Niger Enzyme Research Articles

The Glucoamylase cDNA fromAspergillus oryzae: Its Cloning, Nucleotide Sequence, and Expression inSaccharomyces cerevisiae

A cDNA for Aspergillus oryzae glucoamylase was cloned, using oligodeoxyribonucleotide probes derived from amino sequences of peptide fragments of the enzyme. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae, directed the secretion of active glucoamylase into the culture medium. The complete nucleotide sequence of the cDNA contained an open reading frame encoding 612 amino acid residues. Comparative studies with other fungal glucoamylases showed homologies of 67% with A. niger and 30% with Rhizopus oryzae of the deduced amino acid sequences. In the five conserved regions reported in other fungal glucoamylases, the levels of homologies between those regions of A. oryzae and A. niger enzymes were much higher (78-94%). A. oryzae glucoamylase contained no peptide region abundant in threonine and serine residue (TS-region), like that proposed to adsorb onto raw starch in A. awamori var. kawachii glucoamylase.

The glucoamylase cDNA from Aspergillus oryzae:Its cloning, nucleotide sequence, and expression in Saccharomyces cerevisiae.

A cDNA for Aspergillus oryzae glucoamylase was cloned, using oligodeoxyribonucleotide probes derived from amino sequences of peptide fragments of the enzyme. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae, directed the secretion of active glucoamylase into the culture medium. The complete nucleotide sequence of the cDNA contained an open reading frame encoding 612 amino acid residues. Comparative studies with other fungal glucoamylases showed homologies of 67% with A. niger and 30% with Rhizopus oryzae of the deduced amino acid sequences. In the five conserved regions reported in other fungal glucoamylases, the levels of homologies between those regions of A. oryzae and A. niger enzymes were much higher (78-94%). A. oryzae glucoamylase contained no peptide region abundant in threonine and serine residue (TS-region), like that proposed to adsorb onto raw starch in A. awamori var. kawachii glucoamylase.

Production of β-galactosidase by Aspergillus oryzae in submerged bioreactor cultivation

Eleven strains of Aspergillus oryzae were screened for production of β-galactosidase on media based on lactose and wheat bran. All the strains produced highest levels of activity on the wheat bran medium. Production by the best strain, A. oryzae VTT-D-88353 (ATCC 20423) was considerably better than that obtained with A. niger VTT-D-80144, previously reported as an efficient producer of β-galactosidase, in both shake flask and laboratory bioreactor cultivations. Production on a lactose-based medium in the bioreactor was better in fed-batch than in simple batch cultivation, but considerably poorer than on wheat bran. The role of the organic nitrogen source was also of major importance for β-galactosidase production by the A. oryzae strain. The cultivation time required for maximal production by A. oryzae was longer than that required by A. niger and could not be reduced by the use of surface active agents. In lactose hydrolysis experiments the enzyme of A. oryzae was somewhat more efficient that that of A. niger at near-neutral pH values at 30°C, but at 50°C the A. niger enzyme was more effective at all pH values between 5.0 and 7.0.

Purification and properties of NADP+-dependent glycerol dehydrogenases from Aspergillus nidulans and A. niger

Glycerol dehydrogenase, NADP(+)-specific (EC 1.1.1.72), was purified from mycelium of Aspergillus nidulans and Aspergillus niger using different purification procedures. Both enzymes had an Mr of approximately 38,000 and were immunologically cross-reactive, but had different amino acid compositions and isoelectric points. For both enzymes, the substrate specificity was limited to glycerol and erythritol for the oxidative reaction and to dihydroxyacetone (DHA), diacetyl, methylglyoxal, erythrose and D-glyceraldehyde for the reductive reaction. The A. nidulans enzyme had a turnover number twice that of the A. niger enzyme at pH 6.0, whereas inhibition by NADP+ was less (Ki = 45 microM vs 13 microM). It is proposed that both enzymes catalyse in vivo the reduction of DHA to glycerol and that they are regulated by the anabolic reduction charge.

