glucose from sucrose, while FTS catalyzed exclusively fructosyltransfer reaction producing glucose, 1-kestose, nystose and fructofuranosyl nystose. In addition, at 50% sucrose concentration FTS produced fructooligosaccharides at the yield of 62% against 54% with the crude extract.