Cell Bound α-Amylase in Aspergillus oryzae

It was previously reported that α-amylase accumulation is caused within the mycelium grown in a phosphate deficient medium and the concentration of anions or pH in a surrounding medium is responsible for its liberation. As it was subsequently found that α-amylase liberation from the mycelium of Aspergillus oryzae is stimulated by peptone, an attempt was made on purification of effective substances from it. The present paper describes on purification and properties of phosphopeptides found as an effective substance for α-amylase liberation, and discusses on the stimulation effect, comparing with the effects on pH and concentration of anions which were previously observed.     

Improvement of Heterologous Protein Production in Aspergillus oryzae by RNA Interference with α-Amylase Genes

Aspergillus oryzae RIB40 has three α-amylase genes (amyA, amyB, and amyC), and secretes α-amylase abundantly. However, large amounts of endogenous secretory proteins such as α-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of α-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three α-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of α-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in α-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of α-amylase is effective in heterologous protein production in A. oryzae.     

On the Inducers of α-Amylase Formation In Aspergillus oryzae

In a previous paper it has been described that α-amylase formation in Aspergillus oryzae is stimulated by soluble starch, glycogen and maltose, whereas it is inhibited by glucose, which is added into a growing medium or a secondary incubation medium as the carbon source. The present paper reports that isomaltose and panose are the most effective inducers among a large number of sugars examined here, and suggests the importance of transglucosidase action demonstrated in view of α-amylase formation. The initial action of inducers in this system is also discussed.     

Overproduction of an α-Amylase/Glucoamylase Fusion Protein in Aspergillus oryzae Using a High Expression Vector

The systemic immune response against orally administered antigens is suppressed (oral tolerance), and this has been postulated to avoid excess immunity against dietary constituents which are present in large amounts in the gastrointestinal tract. Taking into consideration that such orally administered protein antigens are subjected to enzymatic degradation in the gastrointestinal tract, we examined whether an enzymatic digest of milk proteins could induce oral tolerance. A tryptic digest of casein, containing mainly fragments smaller than 6000 Da, was fed to mice as a constituent of their diet. Mice fed with the casein-digest diet responded poorly to subsequent immunization with casein, indicating that oral tolerance to casein was induced in these animals. The results suggest the presence of immunosuppressive fragment(s) in the casein digest, which may be of use for preventing milk allergy.     

Independent duplications of α-amylase in different strains of Aspergillus oryzae

Aspergillus oryzae is a filamentous fungus that has arisen through the ancient domestication of Aspergillus flavus for making traditional oriental foods and beverages. In the many centuries A. oryzae has been used for fermenting the starch in rice to simple sugars, it has undergone selection for increased secretion of starch-degrading enzymes. In particular, all A. oryzae strains investigated thus far have two or more copies of a gene encoding α-amylase, whereas A. flavus has only one. Here we investigate the duplications leading to these copies in three A. oryzae strains. We find evidence of at least three separate duplications of α-amylase, an example of parallel evolution in a micro-organism under artificial selection. At least two of these duplications appear to be associated with activity of transposable elements of the Tc1/mariner class. Both involve a 9.1 kb element that terminates in inverted repeats, encodes a putative transposase and another putative protein of unknown function, and contains an unusual arrangement of four short internal imperfect repeats. Although "unusual Mariners" of this size have previously been identified in A. oryzae, Aspergillus fumigatus and Aspergillus nidulans, this is the first evidence we know of that at least some of them are active in modern times and that their activity can contribute to beneficial genetic changes.     

