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Showing papers on "Aspergillus niger published in 2003"


Journal ArticleDOI
TL;DR: Among black aspergilli, A. carbonarius has shown a consistent ability to produce OTA and is the most probable source of this mycotoxin in these substrates.

237 citations


Journal ArticleDOI
TL;DR: This Mini-Review summarizes the current knowledge on the biochemical and physiological events leading to massive citric acid accumulation by Aspergillus niger under industrially comparable conditions, thereby particularly emphasizing the roles of glycolytic flux and its control, excretion ofcitric acid from the mitochondria and the cytosol, and the critical fermentation variables.
Abstract: This Mini-Review summarizes the current knowledge on the biochemical and physiological events leading to massive citric acid accumulation by Aspergillus niger under industrially comparable conditions, thereby particularly emphasizing the roles of glycolytic flux and its control, excretion of citric acid from the mitochondria and the cytosol, and the critical fermentation variables The potential of novel techniques for metabolic analysis and genomic approaches in understanding this fermentation is also discussed

232 citations


Journal ArticleDOI
TL;DR: In this paper, the effect of copper(II), lead(II) and chromium(VI) ions on the growth and bioaccumulation properties of Aspergillus niger was investigated as a function of initial pH and initial metal ion concentration.

201 citations


Journal ArticleDOI
TL;DR: Conidiospores of a ΔmpdA strain were extremely sensitive to a variety of stress conditions, including high temperature, oxidative stress and, to a lesser extent, freezing and lyophilization, indicating that mannitol appears to be essential for the protection of A. niger spores against cell damage under these stress conditions.
Abstract: d-Mannitol is the predominant carbon compound in conidiospores of the filamentous fungus Aspergillus niger and makes up 10 to 15% of the dry weight. A number of physiological functions have been ascribed to mannitol, including serving as a reserve carbon source, as an antioxidant, and to store reducing power. In this study, we cloned and characterized the A. niger mpdA gene, which encodes mannitol 1-phosphate dehydrogenase (MPD), the first enzyme in the mannitol biosynthesis pathway. The mpdA promoter contains putative binding sites for the development-specific transcription factors BRLA and ABAA. Furthermore, increased expression of mpdA in sporulating mycelium suggests that mannitol biosynthesis is, to a certain extent, developmentally regulated in A. niger. Inactivation of mpdA abolished mannitol biosynthesis in growing mycelium and reduced the mannitol level in conidiospores to 30% that in the wild type, indicating that MPD and mannitol 1-phosphate phosphatase form the major metabolic pathway for mannitol biosynthesis in A. niger. The viability of spores after prolonged storage and germination kinetics were normal in an mpdA null mutant, indicating that mannitol does not play an essential role as a reserve carbon source in A. niger conidia. However, conidiospores of a ΔmpdA strain were extremely sensitive to a variety of stress conditions, including high temperature, oxidative stress and, to a lesser extent, freezing and lyophilization. Since mannitol supplied in the medium during sporulation repaired this deficiency, mannitol appears to be essential for the protection of A. niger spores against cell damage under these stress conditions.

200 citations


Journal ArticleDOI
TL;DR: CP-MAS 13C NMR showed marked changes in the chain conformations of LMWC, which are believed to be responsible for its loss of solubility and functionality.

153 citations


Journal ArticleDOI
TL;DR: The results indicated that the identified fungi have enough potential to exploit native organic phosphorus to benefit plant nutrition, after using these organisms as seed inoculants, to help attain higher P nutrition of plants.
Abstract: Seven most efficient phytase and phosphatases producing fungi were isolated from the soils of arid and semi-arid regions of India and tested for their efficiency on hydrolysis of two important organic P compounds: phytin and glycerophosphate. The native soil organic P may be exploited after using these organisms as seed inoculants, to help attain higher P nutrition of plants. The identified organisms belong to the three genera: Aspergillus , Emmericella and Penicillium . Penicillium rubrum released the most acid into the medium during growth. Aspergillus niger isolates were found to accumulate biomass the fastest. A significant negative correlation ( r =−0.593, n =21, p −1 g −1 for glycerophosphate to 0.92–2.10 μg min −1 g −1 for phytin. The trend of efficiency was as follows: Aspergillus sp.> Emmericella sp.> Penicillium sp. The results indicated that the identified fungi have enough potential to exploit native organic phosphorus to benefit plant nutrition.

