Aspergillus Niger Glucoamylase Research Articles

Growth-rate-independent production of recombinant glucoamylase by Fusarium venenatum JeRS 325.

Most recombinant proteins generated in filamentous fungi are produced in fed-batch cultures, in which specific growth rate normally decreases progressively with time. Because of this, such cultures are more suited to the production of products that are produced efficiently at low-growth rates (e.g., penicillin) than to products which are produced more efficiently at high-growth rates (e. g., glucoamylase). Fusarium venenatum A3/5 has been transformed (JeRS 325) to produce Aspergillus niger glucoamylase (GAM) under the control of the Fusarium oxysporum trypsin-like protease promoter. No glucoamylase was detected in the culture supernatant during exponential growth of F. venenatum JeRS 325 in batch culture. In glucose-limited chemostat cultures, GAM concentration increased with decrease in dilution rate, but the specific production rate of GAM (g GAM [g biomass](-1) h(-1)) remained approximately constant over the dilution-rate range 0.05 h to 0.19 h(-1), i.e., the recombinant protein was produced in a growth-rate-independent manner. The specific production rate decreased at dilution rates of 0.04 h(-1) and below. Specific production rates of 5.8 mg and 4.0 mg GAM [g biomass](-1) h(-1) were observed in glucose-limited chemostat cultures in the presence and absence of 1 g mycological peptone L(-1). Compared to production in batch culture, and for the same final volume of medium, there was no increase in glucoamylase production when cultures were grown in fed-batch culture. The results suggested that a chemostat operated at a slow dilution rate would be the most productive culture system for enzyme production under this trypsin-like promoter.

Morphology and enzymatic degradation of thermoplastic starch-polycaprolactone blends

This study's aim was to evaluate the effect of processing conditions on the morphology and enzymatic degradation of 50/50 (w/w) thermoplastic starch–polycaprolactone blends. The blends, produced from native potato starch, glycerol, and polycaprolactone in a melt mixer using different mixing speeds and temperatures, were cocontinuous, and the blends were very homogeneous. Enzymatic hydrolysis was performed using Bacillus licheniformis alpha-amylase and Aspergillus niger glucoamylase on both milled and intact samples. The thin layer of polycaprolactone (≈ 5 μm) formed on the surface of the thermoplastic starch–polycaprolactone blends during compression molding strongly reduced the rate of enzymatic hydrolysis. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 2594–2604, 1999

Steady-state inhibitory kinetic studies on the ligand binding modes of Aspergillus niger glucoamylase.

Inhibitory activities of 1-deoxynojirimycin and gluconolactone on Aspergillus niger glucoamylase were studied in relation to the subsite structure of the enzyme. Although both of these inhibitors are considered to bind at subsite 1 of the enzyme active site, 1-deoxynojirimycin showed competitive type inhibition but gluconolactone was a mixed type (or noncompetitive type) inhibitor for the hydrolysis of p-nitrophenyl alpha-D-glucoside. The former type of inhibition suggested that the main binding mode of the substrate was productive, but the latter, nonproductive. A possible way of explaining these apparent inconsistent results is to assume that the main binding mode of the substrate is productive and gluconolactone forms a nonproductive ternary complex with the enzyme and the substrate.

Possibility for discriminating between two representative non two-state thermal unfolding models of proteins by DSC.

Possible differences between two representative non two-state thermal unfolding mechanisms of protein are discussed concerning differential scanning calorimetry. Numerical simulations showed that, by DSC measurement, it is hard to discriminate between the independent model, which assumes independent unfolding domains in a protein, and the sequential model, which assumes intermediate(s) between native and denatured states, especially when values of molecular weight, denaturation enthalpy, and difference in denaturation temperature of each denaturation process are large. DSC curve analysis of Aspergillus niger glucoamylase based on these two models gave essentially the same thermodynamic parameters.

GENFOLD: a genetic algorithm for folding protein structures using NMR restraints.