Physical characterization of the aldehyde-dehydrogenase-encoding gene of Aspergillus niger

To facilitate a better understanding of the regulation of aldA, the gene encoding aldehyde dehydrogenase (AldDH) in the ascomyeete fungus, Aspergillus niger, the gene has been physically characterized. The complete nucleotide (nt) sequence of the gene and its flanking regions has been determined. Analysis of the gene has revealed the presence of three introns. The homologous gene in the related fungus, Aspergillus nidulans, contains only two introns. The coding regions of these genes, excluding the intron sequences, show 80% homology at the nt level and 82% homology at the amino acid (aa) level. The aa sequence of the A. niger enzyme is significantly homologous to mammalian sequences, particularly around one Cys residue, which is hypothesized to be adjacent to another Cys known to be at the active site of mammalian AldDHs. The 5' region of the gene shows one major and two minor transcription start points and a general structure common to highly expressed fungal genes. The 3' region shows four sites of polyadenylation. Sequence comparisons of the 5' region of the A. niger aldA gene to other Aspergillus genes has shown a common sequence in the 5' regions of several A. nidulans genes, all but one of which are subject to carbon catabolite repression. This sequence may be important for the regulatory mechanism.

Partial purification, and some properties and reactivities of cetraxate benzyl ester hydrochloride-hydrolyzing enzyme.

Debenzylating enzyme from Aspergillus niger enzyme (commercial crude cellulase) catalyzes the hydrolysis of cetraxate benzyl ester hydrochloride (2), a precursor of the antiulcer agent (1). The enzyme was highly purified by three kinds of chromatographies (hydrophobic, ion exchange, gel filtration) with a recovery of 36%. The content of the debenzylating enzyme was about 0.1% in the crude cellulase, but the enzyme showed no cellulase activity. The purified enzyme was inactivated by Hg2+, and diisopropyl phosphorofluoridate (DFP). It was a monomer with a molecular weight of about 35,000, and its isoelectric point was estimated to be 5.3. It showed a debenzylating activity for the phenylpropionic acid benzyl ester moiety of various benzyl ester derivatives, and the benzyl ester of phenylalanine or that of tyrosine was also well hydrolyzed.

A Fructooligosaccharide-producing Enzyme fromAspergillus nigerATCC 20611

The fructosyl transfer to sucrose was investigated, and Aspergillus niger ATCC 20611 was selected as the most suitable strain for fructooligosaccharide production. This strain showed very high enzyme productivity, and its transfructosylating activity was very strong compared to its hydrolyzing activity. Fructooligosaccharides could be more effectively prepared with a higher concentration of sucrose if use could be made of an enzyme having higher transfructosylating ability. Treatment of 50% (w/v) sucrose with the A. niger enzyme afforded a mixture of fructooligosaccharides with inulin-type structures of 1F(1-β-fructofuranosyl)n-sucrose (n = 1 to 3). The individual saccharides could be separated by the combination of a carbon column chromatography and preparative HPLC.

A fructooligosaccharide-producing enzyme from Aspergillus niger ATCC 20611.

The fructosyl transfer to sucrose was investigated, and Aspergillus niger ATCC 20611 was selected as the most suitable strain for fructooligosaccharide production. This strain showed very high enzyme productivity, and its transfructosylating activity was very strong compared to its hydrolyzing activity. Fructooligosaccharides could be more effectively prepared with a higher concentration of sucrose if use could be made of an enzyme having higher transfructosylating ability. Treatment of 50% (w/v) sucrose with the A. niger enzyme afforded a mixture of fructooligosaccharides with inulin-type structures of 1F(1-β-fructofuranosyl)n-sucrose (n = 1 to 3). The individual saccharides could be separated by the combination of a carbon column chromatography and preparative HPLC.

Hydration of cellobial by exo- and endo-type cellulases: evidence for catalytic flexibility of glycosylases.