Expression of Psychrophilic Genes in Mesophilic Hosts: Assessment of the Folding State of a Recombinant α-Amylase

α-Amylase from the antarctic psychrophile Alteromonas haloplanktis is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in Escherichia coli, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18°C.Psychrophilic enzymes possess specific properties, such as high activity at low temperatures and weak thermal stability, which promise to allow the use of these enzymes as industrial biocatalysts, as biotechnological tools, or for fundamental research (6, 8, 11). For instance, substantial energy savings can be obtained if heating is not required during large-scale processes which take advantage of the efficient catalytic capacity of cold-adapted enzymes in the range 0 to 20°C. The pronounced heat lability of psychrophilic enzymes also allows their selective inactivation in a complex mixture, as illustrated by an antarctic bacterial alkaline phosphatase which is available for molecular biology research (7). Finally, psychrophilic enzymes represent the lower natural limit of protein stability (3) and are useful tools for studies in the field of protein folding.Large-scale fermentation of psychrophilic microorganisms suffers from two main drawbacks, however: the low production levels of wild strains and the prohibitive cost of growing wild strains at low temperatures. A possible alternative is to overexpress the gene coding for a psychrophilic protein in a mesophilic host for which efficient expression systems have been designed. In this context, two crucial questions remain to be solved: (i) what is the folding state of an enzyme normally synthesized at 0°C when it is expressed by the mesophilic genetic machinery at higher temperatures, and (ii) is there a temperature at which a compromise can be reached between the stability of the psychrophilic enzyme and the mesophilic growth rate? To address these questions, the heat-labile α-amylase from the antarctic psychrophile Alteromonas haloplanktis (2, 4) was expressed in Escherichia coli at various temperatures.

Construction of the expression vector and α-amylase production.

The α-amylase gene (2) was cloned downstream from the lacZ promoter in pUC12 by ligating the SmaI site of the polylinker to the HpaI site located 60 nucleotides upstream from the formylmethionine codon. This construction is devoid of the C-terminal peptide cleaved by the wild strain following α-amylase secretion. The recombinant enzyme was expressed in E. coli RR1 with the constitutive assistance of lacZ (without IPTG [isopropyl-β-d-thiogalactopyranoside] induction) in a medium containing 16 g of bactotryptone, 16 g of yeast extract, 5 g of NaCl, 2.5 g of K₂HPO₄, 0.1 μM CaCl₂, and 100 mg of ampicillin per liter. The effect of the culture temperature on α-amylase production by E. coli is illustrated in Fig. ​Fig.1.1. Within the range of temperatures used, maximal enzyme production was reached below 18°C, whereas higher temperatures induced a gradual decrease of α-amylase activity in cultures. Three independent cultures were pooled for the purification of the recombinant enzymes produced at 18 and 25°C. Open in a separate windowFIG. 1Temperature dependence of α-amylase production by E. coli. Results are expressed as percent mean maximal activity recorded at 18°C.

α-Amylase purification.

The gram-negative A. haloplanktis was cultivated at 4°C, and α-amylase was purified from the culture supernatants by ion-exchange chromatography on DEAE-agarose followed by gel filtration on Sephadex G-100 and Ultrogel AcA54 as previously described (2, 4). The recombinant α-amylases were purified by the protocol developed for the wild-type enzyme except that concentration by ammonium sulfate precipitation at 70% saturation was required before the first chromatographic step. Recombinant enzyme production at 18 and 25°C ranged between 60 and 100 mg/liter of culture, which corresponds to a 10-fold improvement over production by the wild strain.

Characterization of the recombinant α-amylases.

N- and C-terminal amino acid sequences (determined on an Applied Biosystems Procise analyzer and by carboxypeptidase Y digestion, respectively) of α-amylase produced at 18 and 25°C indicated that the signal peptide is correctly cleaved in E. coli and that no additional posttranslational cleavage occurred. The isoelectric point (5.5) and the molecular mass (49,340 Da as determined from the sequence and 49,342 ± 8 Da as determined from electrospray mass spectroscopy measurements) were identical to the values recorded for the wild-type enzyme. Dynamic light scattering (DynaPro-801; DLS Instruments) also showed that the purified recombinant enzymes are homogeneous, without any evidence of aggregated forms.

Comparison of the wild-type and recombinant α-amylases.

Several properties of the wild-type enzyme produced at 4°C and the recombinant α-amylase expressed in E. coli at 18°C were compared (Table ​(Table1).1).