We report the development and validation of the program GENFOLD, a genetic algorithm that calculates protein structures using restraints obtained from NMR, such as distances derived from nuclear Overhauser effects, and dihedral angles derived from coupling constants. The program has been tested on three proteins: the POU domain (a small three-helix DNA-binding protein), bovine pancreatic trypsin inhibitor (BPTI), and the starch-binding domain from Aspergillus niger glucoamylase I, a 108-residue beta-sheet protein. Structures were calculated for each protein using published NMR restraints. In addition, structures were calculated for BPTI using artificial restraints generated from a high-resolution crystal structure. In all cases the fittest calculated structures were close to the target structure, and could be refined to structures indistinguishable from the target structures by means of a low-temperature simulated annealing refinement. The effectiveness of the program is similar to that of distance geometry and simulated annealing methods, and it is capable of using a very wide range of restraints as input. It can thus be readily extended to the calculation of structures of large proteins, for which few NOE restraints may be available.

Thermal unfolding of the starch binding domain of Aspergillus niger glucoamylase.

A fragment of the starch-binding domain (SBDF) of Aspergillus niger glucoamylase was prepared using recombinant DNA techniques, and its thermal unfolding was investigated by high-sensitivity differential scanning calorimetry (DSC). Thermal unfolding of SBDF was found to be reversible at pH 7 as expected from a DSC study of the whole enzyme molecule [Tanaka A. et al., J. Biochem., 117, 1024-1028 (1995)] but not reversible at acidic region. Numerical analysis of the DSC curves showed that the denaturation was two-state, and some of the SBDF molecules were oligomeric (average degree of oligomerization was 1.2) at pH 7. It was suggested that the denaturation temperature of SBDF was lower than that of the starch-binding domain in the whole enzyme molecule by about 4.5 degrees (decrease in the Gibbs energy change was 5.3 kJ mol-1) indicating a possibility that the starch-binding domain is stabilized by glycosylation of the domain itself, or by the highly glycosylated linker region.

Saccharification of cassava flour starch in a hollow-fiber membrane reactor

The enzymatic saccharification of cassava flour starch with Aspergillus niger glucoamylase was studied in a hollow-fiber enzymatic membrane reactor (HF-EMR). The optimization of the process was also investigated. The substrate was previously gelatinized by extrusion that produced a substrate with a degree of gelatinization and physical properties suitable for the enzymatic process. Typically, continuous saccharification of cassava flour starch in the HF-EMR produced a highly pure product in permeate (i.e., 99.6% glucose) with a global starch to glucose conversion of 97.3%. Results from a factorial experimental design revealed that retention time, enzyme to substrate ratio, and substrate concentration were the variables that most affected the process for starch to glucose conversion and for solid accumulation rate. Empirical models that describe this process were experimentally determined and compared with a theoretical model based on a balance of materials and enzyme kinetics. They were shown to adequately predict the performance of the HF-EMR.

Kinetics and equilibria of condensation reactions between monosaccharide pairs catalyzed by Aspergillus niger glucoamylase

Arabinose, fructose, galactose, myo-inositol, lyxose, mannose, ribose, and xylose were incubated individually and with glucose in the presence of Aspergillus niger glucoamylase at pH 4.5 and 45°C. Glucoamylase condenses galactose, glucose, and mannose individually into disaccharides. It also produces mixed disaccharides when each of the eight carbohydrates is incubated with glucose. Many products were identified by gas chromatography of the derivatized reaction mixtures followed by mass spectroscopy of the individual chromatographic peaks. Galacto-, gluco-, or mannopyranosyl rings appear to be present at the nonreducing ends of all the disaccharides produced. Molecules linked through primary hydroxyl groups have the highest equilibrium constants of all products formed, since these bonds are thermodynamically favored. However, glucoamylase is capable of forming bonds with many available hydroxyl groups, as previously demonstrated when it was incubated with glucose alone. Formation rates of different bonds linking different residues vary widely. These results demonstrate that glucoamylase has a wide selectivity toward residues it will condense into disaccharides and toward bonds it will form between them. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 9–22, 1997.

Kinetics and equilibria of condensation reactions between monosaccharide pairs catalyzed byAspergillus niger glucoamylase

Arabinose, fructose, galactose, myo-inositol, lyxose, mannose, ribose, and xylose were incubated individually and with glucose in the presence of Aspergillus niger glucoamylase at pH 4.5 and 45 degrees C. Glucoamylase condenses galactose, glucose, and mannose individually into disaccharides. It also produces mixed disaccharides when each of the eight carbohydrates is incubated with glucose. Many products were identified by gas chromatography of the derivatized reaction mixtures followed by mass spectroscopy of the individual chromatographic peaks. Galacto-, gluco-, or mannopyranosyl rings appear to be present at the nonreducing ends of all the disaccharides produced. Molecules linked through primary hydroxyl groups have the highest equilibrium constants of all products formed, since these bonds are thermodynamically favored. However, glucoamylase is capable of forming bonds with many available hydroxyl groups, as previously demonstrated when it was incubated with glucose alone. Formation rates of different bonds linking different residues vary widely. These results demonstrate that glucoamylase has a wide selectivity toward residues it will condense into disaccharides and toward bonds it will form between them.