145 citations


Journal ArticleDOI
TL;DR: A pulsed UV-light system was used to inactivate fungal spores of Aspergillus niger in corn meal to maximize the UV fungal disinfection while minimizing the heat generation.
Abstract: Summary Fungal contamination of grains, intended for human and animal consumption, during the pre/post-harvest periods has been a recurring health hazard. A pulsed UV-light system was used to inactivate fungal spores of Aspergillus niger in corn meal. Response surface methodology was utilized for experimental design. The three process parameters evaluated were treatment time (20–100 s), voltage input (2000–3800 V), and distance from the UV strobe (3–13 cm). Optimization of the process parameters was validated by a quadratic regression equation designed to fit the experimental log10 reduction of fungal spores. Model prediction for a 100-s treatment time, 3 cm of distance from the UV strobe, and with 3800 V input gave a 4.93log10 reduction of A. niger. Modification of the pulsed UV-light system was recommended to maximize the UV fungal disinfection while minimizing the heat generation.

137 citations


Journal ArticleDOI
TL;DR: The production of cellulose and hemicellulose-degrading enzymes by cultivation of Aspergillus niger, Botrytis cinerea, Penicillium brasilianum, Schizophyllum commune, and Trichoderma reesei Rut-C30 was studied to obtain an enzyme mixture optimal for enzymatic hydrolysis of wet-oxidised wheat straw.

126 citations


Journal ArticleDOI
Mari Valkonen, Michael Ward1, Huaming Wang1, Merja Penttilä, Markku Saloheimo 
TL;DR: It is shown that the constitutive induction of the UPR pathway in Aspergillus niger var.
Abstract: Unfolded-protein response (UPR) denotes the upregulation of endoplasmic reticulum (ER)-resident chaperone and foldase genes and numerous other genes involved in secretory functions during the accumulation of unfolded proteins into the ER. Overexpression of individual foldases and chaperones has been used in attempts to improve protein production in different production systems. We describe here a novel strategy to improve foreign-protein production. We show that the constitutive induction of the UPR pathway in Aspergillus niger var. awamori can be achieved by expressing the activated form of the transcription factor hacA. This induction enhances the production of Trametes versicolor laccase by up to sevenfold and of bovine preprochymosin by up to 2.8-fold in this biotechnically important fungus. The regulatory range of UPR was studied by analyzing the mRNA levels of novel A. niger var. awamori genes involved in different secretory functions. This revealed both similarities and differences to corresponding studies in Saccharomyces cerevisiae.

125 citations


Journal ArticleDOI
TL;DR: Initial characterisation of the purified enzyme suggested that it could be a potential candidate for use as an animal feed supplement because of its higher thermostability and ability to hydrolyse non-phytate-based substrates.

118 citations


Journal ArticleDOI
TL;DR: In this paper, a solid state fermentation method was used to utilise pineapple, mixed fruit and maosmi waste as substrates for citric acid production using Aspergillus niger DS 1.

Journal ArticleDOI
TL;DR: The recombinant FAEA was tested for wheat straw pulp bleaching, with or without a laccase mediator system and xylanase, and yielded very efficient delignification—close to 75%—and a kappa number of 3.9.
Abstract: A well-known industrial fungus for enzyme production, Aspergillus niger, was selected to produce the feruloyl esterase FAEA by homologous overexpression for pulp bleaching application. The gpd gene promoter was used to drive FAEA expression. Changing the nature and concentration of the carbon source nature (maltose to glucose; from 2.5 to 60 g l-1), improved FAEA activity 24.5-fold and a yield of 1 g l-1 of the corresponding protein in the culture medium was achieved. The secreted FAEA was purified 3.5-fold to homogeneity in a two-step purification procedure with a recovery of 69%. The overproduced protein was characterised and presented properties in good agreement with those of native FAEA. The recombinant FAEA was tested for wheat straw pulp bleaching, with or without a laccase mediator system and xylanase. Best results were obtained using a bi-sequential process with a sequence including xylanase, FAEA and laccase, and yielded very efficient delignification - close to 75% - and a kappa number of 3.9. This is the first report on the potential application of recombinant FAEA in the pulp and paper sector. Chemicals/CAS: hemicellulose, 63100-39-0, 63100-40-3, 9034-32-6; laccase, 80498-15-3; xylan endo 1,3 beta xylosidase, 37278-89-0, 9025-55-2; Carboxylic Ester Hydrolases, EC 3.1.1.-; Endo-1,4-beta Xylanases, EC 3.2.1.8; feruloyl esterase, EC 3.1.1.73; Laccase, EC 1.10.3.2; Recombinant Proteins

