Increased Heterologous Protein Production in Aspergillus niger Fermentation through Extracellular Proteases Inhibition by Pelleted Growth (original) (raw)
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The extracellular protease activity of Aspergillus niger AB4.1[pgpdAGLAGFP]#11, a recombinant strain producing a glucoamylase (GLA)-green fluorescent protein (GFP) fusion protein, was investigated in a 15 l stirred tank reactor and accordingly a pH control strategy was designed to minimize protease activity and increase recombinant yield. By maintaining pH at 6 recombinant protein production was enhanced over 10-fold to 21.0 mg/l compared to growth at acidic pH or without pH control. Protease activity was found to increase after 2 days of culture corresponding to the point where glucose in the culture medium had been completely utilized. When grown at pH 6, A. niger protease activity in the culture was decreased 6-fold to 560 U/l, compared to 3600 U/l under normal, acidic culture conditions. Protease activity at fermentation pH 6 was consistently lower than that at fermentation pH 3 regardless of assay pH, and results indicate that this decrease in activity was a combination of sub-optimal enzyme activity and variation in the spectrum of proteases secreted under the different pH conditions. A comparison of the concentrations of recombinant GLA and GFP demonstrated that high protease activity was responsible for GFP losses. More GFP was secreted in the pH 3 run, but less GFP remained in the broth because of the high protease activity. Although controlling pH at 6 did not completely inhibit the proteases, the GFP concentration in the fermentation broth was increased greatly.
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.
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.
Arabian Journal for Science and Engineering, 2017
The heterologous protein production in Aspergillus niger is often limited by the activity of the host extracellular proteases. To improve heterologous production, a transcription factor controlling expression of several extracellular protease-encoding genes, prtT, was deleted in A. niger PY11 and the mutant (An prtT) characterised. Extracellular proteolytic activity of An prtT was reduced as compared to the wild type, a result that was confirmed by RT-PCR analyses that showed reduced expression levels of several protease gene transcripts. To compare the efficiency of the mutant and parental (PY11) strains as hosts for heterologous protein production, the cutinase gene from Glomerella cingulate, under control of the glucoamylase A promoter, was integrated into each genome. The cutinase activity of PY11 and An prtT harbouring the G. cingulata cutinase gene was increased 20-and 36-fold higher, respectively, than the untransformed parental strains, suggesting that the ability of the mutant to produce heterologous protein is better than the wild type. Cutinase activity in culture filtrates prepared
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...
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.
2011
Aims: The present study was investigated to optimize and partially purify the proteases produced by the food borne bacterial strains. Methodology and Results: Four bacterial strains such as Bacillus cereus, Proteus vulgaris, P. mirabilis and Enterobacter aerogenes were isolated from food wastes. These strains were individually inoculated in to the formulated culture media supplied with three different concentrations (1:1 to 1:3) of raw milk as major substrate. Among the concentrations, 1:2 ratio of substrate supplied medium showed maximum (0.133 to 8.000 IU/mL) protease production by all the tested organisms. After optimization, the organisms were tested for protease production at various pH (3 to 9), and temperature (30 to 80 °C). The result showed that all the organisms were capable of producing maximum protease at pH 6 (8.533 to 10.133 IU/mL) and at 50 °C (8.666 to 10.666 IU/mL). The crude enzymes produced by the tested organisms were individually purified by two different methods viz sodium alginate and ammonium sulphate-butanol methods. The purity of the protease determined in these two methods was ranged between 3.24 to 5.44 I and 3.13 to 5.55 IU/mL respectively. The partially purified enzymes were further analysed through SDS-PAGE; accordingly the molecular weight of protein produced by the test organisms was determined in between 49.44 and 50.98 kDa. Conclusion, significance and impact of study: Among the tested strains P. vulgaris was identified as the major protease producer in optimized culture condition of 50 o C and pH6. The molecular mass of the partially purified protease of P. vulgaris was 50.32 KDa. Further research on optimization of other fermentation parameters using statistical tools with P. vulgaris is needed to scale up the process.
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+ .