Solution structure of the granular starch binding domain of Aspergillus niger glucoamylase bound to beta-cyclodextrin.

Carbohydrate-binding domains are usually small and physically separate from the catalytic domains of hydrolytic enzymes. Glucoamylase 1 (G1) from Aspergillus niger, an enzyme used widely in the food and brewing industries, contains a granular starch binding domain (SBD) which is separated from the catalytic domain by a semi-rigid linker. The aim of this study was to determine how the SBD binds to starch, and thereby more generally to throw light on the role of carbohydrate-binding domains in the hydrolysis of insoluble polysaccharides. The solution structure of the SBD of A. niger G1 bound to beta-cyclodextrin (betaCD), a cyclic starch analogue, shows that the well-defined beta-sheet structure seen in the free SBD is maintained in the SBD-betaCD complex. The main differences between the free and bound states of the SBD are observed in loop regions, in or near the two starch-binding sites. The two binding sites, each of which binds one molecule of betaCD, are structurally different. Binding site 1 is small and accessible, and its structure changes very little upon ligand binding. Site 2 is longer and undergoes a significant structural change on binding. Part of this site comprises a flexible loop, which appears to allow the SBD to bind to starch strands in a range of orientations. The two starch-binding sites of the SBD probably differ functionally as well as structurally; site 1 probably acts as the initial starch recognition site, whereas site 2 is involved in specific recognition of appropriate regions of starch. The two starch strands are bound at approximately 90 degrees to each other. This may be functionally important, as it may force starch strands apart thus increasing the hydrolyzable surface, or alternatively it may localize the enzyme to noncrystalline (more hydrolyzable) areas of starch. The region of the SBD where the linker to the catalytic domain is attached is flexible, allowing the catalytic site to access a large surface area of the starch granules.

Glucoamylase mutants in the conserved active-site segment Trp170-Tyr175 located at a distance from the site of catalysis.

To mimic the structure of the 1.8-fold more active (k(cat)) Rhizopus oryzae glucoamylase (GA), Aspergillus niger GA was subjected to site-directed mutagenesis in the Trp170-Tyr175 segment of the third of the six well-conserved alpha-->alpha connecting loops of the catalytic (alpha/alpha)6-barrel. While the Trp170-->Phe, Gln172-->Asn and Tyr175-->Phe mutants showed an up to 1.7-fold increased k(cat) and Gly174-->Cys GA and approximately 2-fold reduced k(cat) towards maltotriose and longer substrates, Asn171-->Ser, Thr173-->Gly and A.niger wild-type GA had very similar kcat and K(m) values for the hydrolysis of isomaltose and the malto-oligosaccharides of DP 2-7. Crystal structures of pseudotetrasaccharide inhibitor complexes of Aspergillus awamori var. X100 GA, which is 94% identical to A.niger GA, indicate that Tyr175 is located at binding subsite 4, while the preceding target residues and the high-mannose type unit on Asn171 are at a larger distance from the site of catalysis. The mutations had a modest effect on thermostability; the temperature for 50% inactivation, Tm, was thus unchanged for Tyr175 -->Phe GA and reduced by 0.2-2.9 degrees C for the other mutants. The deletion of the N-linked high-mannose unit-in Asn171 -->Ser and Thr173-->Gly GAs-appeared to be of minor importance for enzyme activity and thermostability, and did not increase the sensitivity to proteolysis.