TABLE 1

Kinetic parameters, dissociation constants, and free thiol groups for the wild-type and recombinant α-amylases

Chemical Modification of Amino Groups of Acid-stable α-Amylase and Acid-unstable α-Amylase from Aspergillus niger

Monochlorotrifluoro-p-benzoquinone (CFQ) was used for investigating the state of the amino groups of acid-stable α-amylase and acid-unstable α-amylase. About half of the total amino groups in both enzyme molecules were reacted with the reagent. The unreactive amino groups seemed to exist in a different state from the reactive ones. Both enzymes whose amino groups were modified by CFQ still maintained the α-phenylmaltosidase activity in spite of losing or decreasing the amylase activity. These facts suggest that the amino groups of both enzymes were not in the active site but the modification of them caused steric hindrance.

The pH-stability of the acid-unstable α-amylase whose one or two amino groups were modified with succinic anhydride or 2,4,6-trinitrobenzene-l-sulfonate (TNBS) increased on the acidic side and decreased on the alkaline side, but further modification of them led to decrease the stability on both sides.     

Induction of β-N-acetylhexosaminidase in Aspergillus oryzae

Summary Extracellular -N-acetylhexosaminidase in basic specific activity 1.5 U/mg protein was induced 15 – 35 times (up to 50 U/mg protein) by mixture of chitooligomers (crude chitin hydrolysate), 10 – 20 times (20 – 30 U/mg protein) by N-acetylglucosamine, and 10 times (14 U/mg protein) by chitosan in Aspergillus oryzae. Addition of NaCl (15 – 23 g/l) to the cultivation medium enhanced the induction in 10 – 20 %.     

In Vivo Expression of UDP-N-Acetylglucosamine: α-3-D-Mannoside β-1,2-N-Acetylglucosaminyltransferase I (GnT-1) in Aspergillus oryzae and Effects on the Sugar Chain of α-Amylase

UDP-N-Acetylglucosamine: α-3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) is an essential enzyme in the conversion of high mannose type oligosaccharide to the hybrid or complex type. The full length of the rat GnT-I gene was expressed in the filamentous fungus Aspergillus oryzae. A microsomal preparation from a recombinant fungus (strain NG) showed GnT-I activity that transferred N-acetylglucosamine residue to acceptor heptaose, Man₅GlcNAc₂. The N-linked sugar chain of α-amylase secreted by the strain showed a peak of novel retention on high performance liquid chromatography that was same as a reaction product of in vitro GnT-1 assay. The peak of oligosaccharide disappeared on HPLC after β-N-acetylglucosaminidase treatment. Mass analysis supported the presence of GlcNAcMan₅GlcNAc₂ as a sugar chain of α-amylase from strain NG. Chimera of GnT-I with green fluorescent protein (GFP) showed a dotted pattern of fluorescence in the mycelia, suggesting localization at Golgi vesicles. We concluded that GnT-1 was functionally expressed in A. oryzae cells and that N-acetylglucosamine residue was transferred to N-glycan of α-amylase in vivo. A. oryzae is expected to be a potential host for the production of glycoprotein with a genetically altered sugar chain.     

α-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans

The effect of biomass concentration on the formation of Aspergillus oryzaeα-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of α-amylase formation in chemostats decreased significantly with increasing biomass concentration in the range of approx. 2–12 g dry weight kg⁻¹. When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzaeα-amylase gene (creA) was deleted, no significant decrease in the specific rate of α-amylase formation was observed. On the basis of the experimental results, it is suggested that the low value of the specific α-amylase productivity observed at high biomass concentration is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium. Received: 13 January 2000 / Received revision: 30 June 2000 / Accepted: 1 July 2000     

In vivo imaging of endoplasmic reticulum and distribution of mutant α-amylase in Aspergillus oryzae