Journal ArticleDOI
TL;DR: An extracellular tannase was produced from solid-state cultures of Aspergillus niger and was shown to hydrolyse cellobiose efficiently and be able to remove gallic acid from both condensed and hydrolysable tannins.
Abstract: An extracellular tannase was produced from solid-state cultures of Aspergillus niger. The enzyme was purified to homogeneity from the cell-free culture broth by preparative isoelectric focusing and by FPLC using anion-exchange and gel-filtration chromatography. SDS-PAGE analysis as well as gel localization studies of purified tannase indicated the presence of two enzyme forms, with molecular masses of 90 kDa and 180 kDa. The tannase had an isoelectric point of 3·8, a temperature optimum of 60–70 °C and a pH optimum of 6·0. The substrate specificity of the tannase was determined by HPLC analysis of tannin substrates and products. The enzyme was able to remove gallic acid from both condensed and hydrolysable tannins. Internal sequences were obtained from each of the gel-purified and trypsin-digested tannase forms. The peptide sequences obtained from both forms were identical to sequences within a β-glucosidase from Aspergillus kawachii. The purified tannase was tested for β-glucosidase activity and was shown to hydrolyse cellobiose efficiently. However, no β-glucosidase activity was detected when the enzyme was assayed in the presence of tannic acid.

Journal ArticleDOI
TL;DR: The fermentation kinetics of gluconic acid by Aspergillus niger were studied in a batch system and a simple model appeared to provide a reasonable description for each parameter during the growth phase.

Journal ArticleDOI
TL;DR: In this article, the Aspergillus niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone α-factor secretion signal (MFα1S) and the phosphoglycerate-kinase-1 gene promoter (PGK1P) and terminator.
Abstract: There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; β-d-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone α-factor secretion signal (MFα1S) and the phosphoglycerate-kinase-1 gene promoter (PGK1P) and terminator (PGK1T). The PGK1P-MFα1S-gox-PGK1T cassette (designated GOX1) was introduced into a laboratory strain (Σ1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8–2.0% less alcohol. This was probably due to the production of d-glucono-δ-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.

Journal ArticleDOI
TL;DR: In this paper, the Aspergillus niger fungus was used for cadmium removal from oil field water in the oil industry and a technique for biomass recovery was developed with the objective of determining the capacity of the regenerated biomass to biosorb the metals in solution.
Abstract: Sorption experiments using the Aspergillus niger fungus for cadmium removal were carried out to study the factors influencing and optimizing the biosorption of this metal. The effects of pH, time, biomass concentration, and initial concentration of the heavy metal on the rate of metallic biosorption were examined. An experimental design was also used to determine the values of the under study variables that provided the greatest biosorption efficiency. A technique for biomass recovery was also developed with the objective of determining the capacity of the regenerated biomass to biosorb the metals in solution. This research proved that with a pH of 4.75, a biomass concentration of 0.7 g/L, and a heavy metal concentration varying between 5 and 10 mg/L a biosorption process of biosorption with Aspergillus niger could be successfully used for heavy metal removal from oil field water in the oil industry.

