Purification of glucoamylase fromAspergillus terreus

Glucoamylase from a rice bran culture ofAspergillus terreus was purified by chromatography on DEAE-cellulose and concanavatin A-Sepharose. A homogenous monomer resulted after SDS-PAGE electrophoresis. The enzyme was a glycoprotein, molecular weight, 86,000 with 7.5% (w/w) carbohydrate content.

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Résumé La glucoamylase d'Aspergillus terreus cultivee sur son de riz a été purifiée par chromatographie sur DEAE-cellulose et sur A-sepharose à concavaline. On a obtenu un monomère homogène après électrophorèse sur SDS-PAGE. L'enzyme est une glycoproténe d'un poids moléculaire de 86,000 avec un contenu en polysacchardie de 7.5% (p/p).

     

Multiple forms of a glucoamylase inhibitory factor fromAspergillus niger and its elution after adsorption onto raw wheat starch

An inhibitory factor (IF) fromAspergillus niger, that inhibited the action of glucoamylase on raw starch, was adsorbed tightly onto raw starch but was almost completely desorbed by 0.02m sodium borate. The IF was a glycoprotein and was partially purified by ion exchange chromatography into three active fractions.     

Structure and stability of glucoamylase II fromAspergillus niger: A circular dichroism study

Glucoamylase II (EC 3.2.1.3) fromAspergillus niger has 31 % α-helix, 36 %Β- structure and rest aperiodic structure at pH 4.8 as analysed by the method of Provencher and Glockner (1981,Biochemistry, 20,33). In the near ultra-violet circular dichroism spectrum the enzyme exhibits peaks at 304, 289, 282 and 257 nm and troughs at 285, 277 and 265 nm respectively. The enzyme activity and structure showed greater stability at pH 4.8 than at pH 7.0, were highly sensitive to alkaline pH but less sensitive to acid pH values. The enzyme retained most of its catalytic activity and structure even on partial removal of carbohydrate moieties by periodate treatment but was less stable at higher temperatures and storage at 30‡C. Reduction of the periodate treated enzyme did not reverse the loss of stability. Binding of the synthetic substrate,p-nitrophenyl-α-D-glucoside, perturbed the environment around aromatic amino acids and caused a decrease in the ordered structure.     

Phytase fromAspergillus niger

132 microorganisms, isolates from soil and decayed fruits, were tested for phytase production. All isolates intensively producing active extracellular phytase were of fungal origin. The most active fungal isolates with phytase activity were identified asAspergillus niger. At the end of the growth phase, the extracellular phytase activity produced byA. niger strain 92 was 132 nkat/mL, with strain 89 it was 53 nkat/mL. In both strains the extracellular enzyme activity exhibited two marked activity optima at pH 1.8 and 5.0 and a temperature optimum at 55°C.     

Purification and some properties of three forms of glucoamylase from a Rhizopus species.

1. Three forms of glucoamylase [EC 3.2.1.3] were simultaneously purified from a Rhizopus species by (NH4)2SO4 fractionation and successive chromatographies on Sephadex G-75, DEAE-Sephadex, and CM-Sephadex, and were finally separated from each other by means of recycling chromatography on Bio-Gel P-150. The purification achieved was 3--4 fold from crude extract with respect to each glucoamylase; the yields of the three glucoamylases, designated as Gluc1, Gluc2, and Gluc3 in order of content, were 39, 7, and 0.4%, respectively. All the purified enzymes were homogeneous in polyacrylamide gel electrophoresis, isoelectric focusing, and ultracentrifugation. 2. The three glucoamylases were glycoproteins differing in both amino acid composition and carbohydrate content, but showed a common antigenicity in immunodiffusion. The molecular weights of Gluc1, Gluc2, and Gluc3 were estimated to be 74,000, 58,600, and 61,400, respectively, by sedimentation equilibrium and these values were verified by SDS-polyacrylamide gel electrophoresis. The specific activities of the three enzymes toward starch were in the opposite order to their molecular weights. 3. The three glucoamylases had the same broad pH optima in the range pH 4.5--5.0 and shared a common susceptibility to inactivation by heat, extreme pH, and such divalent cations as Hg2+, Pb2+, and Mn2+, indicating close similarity in enzymatic properties.     

Purification and the effect of manganese ions on the activity of carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea

Carboxymethylcellulases (CMCases) fromAspergillus niger andCellulomonas biazotea were purified by a combination of ammonium sulfate precipitation, anion-exchange and gel-filtration chromatography with a 12- and 9-fold increase in the purification factor. The native and subunit molar mass of CMCase fromA. niger were 40 and 25–57 kDa, respectively, while those fromC. biazotea were 23 and 20–30 kDa, respectively. Low concentrations of Mn²⁺ activated the enzymes from both organisms (mixed activation) with apparent activation constants of 0.80 and 0.45 mmol/L of CMCases fromA. niger andC. biazotea, respectively, while at higher CMC concentrations Mn²⁺ inhibited the enzymes (mixed and partial uncompetitive inhibition). The reason for this complex behavior is that more than one Mn²⁺ bind to the same enzyme form with the apparent average inhibition constants of 2.7 and 1.3 mmol/L for CMCases fromA. niger andC. biazotea, respectively.     