New insight has been obtained into the catalytic capabilities of cellulase. Essentially homogeneous preparations of exo- (or Avicelase-) type and endo- (or CMCase-) type cellulases from Irpex lacteus and Aspergillus niger, respectively, were shown to hydrate the enolic bond of cellobial to form 2-deoxycellobiose. The A. niger enzyme also synthesized a small amount of a 2-deoxycellobiosyl-transfer product from cellobial. By use of digests conducted in deuterated buffer and 1H NMR spectra for product analysis, both cellulases were found to protonate (deuterate) the double bond of cellobial from below the si face of the D-glucal moiety, i.e., from a direction opposite that assumed for protonation of the beta-D-glycosidic linkages of cellulose and cellodextrins. The exo enzyme, which hydrolyzes the latter substrates primarily to cellobiose, rapidly catalyzed cellobial hydration to produce the beta-anomer of beta-D-glucopyranosyl(1----4)-2-deoxy-D-glucose-2(e)-d. The A. niger cellulase produced the same 2-deoxycellobiose-d from cellobial, though too slowly for its configuration to be determined. However, evidence was obtained for the formation of a beta-2-deoxycellobiosyl-d-D-glucose-transfer product by the enzyme. Thus, it is likely that all of the observed reactions with cellobial represent trans additions at the double bond. In any case, the anomeric configuration of products is created de novo. Separate mechanisms are described for the reaction of cellobial hydration and for the stereochemically different reaction of cellulose hydrolysis catalyzed by the present enzymes, assuming an arrangement of their catalytic groups analogous to that found in lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Aconitase and citric acid fermentation by Aspergillus niger.

In view of the often-cited theory that citric acid accumulation is caused by an inhibition of aconitase activity, the equilibrium of the reaction of aconitase was investigated by comparing in vivo steady-state concentrations of citrate and isocitrate in Aspergillus niger grown under various citric acid-producing conditions. With the equilibrium catalyzed by the A. niger enzyme in vitro, similar values were obtained. The validity of our in vivo measurements was verified by the addition of the aconitase inhibitor fluorocitrate, which appreciably elevated the citrate:isocitrate ratio. The results strongly argue against an inhibition of aconitase during citric acid fermentation.

Isolation, Purification, and Characterization of Lipase Isoenzymes from a Technical Aspergillus niger Enzyme

ABSTRACTA qualitative screening revealed the occurrence of lipase, esterase, protease, amylase, endo‐1,4‐β‐D‐glucanase, xylanase, pectinmethylesterase, polygalacturonase, catalase, β‐D‐glucosidase and β‐D‐galactosidase activities in the technical Aspergillus niger enzyme under study (Lipase 2212 D, Röhm). The isolation and purification of lipolytic activities were performed by combination of DEAE‐Trisacryl M ion exchange chromatography, Sephadex G 50 gel filtration and hydrophobic chromatography using Phenylsepharose CL‐4B. The individual purification steps were checked by specific enzyme visualization in ultrathin agar gels after ultrathin‐layer isoelectric focusing (UIEF). Two UIEF homogeneous lipase isoenzymes (I and II) were isolated and characterized by the following parameters: isoelectric points (I: 4.0; II. 3.5); molecular weights (I: 31000 daltons; II: 19000 daltons); carbohydrate contents (I: 6%; II: 9%) and compositions; pH optima (I, II: 5‐6); substrate specificities and various effectors.

Kinetics of cellobiose hydrolysis using cellobiase composites from Ttrichoderma reesei and Aspergillus niger

The enzymatic hydrolysis of cellulose to glucose involves the formation of cellobiose as an intermediate. It has been found necessary(1) to add cellobiase from Aspergillus niger (NOVO) to the cellobiase component of Trichoderma reesei mutant Rut C-30 (Natick) cellulase enzymes in order to obtain after 48 h complete conversion of the cellobiose formed in the enzymatic hydrolysis of biomass. This study of the cellobiase activity of these two enzyme sources was undertaken as a first step in the formation of a kinetic model for cellulose hydrolysis that can be used in process design. In order to cover the full range of cellobiose concentrations, it was necessary to develop separate kinetic parameters for high- and low-concentration ranges of cellobiose for the enzymes from each organism. Competitive glucose inhibition was observed with the enzymes from both organisms. Substrate inhibition was observed only with the A. niger enzymes.