Properly folded proteins destined for secretion exit through a specific subdomain of the endoplasmic reticulum (ER) known as transitional ER (tER) sites or ER exit sites (ERES). While such proteins in filamentous fungi localize at the hyphal tips overlapping the Spitzenk?rper, the distribution of misfolded proteins remains unknown. In the present study, we analyzed the distribution of mutant protein as well as ER and tER sites visualized by expression of AoClxA and AoSec13 fused with fluorescent protein, respectively, in the filamentous fungus Aspergillus oryzae. Discrete tER subdomains were visualized as the punctate dots of AoSec13 overlapping or associated with AoClxA distribution. Both ER and tER sites were concentrated near hyphal tips and formed apical gradients. Interestingly, while the expression of wild-type α-amylase fusion protein (AmyB-mDsRed) showed its localization coinciding with the Spitzenk?rper, a disulfide bond-deletion in AmyB causing its misfolding resulted in its accumulation in the subapical and basal ER, creating a reciprocal gradient to the tER sites. Furthermore, the reciprocal gradient enabled a clear distinction between the tER sites and the mutant AmyB accumulation sites near the apex. Based on these findings, we conclude that A. oryzae accumulates aberrant proteins toward basal hyphae while maintaining polarized tER sites for secretion of properly folded proteins at the hyphal tip.     

Cloning,Sequencing, and Expression of the Genes Encoding an Isocyclomaltooligosaccharide Glucanotransferase and an α-Amylase from a Bacillus circulans Strain

The gene for a novel glucanotransferase, isocyclomaltooligosaccharide glucanotransferase (IgtY), involved in the synthesis of a cyclomaltopentaose cyclized by an α-1,6-linkage [ICG5; cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}] from starch, was cloned from the genome of B. circulans AM7. The IgtY gene, designated igtY, consisted of 2,985 bp encoding a signal peptide of 35 amino acids and a mature protein of 960 amino acids with a calculated molecular mass of 102,071 Da. The deduced amino-acid sequence showed similarities to 6-α-maltosyltransferase, α-amylase, and cyclomaltodextrin glucanotransferase. The four conserved regions common in the α-amylase family enzymes were also found in this enzyme, indicating that this enzyme should be assigned to this family. The DNA sequence of 8,325-bp analyzed in this study contained two open reading frames (ORFs) downstream of igtY. The first ORF, designated igtZ, formed a gene cluster, igtYZ. The amino-acid sequence deduced from igtZ exhibited no similarity to any proteins with known or unknown functions. IgtZ was expressed in Escherichia coli, and the enzyme was purified. The enzyme acted on maltooligosaccharides that have a degree of polymerization (DP) of 4 or more, amylose, and soluble starch to produce glucose and maltooligosaccharides up to DP5 by a hydrolysis reaction. The enzyme (IgtZ), which has a novel amino-acid sequence, should be assigned to α-amylase. It is notable that both IgtY and IgtZ have a tandem sequence similar to a carbohydrate-binding module belonging to a family 25. These two enzymes jointly acted on raw starch, and efficiently generated ICG5.     

Cloning and High-Level Expression of α-Galactosidase cDNA from Penicillium purpurogenum