Expression of human recombinant β 2-microglobulin by Aspergillus nidulans and its activity

The light chain of HLA class I protein (β 2m) has been expressed in Aspergillus nidulans. The cDNA of β 2m was modified using the polymerase chain reaction to include overlapping extensions for its subsequent fusion into an Aspergillus vector. This fusion resulted in β 2m cDNA being flanked by the Aspergillus awamori glucoamylase promoter and the Aspergillus niger glucoamylase terminator. Expression of β 2m was induced by the addition of starch to the culture medium. In preliminary mass culture trials, 117 μg/liter of fβ 2m were obtained in 60-liter fermentations. N-terminal sequencing of purified human β 2m produced in fungi (fβ 2m) revealed that 28% of the purified protein was of proper sequence and 61% of the protein had an additional serine and lysine residue derived from the C-terminus of the fungal leader. Purified fβ 2m from culture supernatants appeared biochemically similar to β 2m obtained from human urine (uβ 2m) as seen in immunoblot analysis. Functionally, fβ 2m effectively interacted as a subunit of class I MHC molecules. This was seen both in a sandwich ELIS A for detecting properly folded HLA class I heavy chain and in assays showing cell-surface β 2m exchange into the mouse class I MHC H-2K d. In these experiments the biological activity of fβ 2m was indistinguishable from uβ 2m. The successful expression of biologically active β 2m in A. nidulans suggests that fungal systems might be useful for the production of other active components of the HLA class I MHC complex.

Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains.

A study was carried out to obtain more insight into the parameters that determine the secretion of heterologous proteins from filamentous fungi. A strategy was chosen in which the mRNA levels and protein levels of a number of heterologous genes of different origins were compared. All genes were under control of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) expression signals and were integrated in a single copy at the A. awamori pyrG locus. A Northern (RNA) analysis showed that large differences occurred in the steady-state mRNA levels obtained with the various genes; those levels varied from high values for genes of fungal origin (A. awamori 1,4-beta-endoxylanase A, Aspergillus niger glucoamylase, and Thermomyces lanuginosa lipase) to low values for genes of nonfungal origin (human interleukin 6 and Cyamopsis tetragonoloba [guar] alpha-galactosidase). With the C. tetragonoloba alpha-galactosidase wild-type gene full-length mRNA was even undetectable. Surprisingly, small amounts of full-length mRNA could be detected when a C. tetragonoloba alpha-galactosidase gene with an optimized Saccharomyces cerevisiae codon preference was expressed. In all cases except human interleukin 6, the protein levels corresponded to the amounts expected on basis of the mRNA levels. For human interleukin 6, very low protein levels were observed, whereas relatively high steady-state mRNA levels were obtained. Our data suggest that intracellular protein degradation is the most likely explanation for the low levels of secreted human interleukin 6.

Steady-state kinetic and calorimetric studies on the binding of Aspergillus niger glucoamylase with gluconolactone, 1-deoxynojirimycin, and beta-cyclodextrin.

Binding equilibria of Aspergillus niger glucoamylase with its several ligands were observed to analyze the binding modes of the ligands. Steady-state kinetic studies using p-nitrophenyl alpha-glucoside as a substrate showed that 1-deoxynojirimycin, which is a mixed type inhibitor for Rhizopus glucoamylase, was a competitive type inhibitor bound at the active site of the enzyme, but gluconolactone, which is also a mixed type inhibitor for Rhizopus glucoamylase, was a non-competitive type inhibitor forming a nonproductive ternary complex with the enzyme and the substrate. beta-Cyclodextrin, which binds to the starch-binding domain of the enzyme, did not inhibit the enzyme activity, showing that there was no interaction between the catalytic domain and the starch-binding domain for the binding of the substrate and beta-cyclodextrin. Isothermal titration calorimetry showed that one 1-deoxynojirimycin molecule and two beta-cyclodextrin molecules bind to the catalytic domain and the starch-binding domain of the enzyme, respectively, and there is no significant interaction between the binding of these ligands.

Chemoenzymatic synthesis of [formula omitted]: their binding to Aspergillus niger glucoamylase G1 and its starch-binding domain

A coupling reaction of cyclodextrin glucosyltransferase (CGTase) with glucose and 6-deoxy-6-iodo-cyclomaltoheptaose ( 1), in the presence of glucoamylase, followed by acetylation, led to a convenient synthesis of acetylated 6 III-deoxy-6 III-iodo-maltotriose ( 2) and 6 IV-deoxy-6 IV-iodo-maltotetraose ( 3). Nucleophilic displacement of the iodine atom of these protected maltotriose and maltotetraose analogs by the activated form of 2,3,4,6-tetra-O- acetyl-1-S- acetyl-1-thio-α- d-glucose ( 4) afforded peracetylated 6 III -S- α- d-glucopyranosyl-6 III-thiomaltotriose ( 5) and 6 IV -S- α- d-glucopyranosyl-6 IV-thiomaltotetraose ( 6) in high yield. The interaction of OH-free tetra- and penta-saccharides ( 7 and 8) with both glucoamylase G1 from Aspergillus niger as well as its isolated starch-binding domain fragment were studied by UV difference spectroscopy. It was found that the starch-binding domain has higher affinity for 7 and 8 than for maltotetraose and maltopentaose.

