Comparative studies on extracellular protease secretion and glucoamylase production by free and immobilized Aspergillus niger cultures (original) (raw)
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Applied Microbiology and Biotechnology, 2008
Although host proteases are often considered to have a negative impact upon heterologous protein production by filamentous fungi, relatively little is known about the pattern of their appearance in recombinant fungal bioprocesses. In the present study, we investigated extracellular proteases from a filamentous fungus, Aspergillus niger B1-D, genetically modified to secrete hen egg white lysozyme (HEWL). Our findings indicate that extracellular protease activity is only detected after the carbon source is completely utilised in batch cultures. The proteases are predominantly acid proteases and have optimal temperature for activity at around 45°C. Their activity could be partially inhibited by protease inhibitors, indicating the existence of at least four kinds of proteases in these culture fluids, aspartic-, serine-, cysteine-, and metallo-proteases. Oxygen enrichment does not have any noticeable effects on extracellular protease activity except that the onset of protease activity appears earlier in oxygen enrichment runs. Oxygen enrichment stimulates HEWL production substantially, and we propose that it is related to fungal morphology. Thermal stress imposed by raising process temperature (from 25 to 30 and 35°C) in early exponential phase, led to appearance of protease activity in the medium following the heat shock. Continued cultivation at high temperatures significantly reduced HEWL production, which was associated with increased activity of the extracellular proteases in these cultures.
Applied Microbiology and Biotechnology, 1991
A number of culture conditions for protease production by Aspergillus oryzae NRRL 2160 on solid substrates were investigated. The pH of the medium and the substrate markedly affected protease production. High protease yield was obtained when the fungus was cultivated for 72–96 h on rice hulls: rice bran (7:3), at an initial pH of 7.0. Maximal protease production was achieved at an initial moisture content of 35–40%, corresponding to a water activity range of 0.982–0.986. Casein and gluten were effective inducers. Polyethylene bags proved to be promising containment systems for solid state cultivation.
World Journal of Microbiology and Biotechnology, 2005
While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.
Biotechnology Progress, 2000
The dependence of filamentous fungal protease secretion on morphology was investigated by employing the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP] which contains a gene for the glucoamylase-GFP (green fluorescence protein) fusion protein. Different inoculum levels were used to obtain different sizes of pellet or free mycelia. The extracellular protease activity of the cultures varied with the pellet size and decreased dramatically when the morphology was changed from free mycelia to pellets. The culture with an optimal pellet size of 1.6 mm was obtained from an inoculum of 4 × 10 6 spores/mL. It resulted in a specific protease activity of 158 units/ L, only one-third of that in free mycelial growth, and a maximum specific GFP yield of 0.98 mg/g (cell mass) compared to 0.29 mg/g for free mycelial growth with an inoculum of 10 7 spores/mL. The results indicate that this bioprocessing strategy can be effectively used to inhibit protease activity in filamentous fungal fermentation and thereby to enhance heterologous protein production.
The ability of a raw starch digesting amylase producer, Aspergillus carbonarius for protease production was evaluated in this study using standard methods. The fungus grew and produced appreciable levels of protease using different carbon and nitrogen sources. Sources of carbon and nitrogen significantly (p < 0.001) influenced protease production by this fungus. Soybean meal and glucose were the best nitrogen and carbon sources, respectively for the production of protease by A. carbonarius. Maximum protease production was achieved with 5% glucose and 5% soybean meal in combination with 0.1% peptone (as carbon and nitrogen sources, respectively), 0.04% FeSO 4 , 0.1% NaCl, 0.1% (v/v) Tween 80 and initial pH 6.0. Time course of enzyme synthesis by the fungus showed that the enzyme production occurred through the logarithmic to stationary growth phases with maximum enzyme yield being obtained on the 9th day of fermentation corresponding to the final culture pH of 4.6. The results sug...