The cDNA coding for Penicillium purpurogenum α-galactosidase (αGal) was cloned and sequenced. The deduced amino acid sequence of the α-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic αGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides.α-Galactosidase (αGal) (EC 3.2.1.22) is of particular interest in view of its biotechnological applications. αGal from coffee beans demonstrates a relatively broad substrate specificity, cleaving a variety of terminal α-galactosyl residues, including blood group B antigens on the erythrocyte surface. Treatment of type B erythrocytes with coffee bean αGal results in specific removal of the terminal α-galactosyl residues, thus generating serological type O erythrocytes (8). Cyamopsis tetragonoloba (guar) αGal effectively liberates the α-galactosyl residue of galactomannan. Removal of a quantitative proportion of galactose moieties from guar gum by αGal improves the gelling properties of the polysaccharide and makes them comparable to those of locust bean gum (18). In the sugar beet industry, αGal has been used to increase the sucrose yield by eliminating raffinose, which prevents normal crystallization of beet sugar (28). Raffinose and stachyose in beans are known to cause flatulence. αGal has the potential to alleviate these symptoms, for instance, in the treatment of soybean milk (16).αGals are also known to occur widely in microorganisms, plants, and animals, and some of them have been purified and characterized (5). Dey et al. showed that αGals are classified into two groups based on their substrate specificity. One group is specific for low-M_r α-galactosides such as pNPGal (p-nitrophenyl-α-d-galactopyranoside), melibiose, and the raffinose family of oligosaccharides. The other group of αGals acts on galactomannans and also hydrolyzes low-M_r substrates to various extents (6).We have studied the substrate specificity of αGals by using galactomanno-oligosaccharides such as Gal³Man₃ (6³-mono-α-d-galactopyranosyl-β-1,4-mannotriose) and Gal³Man₄ (6³-mono-α-d-galactopyranosyl-β-1,4-mannotetraose). The structures of these galactomanno-oligosaccharides are shown in Fig. ​Fig.1.1. Mortierella vinacea αGal I (11) and yeast αGals (29) are specific for the Gal³Man₃ having an α-galactosyl residue (designated the terminal α-galactosyl residue) attached to the O-6 position of the nonreducing end mannose of β-1,4-mannotriose. On the other hand, Aspergillus niger 5-16 αGal (12) and Penicillium purpurogenum αGal (25) show a preference for the Gal³Man₄ having an α-galactosyl residue (designated the stubbed α-galactosyl residue) attached to the O-6 position of the third mannose from the reducing end of β-1,4-mannotetraose. The M. vinacea αGal II (26) acts on both substrates to almost equal extents. The difference in specificity may be ascribed to the tertiary structures of these enzymes. Open in a separate windowFIG. 1Structures of galactomanno-oligosaccharides.Genes encoding αGals have been cloned from various sources, including humans (3), plants (20, 32), yeasts (27), filamentous fungi (4, 17, 24, 26), and bacteria (1, 2, 15). αGals from eukaryotes show a considerable degree of similarity and are grouped into family 27 (10).Here we describe the cloning of P. purpurogenum αGal cDNA, its expression in Saccharomyces cerevisiae, and the purification and characterization of the recombinant enzyme.     

Stepwise Introduction of Regulatory Genes Stimulating Production of α-Amylase into Bacillus subtilis : Construction of an α-Amylase Extrahyper Producing Strain

The production of extracellular α-amylase in Bacillus subtilis is probably regulated by many genetic elements, such as amyR, tmrA7, pap, amyB and sacU. Additional genetic elements, C-108 and A-2 for production of the α-amylase were found in D-cycloserine and ampicillin resistant mutants (C108 and A2) of B. subtilis 6160, respectively. Strain C108 increased the production of α-amylase about 5 times and protease about 80 times compared to parental 6160 strain. Strain A2 showed a nearly 6-fold increased α-amylase production.

These genetic elements displayed a synergistic effect with other genetic factors in production of extracellular α-amylase when these elements were transferred by DNA mediated transformation. By stepwise introduction of these and other genetic elements into B. subtilis 6160 by transformation and mutation, strains with higher α-amylase producing activity were obtained. The finally obtained strain, T2N26, produced about 1,500-2,000 times more α-amylase than parental 6160 strain.     

Sequence Variations and Haplotype Identification of Wheat Dimeric α-Amylase Inhibitor Genes in Einkorn Wheats

This study characterizes 80 dimeric alpha-amylase inhibitor genes from 68 accessions of the einkorn wheats Triticum urartu, T. boeoticum, and T. monococcum. The mature protein coding sequences of WDAI genes were analyzed. Nucleotide sequence variations in these regions resulted from base substitution and/or indel mutations. Most of the WDAI gene sequences from T. boeoticum and all sequences from T. monococcum had one nucleotide insertion in the coding region, such that these alpha-amylase inhibitor sequences could not encode the correct mature proteins. We identified 21 distinct haplotypes from the diploid wheat WDAI gene sequences. A main haplotype was found in 15 gene samples from the A(u) genome and 35 gene samples from the A(m) genome. The T. monococcum and T. boeoticum accessions shared the same main haplotype, with 25 samples from T. monococcum and 10 from T. boeoticum. The WDAI gene sequences from the A(u) and A(m) genomes could be obviously clustered into two clades, but the sequences from the A(m) genome of T. boeoticum and T. monococcum could not be clearly distinguished. The phylogenetic analysis revealed that the WDAI gene sequences from the A(m) genome had accumulated fewer variations and evolved at a slower rate than the sequences from the A(u) genome. Although some accessions from only one or two areas had unique mutations at the same position, the diversity of WDAI gene sequences in diploid wheat showed little relationship to the origin of the accessions.     