Differential Scanning Calorimetric Studies on the Domain Structure of Aspergillus Glucoamylase1

Thermal unfolding of two forms of Aspergillus niger glucoamylase was observed by adiabatic differential scanning calorimetry (DSC) at pH 7. The DSC traces of the larger form of the enzyme, G1, and the shorter form which lacks the C-terminal starch-binding domain of G1, G2, could be resolved by assuming two-state unfolding of five and four independent components, respectively. The thermal unfolding of the starch-binding domain was found to be reversible, but that of the catalytic domain was irreversible. The DSC observation of G1 and G2 in the presence of beta-cyclodextrin or 1-deoxynojirimycin showed that there was no domain interaction between the starch-binding domain and catalytic domain.

Biodegradation of Starch-Based Materials

Abstract The aim of this study was to compare three different test methods for assaying the biodegradability of starch-based materials. The materials tested included some commercial starch-based materials and thermoplastic starch film prepared by extrusion from native potato starch and glycerol. Enzymatic hydrolysis was performed using excess Bacillus licheniformis α-amylase and Aspergillus niger glucoamylase at 37°C. The degree of degradation was assayed by measuring the dissolved carbohydrates and the weight loss of the samples. The head-space test was based on carbon dioxide evolution using sewage sludge as an inoculum. The composting experiments were carried out in an insulated commercial composter bin. The degradation was evaluated visually at weekly intervals, and the weight loss of the samples was measured after composting. Good correlation was found among the three different test methods.

Expression and secretion of Aspergillus niger glucoamylase in Saccharomyces cerevisiae

Aspergillus niger glucoamylase GA 1 cDNA was inserted in between the yeast PGK promoter and terminator on plasmid pMA91. The resultant plasmid pMAG69 was introduced into Saccharomyces cerevisiae GRF18 by protoplast transformation. The A niger GA I cDNA was expressed efficiently under the contiol of PGK promoter and 99% of the gene products were secreted into the culture medium using its own signal sequence The recombmant yeast can digest 87% of starch in 2 d in the medium containing 10% starch. The recombinant plasmid pMAG69 can exist stably in 5. cerevisiae.

Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase.

Both native Aspergillus niger glucoamylase and wild-type Aspergillus awamori glucoamylase expressed in Saccharomyces cerevisiae, which have identical primary structures, undergo hydrolysis at aspartyl bonds at low pH values and elevated temperatures. In native A.niger enzyme the Asp126-Gly127 bond was preferentially cleaved at pH 3.5, while at pH 4.5 cleavage of the Asp257-Pro258 and Asp293-Gly294 bonds was dominant. In wild-type A.awamori glucoamylase, cleavage of the latter was dominant at both pH 3.5 and 4.5. Site-directed mutations Asp126-->Glu and Gly127-->Ala in wild-type enzyme decreased specific activities by approximately 60 and 30%, respectively, and increased irreversible thermoinactivation rates 3- to 4-fold at pH 4.5. Replacement of Asp257 with Glu and Asp293 with Glu or Gln decreased specific activities by approximately 20%, but greatly reduced cleavage of the Asp257-Pro258 and Asp293-Gly294 bonds. The Asp257-->Glu mutant was produced very slowly and was more thermostable than wild-type glucoamylase at pH 4.5 up to 70 degrees C. Replacement of Asp293 with either Glu or Gln significantly raised protein production and slightly increased thermostability at pH 3.5 and 4.5, but not at pH 5.5.

A novel glucoamylase preparation for grain mash saccharification

The capacity to saccharify barley grain mash of Hormoconis resinae glucoamylase P produced by a heterologous host, Trichoderma reesei, was compared with that of Aspergillus niger glucoamylase. The results showed that the glucoamylase P secreted by T. reesei produces more fermentable sugars from mash and thus makes a higher ethanol yield possible in fermentation.