Production and properties of Aspergillus niger inulinase

AbstractAn inulinase producing Aspergillus niger strain was isolated from Compositae rhizosphere soil samples. High inulinase levels were produced on a corn steep liquor (CSL)‐maltose medium in the absence of inulin at 28°C within 110 h of fermentation. Media based on CSL‐sucrose yielded high cell‐bound inulinase activity; on inulin‐based media the enzyme was mainly extracellularly produced. Both crude extra‐and intracellular inulinase preparations displayed identical pH and temperature optima with maximal activity at pH 4.3–4.4 and at 55–56°C. These properties are favourable in view of large scale inulinase application for pure fructose production. High operation temperatures would avoid microbial contamination of reactors and would allow the use of high inulin‐substrate concentrations, a limiting factor in obtaining high conversion ratios. The remarkably low pH optimum prevents colour formation and undesirable chemical side reactions. An advantageous low ratio of invertase to inulinase activity (S/I value) of 0.85 was found for the crude extracellular enzyme preparation. Crude inulin (chicory) extracts are hydrolysed faster than pure inulin. Apart from inulin (100% hydrolysis), sucrose (45%) and raffinose (20%) are also hydrolysed, and no liberation of oligomers or of sucrose from inulin was observed. These facts indicate that the A. niger enzyme is an exo‐acting inulinase. The above characteristics make this A. niger inulinase an industrially attractive enzyme for the preparation of pure fructose from inulin‐containing agricultural crops.

Studies onAspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grown Aspergillus niger was increased 3-5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH4 + , and further, the enzyme is repressed by increasing concentrations of NH4 +. In contrast to other micro-organisms, the Aspergillus niger enzyme was neither specifically inactivated by NH4+ or L-glutamine nor regulated by covalent modification.Glutamine synthetase from Aspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn2+ or Mg2+-dependent synthetase and Mn2+-dependent transferase activity.Aspergillus niger glutamine synthetase was completely inactivated by two mol of phenylglyoxal and one mol of N-ethylmaleimide with second order rate constants of 3·8 M–1 min–1 and 760 M–1 min–1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH4+, Mn2+ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.

Production of ethanol from raw cassava starch by a nonconventional fermentation method

AbstractRaw cassava root starch was transformed into ethanol in a one‐step process of fermentation, in which are combined the conventional processes of liquefaction, saccharification, and fermentation to alcohol. Aspergillus awamori NRRL 3112 and Aspergillus niger were cultivated on wheat bran and used as Koji enzymes. Commercial A. niger amyloglucosidase was also used in this experiment. A raw cassava root homogenate–enzymes–yeast mixture fermented optimally at pH 3.5 and 30°C, for five days and produced ethanol. Alcohol yields from raw cassava roots were between 82.3 and 99.6%. Fungal Koji enzymes effectively decreased the viscosity of cassava root fermentation mashes during incubation. Commercial A. niger amyloglucosidase decreased the viscosity slightly. Reduction of viscosity of fermentation mashes was 40, 84, and 93% by commercial amyloglucosidase, A. awamori, and A. niger enzymes, respectively. The reduction of viscosity of fermentation mashes is probably due to the hydrolysis of pentosans by Koji enzymes.

Comparative studies on glucoamylases from three fungal sources

Five commercial preparations of glucoamylases (three fromAspergillus niger, one each fromAspergillus foetidus andAspergillus candidus) were purified by ultrafiltration, Sepharose-gel filtration and DEAE-sephadex chromatography. Two forms of the enzyme, namely glucoamylase I and glucoamylase II were obtained from the fungi except from one strain ofA. Niger. All the enzymes appeared homogeneous by electrophoresis and ultracentrifugation. The specific activities varied between 85 and 142 units. The pH and temperature optima were between 4 and 5, and 60‡C respectively. The molecular weight as determined by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis ranged from 75,000 to 79,000 for glucoamylase I and 60,000 to 72,000 for glucoamylase II. OnlyA. niger glucoamylases contained phenylalanine at the N-terminal end. The amino acid composition of the enzymes was generally similar. However,A. niger andA. foetidus glucoamylases, in contrast toA. candidus enzymes, contained greater percentage of acidic than of basic amino acids. The enzymes contained 15 to 30% carbohydrate and 49 to 57 residues of monosaccharides per mol.A. niger enzymes contained mannose, glucose, galactose, xylose and glucosamine but theA. candidus enzyme lacked xylose and glucose and only xylose was absent inA, foetidus enzymes. Majority of the carbohydrate moieties were O-glycosidically linked through mannose to the hydroxyl groups of seline and threonine of the polypeptide chain.