Journal ArticleDOI
TL;DR: In this article, Aspergillus niger produces citric acid from whey with different concentrations of sucrose, glucose, fructose, galactose, riboflavin, tricalcium phosphate and methanol.
Abstract: Citric acid (CA) production by Aspergillus niger ATCC9642 from whey with different concentrations of sucrose, glucose, fructose, galactose riboflavin, tricalcium phosphate and methanol in surface culture process was studied. It was found that whey with 15% (w/v) sucrose with or without 1% methanol was the most favourable medium producing the highest amount (106.5 g/l) of citric acid. Lower CA was produced from whey with other concentrations of sugars and other additives used. Highest biomass ofA. niger was produced with the addition of riboflavins. In general, extension of the fermentation (up to 20 days) resulted in an increase in CA and biomass, and decrease in both residual sucrose and pH values. Key words: Citric acid, Aspergillus niger, whey fermentation, surface culture.

Journal ArticleDOI
TL;DR: The bioaccumulation of Cu(II and Cd(II) ions by viable Rhizopus arrhizus and Aspergillus niger was studied as a function of initial pH and initial metal ion concentration in this paper.

Journal ArticleDOI
TL;DR: Polygalacturonase and pectinmethylesterase were found to be inducible by polygalacturonic acid and pECTin in the medium, respectively, and ammonium sulphate was the best nitrogen source for the production of both enzymes.

Journal ArticleDOI
TL;DR: The essential oil fraction of P. graveolens and its main components, geraniol and citronellol, exhibited additive effects with amphotericin B and with ketoconazole against both Aspergillus species, resulting in fractional inhibitory concentration (FIC) indices ranging from 0.52 to 1.00.
Abstract: The essential oils from Cedrus atlantica, Styrax tonkinensis, Juniperus communis, Lavandula angustifolia, Melaleuca alternifolia, Pelargonium graveolens, Pogesternon patchouli and Rosmarinus officinalis were analyzed by GC-MS. Antifungal activities of the oils were investigated by disk diffusion assay and the broth dilution method against Aspergillus niger and A. flavus. The effects of geraniol and the essential oil fraction from P. graveolens on the antifungal activity of amphotericin B and ketoconazole were examined using a checkerboard microtiter assay against both Aspergillus fungi. Most of the tested essential oils, with the exception of C. atlantica, J. communis, and P. patchouli, significantly inhibited growth of A. niger and to a lesser extent that of A. flavus, with MICs (minimal inhibitory concentrations) in the range 0.78-12.5 mg/mL. The essential oil fraction of P. graveolens and its main components, geraniol and citronellol, exhibited additive effects with amphotericin B and with ketoconazole against both Aspergillus species, resulting in fractional inhibitory concentration (FIC) indices ranging from 0.52 to 1.00.

Journal ArticleDOI
TL;DR: This study suggests the possible use of pectinase isozyme in place of chitosanase, which is expensive and unavailable in bulk quantity for the production of LMWC and chitooligomers.

Journal ArticleDOI
TL;DR: Sequencing and hybridization showed reliable results for A. fumigatus, which proved to be the most common agent in fungus balls of the maxillary sinus, and other Aspergillus species and other genera were rarely found.
Abstract: The aim of this study was to find a reliable method for the detection and identification of fungi in fungus balls of the maxillary sinus and to evaluate the spectrum of fungi in these samples. One hundred twelve samples were obtained from patients with histologically proven fungal infections; 81 samples were paraffin-embedded tissue sections of the maxillary sinus. In 31 cases, sinus contents without paraffin embedding were sent for investigation. PCR amplification with universal fungal primers for 28S ribosomal DNA and amplicon identification by hybridization with species-specific probes for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus glaucus, Pseudallescheria boydii, Candida albicans, and Candida glabrata were performed for all samples. Furthermore, PCR products were sequenced. Fresh samples were also cultivated. Fungal DNA was detected in all of the fresh samples but only in 71 paraffin-embedded tissue samples (87.7%). Sequence analysis was the most sensitive technique, as results could be obtained for 28 (90.3%) fresh samples by this method in comparison to 24 (77.4%) samples by hybridization and 16 (51.6%) samples by culture. However, sequence analysis delivered a result for only 36 (50.7%) of the paraffin-embedded specimens. Hybridization showed reliable results for A. fumigatus, which proved to be the most common agent in fungus balls of the maxillary sinus. Other Aspergillus species and other genera were rarely found.

