Enhanced heterologous protein production in Aspergillus niger through pH control of extracellular protease activity

O’Donnell, Dara; Wang, Liping; Xu, Jianfeng; Ridgway, Darin; Gu, Tingyue; Moo‐Young, Murray

doi:10.1016/s1369-703x(01)00102-4

Cited by 51 publications

(25 citation statements)

References 28 publications

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“…Culture pH itself has often been indicated as an important environmental parameter in controlling extracellular protease activity. It affects both the activity of the secreted proteases (O'Donnell et al, 2001) as well as the expression of protease van den Hombergh et al, 1997b). Also, in our experiments we observed that extracellular protease activities were higher at a culture pH of 4 than at pH 5 or 6 (Table 1).…”

Section: Discussionsupporting

confidence: 59%

“…Complementary to strain improvement, manipulation of environmental conditions can help to reduce protease secretion and thus improve heterologous protein production, but this has not been investigated systematically (Braaksma & Punt, 2008). Examples of bioprocess parameters which have been investigated for their influence on extracellular protease activity include fungal morphology manipulation (Liu et al, 1998;Xu et al, 2000;, pH control (O'Donnell et al, 2001), oxygen enrichment and cultivation temperature (Li et al, 2008). Studies on the effect of medium components have mainly focused on the derepression of protease genes when transferring mycelia to medium lacking either carbon or nitrogen sources (Cohen, 1981;.…”

Section: Introductionmentioning

confidence: 99%

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The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger

et al. 2009

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Proteolytic degradation by host proteases is one of the key issues in the application of filamentous fungi for non-fungal protein production. In this study the influence of several environmental factors on the production of extracellular proteases of Aspergillus niger was investigated systematically in controlled batch cultures. Of all factors investigated in a series of initial screening experiments, culture pH and nitrogen concentration in particular strongly affected extracellular protease activities. For instance, at a culture pH of 4, protease activity was higher than at pH 5, and protease activity increased with increasing concentrations of ammonium as nitrogen source. Interestingly, an interdependence was observed for several of the factors studied. These possible interaction effects were investigated further using a full factorial experimental design. Amongst others, the results showed a clear interaction effect between nitrogen source and nitrogen concentration. Based on the observed interactions, the selection of environmental factors to reduce protease activity is not straightforward, as unexpected antagonistic or synergistic effects occur. Furthermore, not only were the effects of the process parameters on maximum protease activity investigated, but five other protease-related phenotypes were studied as well, such as maximum specific protease activity and maximum protease productivity. There were significant differences in the effect of the environmental parameters on the various protease-related phenotypes. For instance, pH significantly affected final levels of protease activity, but not protease productivity. The results obtained in this study are important for the optimization of A. niger for protein production.

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Section: Discussionsupporting

confidence: 59%

Section: Introductionmentioning

confidence: 99%

The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger

et al. 2009

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“…Acidic proteases have been isolated from various species of Aspergillus (O'Donnell et al, 2001;Tremacoldi et al, 2004), Mucor (Escobar and Barnett, 1993;Fernandez-Lahore et al, 1999) and Rhizopus (Kumar et al, 2005). Fu et al (2008) obtained an acid protease from Bacillus megaterium KLP-98 which had an optimum pH of 5.5 and was stable in the pH range of 4.5 -6.0.…”

Section: Effect Of Ph On Protease Activitymentioning

confidence: 99%

Partial Purification and Characterization of Extracellular Protease from <i>Pedicoccus acidilactici</i>

Oke¹,

Onilude²

2014

Nig J Bas App Sci

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Microbial proteases have wide industrial applications and proteases of the lactic acid bacteria (LAB) have received special attention because of their importance in the food and dairy industry. Of all the LAB, the proteolytic system of the pediococci is the least studied. Therefore, this study was aimed at characterizing and purifying the protease produced by Pediococcus acidilactici grown in de Man, Rogosa and Sharpe (MRS) broth. Casein concentration of 2% (w/v) and 2.5 ml of the crude enzyme were optimal for the activity of the protease. The crude protease had temperature and pH optima of 28 o C and 4.0 respectively thus indicating that the enzyme is a mesophilic and acidic protease. Purification of the enzyme by gel filtration chromatography on Sephadex G75 following ammonium sulphate precipitation gave 2.26 fold increase in purification with specific activity of 46.13 units/mg protein while purification on Sephadex CM50 resulted in reduced purification fold (1.24 -1.59) with specific activity ranging between 25.39 -32.54 units/mg protein. The protease showed a characteristic band on SDS-PAGE and the molecular weight was estimated between 45 and 66 kDa. The potential applications of the protease are discussed.

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“…Though commercially interesting levels of recombinant bovine chymosin, an enzyme used for cheese manufacture, have already been reported in the early nineties [4,5], other proteins, for example, biopharmaceuticals such as interleukin-6 [3], tissue plasminogen activator [6], single-chain antibodies [7], and hepatitis B pseudoviral particles [8] are still just found in milligram per liter quantities in the culture medium. Unsatisfactory low yields are often caused by proteolytic degradation of the target protein [6,9], with improvements being obtained by modifying culture conditions [6,10,11] and developing strains with less proteases [12,13]. Moreover, the complex and not well understood impact of environmental conditions on fungal morphology and resulting effects on protein production and excretion imposes an additional handicap for a more widespread distribution of Aspergillus as a recombinant protein producer.…”

Section: Introductionmentioning

confidence: 99%

Agitation effects on morphology and protein productive fractions of filamentous and pelleted growth forms of recombinant Aspergillus niger

El‐Enshasy

Kleine

Rinas

2006

Process Biochemistry

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Recombinant Aspergillus niger genetically engineered to produce glucose oxidase using the constitutive gpdA promoter and the glucoamylase signal sequence for secretion was grown in batch cultures at agitation speeds of 200 -800 rpm covering the industrial relevant power input range of 0.1 -5 W kg -1 . The growth morphology ranged from large pellets with an average diameter of 1500 µm at low power input up to micropellets embedded in a filamentous network at high power input. A correlation of agitation intensity with growth morphology and glucose oxidase production revealed an increase of the protein production capability with the change of the growth morphology from pelleted to filamentous growth forms. However, the exposure to higher shear stress with increasing power input also resulted in lower biomass yields as well as increased transient formation of polyol (xylitol) and higher final concentrations of oxalic acid. The highest specific production rates were found in young filamentous growth forms at high power input. Although intermediate agitation intensity leading to small pellets became more favorable during prolonged cultivation. An acridine orange staining procedure discriminating between RNA rich (red) and RNA poor regions (green) of the fungal biomass proved that active protein production is restricted to filamentous growth forms and the outer layer of fungal pellets. A correlation between the RNA rich fraction of the biomass determined by image analysis and the productivity is shown.

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Enhanced heterologous protein production in Aspergillus niger through pH control of extracellular protease activity

Cited by 51 publications

References 28 publications

The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger

The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger

Partial Purification and Characterization of Extracellular Protease from <i>Pedicoccus acidilactici</i>

Agitation effects on morphology and protein productive fractions of filamentous and pelleted growth forms of recombinant Aspergillus niger

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