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-α- 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-galacturonans by the terminal action pattern, in not clearly preferring low-molecular substrates. It is therefore classified as an exo- d-galacturonanase.

Untersuchungen über den Abbaumechanismus einer gereinigten Polygalakturonase aus Aspergillus niger

Using an Aspergillus niger enzyme preparation, a pure polygalacturonase has been isolated by chromatography on cellulose phosphate. The alkali‐labile enzyme has an isoelectric point of 4.5 and a pH optimum of 5.5; its activation energy is 8.3 kcal/mole. The enzyme hydrolyzes 48% of the glycosidic bonds in pectic acid. Besides di‐ and monogalacturonic acid, the hydrolysate mostly contains trigalacturonic acid. Depending on the degree of esterification, the methyl ester of pectic acid is degraded with varying intensity. If the degree of esterification exceeds 75%, pectin is no longer degraded. Acetylation of the secondary alcohol groups, up to 70%, has little effect. Di‐ and trigalacturonic acid are not hydrolyzed. Tetragalacturonic acid is split into mono‐and trigalacturonic acid, penta‐ and hexagalacturonic acid into tri‐, di‐ and monogalacturonic acid. The isolated enzyme is an endopolygalacturonase. The study of the degradation products strongly suggests that two carboxyl groups of pectic acid become attached to the binding sites of the enzyme molecule. These two groups are separated from the active center by three and six galacturonic acid units respectively. Blocking of the binding sites at higher substrate concentrations causes a decrease in enzyme activity.

The use of 6- O-methylamylose as a substrate for amylases

A type of chemically modified amylose, in which a fraction of the primary hydroxyl groups is specifically methylated, has been prepared by the following sequence of reactions: amylose → 6- O-tritylamylose → 2,3-di- O-benzoyl-6- O-tritylamylose → 2,3-di- O-benzoylamylose → 2,3-di- O-benzoyl-6- O-methylamylose → 6- O-methylamylose. Two such preparations of 6- O-methylamylose, having D.S. 0.2 and D.S. 0.4, were examined as a model substrates for a number of starch-degrading enzymes. Neither was detectably attacked by sweet-potato β-amylase or potato phosphorylase. Some hydrolysis (much greater for the 6- O-methylamylose of D.S. 0.2 than for that of D.S. 0.4) did occur with crystalline α-amylase preparations from hog pancreas and Bacillus subtilis. Relatively extensive hydrolysis was effected by an Aspergillus niger enzyme preparation containing both α-amylase and amyloglucosidase. Products of the action of the pancreatic and Bacillus subtilis amylases included maltose and higher saccharides, but neither d-glucose nor 6- O-methyl- d-glucose was detected. Products obtained with the A. niger enzyme preparation included d-glucose and a saccharide tentatively identified as 4- O-(6- O-methyl-α- d-glucopyranosyl)- d-glucose; 6- O-methyl- d-glucose was not detected. The failure of the exo-enzymes to effect any hydrolysis, and the extent of hydrolysis and the nature of the products formed by the two crystalline α-amylases, demonstrate that hydrolysis is blocked in the vicinity of the methylated primary hydroxyl groups. The results suggest that those limitations on amylase action normally attributed to the bulky (1→6)- d-glucose branch are also imposed by a small group, namely, the methyl group.

Inhibition of the condensing enzyme of Aspergillus niger by magnesium.

Stern and Ochoa1,2 found that magnesium (or manganese) was required for maximum activity of the ‘condensing enzyme’ (oxalacetate + acetylcoenzyme A ⇌ citrate + coenzyme A) isolated from pigeon liver, and used an assay system containing 4 µM magnesium chloride per ml. The same or similar systems have been used by others in comparing enzyme-levels in different organisms2–5 and by us in studies of the ‘condensing enzyme’ of Aspergillus niger 6,7. In later work with the A. niger enzyme, we found that magnesium ions were not required and that they were actually inhibitory. Inhibition of the A. nigerenzyme by magnesium ions differentiates it from the avian enzyme and suggests that some of the lower specific activities reported for enzyme preparations from other organisms3,5 may reflect the same phenomenon.