Journal ArticleDOI
TL;DR: Metal-dye detection HPLC analysis of inositol phosphate degradation in flour from transgenic wheat materials possessing wheat endogenous 6-phytase and Aspergillus 3-phyTase activities is reported, finding that after 50 min incubation virtually all InsP5, InsP4 and InsP3 isomers are hydrolysed.
Abstract: Expression of heterologous phytases in crops offers a great potential for improving phosphate and mineral bioavailability in food and feed. In this context it is of relevance to describe the concerted action of endogenous and hetrologous phytases on the transgenic seed inositol phosphate profile. Here we report metal-dye detection HPLC analysis of inositol phosphate degradation in flour from transgenic wheat materials possessing wheat endogenous 6-phytase [EC 3.1.3.26] and Aspergillus 3-phytase [EC 3.1.3.8] activities under the control of the maize ubiquitin-1 promoter and the wheat high molecular weight glutenin subunit 1DX5 promoter respectively. During 50 min incubation there is an accumulation of InsP5 to InsP2 breakdown products in non-transgenic material. Aspergillus niger phytase specific breakdown products are transiently detected in transgenic material but after 50 min incubation virtually all InsP5, InsP4 and InsP3 isomers are hydrolysed.

Journal Article
TL;DR: The enzyme production was performed and optimized for the highest -glucosidase yield in cofermentation with a Trichoderma strain to support the degradation of cellulose and to provide the non-cellulolytic Aspergillus with water soluble carbon source.
Abstract: Summary The aim of the present study was to investigate a new approach to -glucosidase production of an Aspergillus strain using cheap lignocellulosic material i.e. waste paper in order to substitute glucose, a generally used carbon source, and thereby reduce the production cost. The enzyme production was performed and optimized for the highest -glucosidase yield in cofermentation with a Trichoderma strain to support the degradation of cellulose and to provide the non-cellulolytic Aspergillus with water soluble carbon source. Batch fermentation experiments of Aspergillus niger BKMF 1305 and Trichoderma reesei RUT C30 were carried out in shake flask cultures. The factors influencing the enzyme production, such as the concentrations of nutrients and carbon source, the inoculum ratio of the two species, and the delay in A. niger inoculation were investigated using a 2 3 full factorial design. The results were analyzed with the response surface methodology using commercially available software, Statistica for Windows. All three examined factors were found significant. The highest -glucosidase activity of 3.07 IU/mL was obtained after 7 days of incubation, if 3.3 % Aspergillus and 6.7 % Trichoderma inoculum were added at the same time to modified Mandels’ medium, in which the concentration of nutrients was doubled compared to normal Mandels’ medium and the carbon source concentration was set to 20 g/L waste paper.

Journal ArticleDOI
TL;DR: Two sulfonylurea herbicides, chlorsulfuron and metsulfuron‐methyl, were studied under laboratory conditions, in order to elucidate the biodegradation pathway operated by Aspergillus niger, a common soil fungus, which is often involved in the degradation of xenobiotics.
Abstract: Two sulfonylurea herbicides, chlorsulfuron and metsulfuron-methyl, were studied under laboratory conditions, in order to elucidate the biodegradation pathway operated by Aspergillus niger, a common soil fungus, which is often involved in the degradation of xenobiotics. HPLC-UV was used to study the kinetic of degradation, whereas LC-MS was used to identify the metabolites structure. In order to avoid the chemical degradation induced by a decrease in pH, due to the production of citric acid by the fungus, the experiments were performed in a buffered neutral medium. No significant degradation for both compounds was observed in mineral medium with 0.2% sodium acetate. On the contrary, in a rich medium, after 28 days the degradations, chemical degradation excluded, were about 30% for chlorsulfuron and 33% for metsulfuron-methyl. The main microbial metabolites were obtained via cleavage of the sulfonylurea bridge. In addition the fungus seems to be able to hydroxylate the aromatic ring of chlorsulfuron. In the case of metsulfuron-methyl the only detected metabolite was the triazine derivative, while the aromatic portion was completely degraded. Finally, the demethylation of the methoxy group on the triazine ring, previously observed with a Pseudomonas fluorescens strain, was not observed with A. niger.