Expression of α-amylase in response to wounding in mung bean

The activity of -amylase (EC 3.2.1.1) in mung bean (Vigna radiata (L.) Wilczek) cotyledons increased markedly in response to wounding. The changes in enzyme activity were in parallel with those in enzyme content. The level of -amylase mRNA also notably increased in wounded cotyledons and attained its maximum level during the period between 1 and 2 d after wounding. The level of mRNA for phenylalanine ammonia-lyase, which is one of the well-characterized stress-inducible proteins, also increased after wounding, but the increase in mRNA level was faster than that of -amylase mRNA. On the other hand, the content of mRNA for actin, a housekeeping protein, was almost the same in wounded and unwounded cotyledons. The increase in -amylase mRNA level in wounded cotyledons was severely inhibited by -amanitin and cordycepin. -Amylase expression in the first leaves of mung-bean seedlings was also induced by wounding.Abbreviations PAL phenylalanine ammonia-lyase - SSC standard saline citrate We greatly acknowledge Prof. Richard Meagher, Department of Genetics, University of Georgia, Athens, USA for the gift of soybean actin gene clone. We also thank Mr. Kaoru Ishiwata for technical assistance.     

High-Level Expression and Purification of a Recombinant Human α-1,3-Fucosyltransferase in Baculovirus-Infected Insect Cells

A human α-1,3-fucosyltransferase (Fuc-TVII) was expressed by recombinant baculovirus-infected insect Sf9 cells as a secretory fusion protein. The fusion protein consisted of the human granulocyte colony-stimulating factor signal peptide followed by an IgG-binding domain of protein A, a Fuc-TVI-derived peptide, and the putative catalytic domain of Fuc-TVII. The signal peptide was correctly cleaved and the recombinant Fuc-TVII was secreted into the culture medium at a concentration of 10 μg/ml. The recombinant Fuc-TVII could be highly purified in a single-step purification procedure, i.e., IgG–Sepharose column chromatography. The enzymatic properties of the Sf9-produced Fuc-TVII were compared with the properties of that expressed by a human B-cell line, Namalwa KJM-1, transfected with an episomal plasmid carrying the fusion Fuc-TVII cDNA. Both recombinant proteins showed α-1,3-fucosyltransferase activity toward a type II oligosaccharide with a terminal α-2,3-linked sialic acid among various acceptors. The apparentK_mvalues of Sf9-produced Fuc-TVII for GDP-fucose and its acceptor substrate were slightly lower than those of the Fuc-TVII produced by Namalwa KJM-1 cells. Sf9-produced Fuc-TVII has N-linked carbohydrate chains whose molecular weights are lower than those linked to Namalwa KJM-1-produced Fuc-TVII. This difference in carbohydrate structure hardly affects the thermal stability of Fuc-TVII. The baculovirus expression system is available for high-level expression of stable and enzymatically active secretory Fuc-TVII.     

Indigestible dextrin is an excellent inducer for α-amylase, α-glucosidase and glucoamylase production in a submerged culture of Aspergillus oryzae

α-Amylase activities of Aspergillus oryzae grown on dextrin or indigestible dextrin were 7·8 and 27·7 U ml⁻¹, respectively. Glucoamylase activities of the cultures grown on dextrin or indigestible dextrin were 5·4 and 301 mU ml⁻¹, respectively. The specific glucoamylase production rate in indigestible dextrin batch culture reached 1·35 U g DW⁻¹ h⁻¹. In contrast, biomass concentration of A. oryzae in indigestible dextrin culture was 35% of that in dextrin culture. Thus, the culture method using indigestible dextrin has the potential to improve amylolytic enzyme production and fungal fermentation broth rheology.