Archives of Applied Science Research, 2012
The study aimed at isolating a potential acid protease producing fungal strain from local soil source. The fungal strains were isolated from garden soil on the basis of clearance zone on casein-glycerol agar flooded by coomassie blue stain. Aspergillus sp. showing maximum clearance on casein agar plates (pH 5) was selected for further studies. Optimization of various factors influencing maximum enzyme production by Aspergillus sp. using solid state fermentation was investigated. Optimum fermentation conditions for enzyme production were-substrate (wheat bran & gelatin; 1%, w/v), fermentation time (120 h), moisture content (20 %), growth pH (5.0) and temperature (30ºC). Wheat bran supplemented with nitrogen sources viz., gelatin and potassium nitrate showed 15-17 % increase in enzyme productivity. However, supplementation with additional carbon sources or salt solution had no profound influence on enzyme productivity. The crude acid protease of Aspergillus sp. showed pH and temperature optima of 5 and 50°C respectively. An increased enzyme activity was observed in presence of Ca 2+ and Mg 2+. The enzyme showed broad substrate specificity. Thermostability of this enzyme at 50°C for 30 min throws light on its potential for applications in food industries.
Optimization of Culture Condition for Protease Production by Aspergillus Niger
Proteases are used in various biotechnological industries such as detergent, food, pharmaceutical and cosmetics. Due to high demand, the production of protease by Aspergillus niger was studied on the basis of various fermentation parameters such as incubation time, carbon sources, nitrogen sources, pH and temperature. On the basis of present study, it was noted that Aspergillus niger secreted higher yield of protease (0.131mg/ml) when grown on mineral medium containing 0.3% glucose and 0.02% yeast extract as a carbon and nitrogen sources in comparison to other carbon and nitrogen sources at 30±2°C for 96 hour. Whereas pH 7.0 and 35°C was found favorable and the yield of protease increased up to 0.199 mg/ml by Aspergillus niger.
Protease production under solid state fermentation (SSF) was investigated using isolated Aspergillus flavus. Different agroindustrial waste products were evaluated to check the possibility of potential utilization of substrates in SSF for protease production by Aspergillus flavus using wheat bran as a substrate. The results showed that the optimum conditions for maximum protease production were found to be 7 th day of incubation at pH 5.0, temperature 30 o C; inoculum size 3%; substrate concentration 3% and 3% KNO 3 as nitrogen source. The purified enzyme produced 5.8 fold with recovery of 3.2% by DEAE-column chromatography and the molecular weight was estimated to be 46kDa by SDS-PAGE. It has a V max value of 60.0 U/mg and K m value of 0.6 mg/ml at pH of 7. The enzyme activity was found to be stable at 50 0 C and it was stimulated by metal ions like Cu 2+ and Zn 2+ and inhibited by Ca 2+ and Mg 2+ .
Enzyme and Microbial Technology, 2002
In order to identify factors responsible for production of multiple forms of glucoamylase (GA) by Aspergillus niger Bo-1, the fungus was cultured in both complex and defined media in pH-controlled batch fermenters and chemostats. At all culture conditions three forms of GA were produced with molecular weights of approx. 91 (GAI), 73 (GAII), and 59 kDa (GAIII). Data from batch fermentations with constant pH 3.0 and 5.0 showed a uniform distribution of extracellular GA forms throughout the fermentations and independent of culture growth phases. Furthermore, steady-state data from chemostat cultivations at constant pH 3.0 and 5.0 showed a similar distribution of extracellular GA forms and established that the nitrogen concentration of the medium (C/N ratio) did not affect the distribution of multiple forms of GA. The extracellular acid protease activity was only moderate when the fungus was cultivated in batch and continuous fermentations with a constant pH of 3.0 or 5.0, whereas acidification of both complex and defined batch culture media after 20 h of growth induced a significant secretion of acid protease(s). The protease(s) present in the complex medium catalysed modification of the extracellular profile of the multiple forms of GA by degradation of GAI to GAII, whereas no proteolytic processing of the GA profile was observed in the defined medium. In vitro experiments confirmed that the pH-induced modifications of the GA multiple-form profile were caused by proteolytic and not spontaneous degradation of the GA forms at low pH. It was concluded that the observed modifications of the extracellular profile of GA isoforms in A. niger Bo-1 are due to changes in pH and medium composition.