Journal ArticleDOI
TL;DR: In this article, the feasibility of using the fungus Aspergillus niger for bio-leaching metals from oxide low-grade ore was investigated and the fungus produced a variety of organic acids.
Abstract: A study was initiated to determine the feasibility of using the fungus Aspergillus niger for bioleaching metals from oxide low-grade ore. Large quantities of the metals are embodied in the low-grade ores and mining residues that can be recovered. Presently available techniques (pyrometallurgical and hydrometallurgical) are expensive or may have a negative impact on the environment. An oxidized mining ore containing mainly copper (7245 mg kg−1 residue) was studied. In this study, the fungus A niger produced a variety of organic acids. Addition of small quantities of sulfuric acid enhanced the organic acids, efficiency. Various agricultural wastes were evaluated as substrates and a maximum solubilization of 68% for copper for a medium containing potato peels was achieved. In conclusion, leaching of copper from a mining ore is technically feasible using A niger. Further research must be performed to increase the rate of copper removal. Copyright © 2003 Society of Chemical Industry

Journal ArticleDOI
TL;DR: The antimicrobial assay showed that this compound was inhibitory to the growth of Bacillus subtilis, Staphylococcus aureus, Aspergillus niger and Candida albicans, with minimum inhibitory concentrations (MICs) of 20, 50, 30 and 10 microg/ml, respectively.

Journal ArticleDOI
TL;DR: The present investigation was carried out for increasing the yield of tannase of Aspergillus niger and the physico‐chemical characterization of this enzyme, and tannic acid was the best substrate for three substrates tested, followed by methyl gallate and propyl gallate.
Abstract: The present investigation was carried out for increasing the yield of tannase of Aspergillus niger and the physico-chemical characterization of this enzyme. the extraction of enzyme protein. However, extraction of fungal pigments and proteins was observed to have high pH dependence, and maximum enzyme extraction was obtained at pH 5.5. The two-step purification protocol gave 51-fold purified enzyme with a yield of 20%. The total tannase activity was made up of nearly equal activity of esterase and depsidase. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of purified tannase protein indicated it to be made up of two polypeptides of molecular weight 102 and 83 kDa. Based on the Michaelis-Menten constant (Km) of tannase for three substrates tested, tannic acid was the best substrate with Km of 2.8 x 10(-4) M, followed by methyl gallate and propyl gallate. The inhibition was maximum for CaCl2 (58%) whereas EDTA had no modulatory effect on tannase activity. The inhibitor binding constant (KI) of CaCl2 was 5.9 x 10(-4) M Homogenization and detergent pretreatments did not have any remarkable effect on and the inhibition was of noncompetitive type.

Journal ArticleDOI
TL;DR: Tannase from Aspergillus niger van Teighem has been used for synthesis of food additive antioxidant propyl gallate by direct transesterification of tannic acid by using simultaneously pH tuned enzyme and immobilized on Celite.

Journal ArticleDOI
TL;DR: It was found that agitation affected the GFP production most significantly, and protease activity was most influenced by initial glucose concentration and DO, and fungus morphology was also affected by these parameters.
Abstract: Filamentous fungi such as Aspergillus niger are attractive hosts for recombinant DNA technology because of their ability to secrete bioactive proteins with post-translational processing such as glycosylation. Foreign genes can be incorporated into the chromosomes of the filamentous fungi, providing superior long-term genetic stability. However, heterologous protein production is often severely hampered by fungal proteases. In this work, a recombinant Aspergillusniger strain AB4.1(pgpdAGLAGFP)#11which carries a glucoamylase (GLA)-green fluorescent protein (GFP) fusion gene was selected as a model system to study the effects of bioprocess parameters—agitation intensity, initial glucose concentration, initial yeast extract concentration, and dissolved oxygen tension (DO)—on extracellular protease inhibition and heterologous protein production. Based on previous experimental experience and results, a 2 4-1 fractional factorial design was applied to the experiments. Each parameter was tested at two levels. It was found that agitation affected the GFP production most significantly. Higher agitation rate resulted in higher GFP production. Protease activity was most influenced by initial glucose concentration and DO. Fungal morphology was also affected by these parameters. The effects of these parameters on pellet size and pellet porosity are discussed.  2003 Society of Chemical Industry