Purification and properties of two forms of rat alpha-lactalbumin.

Two species of alpha-lactalbumin, alpha-lactalbumin1 and alpha-lactalbumin2, were separated from rat milk and purified to homogeneity by gel filtration, followed by the DEAE-cellulose chromatography. alpha-Lactalbumin1 is a bigger molecule in contrast to other known alpha-lactalbumins, and has a molecular weight of 21,500 as determined by sedimentation equilibrium and sodium dodecyl sulfate-polyacrylamide gel analysis. alpha-Lactalbumin2 has a molecular weight of 16,000 measured by sedimentation analysis. alpha-Lactalbumin2, however, exhibits abnormally high molecular weight of 22,500 on sodium dodecyl sulfate polyacrylamide gels. Both alpha-lactalbumins are active in lactose synthase assay and are glycoproteins containing 7 to 9% carbohydrate. Antiserum raised against alpha-lactalbumin1 cannot discriminate between the two species in a radioimmunoassay.     

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.     

Amino acid sequence of cytochrome c fromAspergillus niger

Cytochrome c fromAspergillus niger consists of two forms, a major one (80%) with 111 amino acid residues and a minor one (20%) with 108 residues, missing the three N-terminal residues of the major one. The primary sequence ofA. niger cytochrome c was determined by standard spinning-cup Edman degradation of purified peptides and of pairs of peptides, from which the desired sequence was readily deduced by subtraction of common sequencies. Except for the extension and some variability at the N-terminal sequence, theA. niger protein conforms well with other cytochrome c structures.     

Regioselectivity enhancement by partial purification of lipase fromAspergillus niger

Lipase fromAspergillus niger was partially purified by a single step of ion-exchange chromatography and two major fractions I and II could be obtained. Two substrates, peracetylated 1-methyl and 1-thioethyl -D-glucopyranosides, were deacetylated by fraction II to give 3-OH derivatives as sole products with very high yield, respectively.     

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.     

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.     

Purification and properties of two multiple forms of dihydrodiol dehydrogenase from guinea-pig testis

NADP+-dependent dihydrodiol dehydrogenase (trans-1,2-dihydrobenzene-1,2-diol: NADP+ oxidoreductase, EC 1.3.1.20) activity in the cytosol of guinea-pig testis was separated into two major and two minor peaks by Q-Sepharose chromatography; one minor form was immunologically cross-reacted with hepatic aldehyde reductase. The two major enzyme forms were purified to homogeneity. One form, which had the highest amount in the tissue, was a monomeric protein with a molecular weight of 32,000 and isoelectric point of 4.2, showed strict specificity for benzene dihydrodiol and NADP+, and reduced pyridine aldehydes, glyceraldehyde and diacetyl at low rates. Another form, with a molecular weight of 36,000 and isoelectric point of 5.0, oxidized n-butanol, glycerol and sorbitol as well as benzene dihydrodiol in the presence of NADP+ or NAD+, and exhibited much higher reductase activity towards various aldehydes, aldoses and diacetyl. The pI 5.0 form was more sensitive to inhibition by sorbinil and p-chloromercuriphenyl sulfonate than the pI 4.2 form and was activated by sulfate ion. The two enzymes did not catalyze the oxidation of hydroxysteroids and xenobiotic alicyclic alcohols and were immunologically different from hepatic 17 beta-hydroxysteroid-dihydrodiol dehydrogenase. The results indicate that guinea-pig testis contains at least two dihydrodiol dehydrogenases distinct from the hepatic enzymes, one of which, the pI 5.0 enzyme form, may be identical to aldose reductase.     

Stability and identification of active-site residues of carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea

Determination of the apparent pK _a's of purified carboxymethylcellulases fromAspergillus niger andCellulomonas biazotea at different temperatures and in the presence of dioxane indicated two side chain carboxyl groups which controlled the limiting rate in both organisms. The thermostability of both enzymes slightly decreased with increasing pH from 5 to 7.5 but was unaffected in the presence of 0.5 mmol/L Mn²⁺. The CMCase fromC. biazotea had an activation energy of 35 kJ/mol and a half-life of 89 min in the presence of 8 mol/L urea at 40°C. The half-life of CMCase fromA. niger in 8 mol/L urea and at 37°C was 125 min as determined by a 0–9 mol/L transverse urea gradient PAGE. The CMCases fromA. niger andC. biazotea had the same thermostabilities in the absence of CMC although the enzyme from the former was more thermostable in the presence of the substrate. The CMCase fromA. niger was also more efficient in hydrolyzing CMC than the enzyme fromC. biazotea.