Optimization of glucose oxidase production by Aspergillus niger using genetic-and process-engineering techniques

Hellmuth, Karsten; Pluschkell, Stefanie; Jung, J. K.; Ruttkowski, Edeltraud; Rinas, Ursula

doi:10.1007/bf00166912

Cited by 34 publications

(18 citation statements)

References 22 publications

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“…Sample preparation and analysis of cell dry mass (CDM), and endo-and exocellular GOD activities were carried out as described previously [40,42]. The quantitative determination of xylose, organic acids and xylitol were carried out by HPLC using an Aminex HXP-87H column (BioRad) for separation (T=25°C) and ultravilotet (UV) and refractive index detectors …”

Section: Methodsmentioning

confidence: 99%

“…The construction of recombinant A. niger NRRL-3 (GOD 3-18), carrying multiple copies of the god gene fused to the α-amylase signal sequence and controlled by the constitutive gpdApromoter has been described before [40].…”

Section: Strainmentioning

confidence: 99%

“…Inoculation was carried out by 1×10 7 spores mL -1 obtained from a densely conidiating culture grown on CM agar medium for 48-72 h. CM-agar plates for sporulation were prepared as described previously [40]. The composition of the culture medium was as follows [g L -1 The digital control unit of the Biostat MD bioreactor, mass flow meter, balance of NaOH solution, pumps and exhaust-gas analysis system were interfaced to a VME-bus microcomputer using UBICON (Universal Bio-Process Control System) software for data acquisition and processing.…”

Section: Culture Conditionsmentioning

confidence: 99%

“…In this work, we employed a recombinant strain of Aspergillus niger genetically engineered to produce glucose oxidase using the constitutive gpdA promoter and the glucoamylase signal sequence for secretion [40]. This strain produces glucose oxidase during growth on glucose but also during growth on other carbon sources such as fructose or xylose [41].…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

Section: Strainmentioning

confidence: 99%

Section: Culture Conditionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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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“…The enzyme activity in the cell-free culture broth was determined according to Hellmuth et al (1995) with minor modi®cations (Kapat et al 1998b). The speci®c production of GOD was expressed in terms of Units per milligram of dry cell mass (U mg )1 DCM).…”

Section: Methodsmentioning

confidence: 99%

Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition

2001

Self Cite

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Aims: To obtain an optimal combination of agitation speed and aeration rate for maximization of speci®c glucose oxidase (GOD) production in recombinant Saccharomyces cerevisiae, and to establish a correlation between k L a vis-a Á-vis oxygen transfer condition and speci®c glucose oxidase production. Methods and Results: The oxygen transfer condition was manifested indirectly by manipulating the impeller speed and aeration rate in accordance with a Central Composite Rotatory Design (CCRD). The dissolved oxygen concentration and the volumetric oxygen transfer coef®cient (k L a) 1 were determined at corresponding combinations of impeller speed and aeration rate. The maximal speci®c extracellular glucose oxidase production (3á17 U mg ±1 dry cell mass) was achieved when the initial dissolved oxygen concentration was 6á83 mg l ±1 at the impeller speed of 420 rev min ±1 and at the rate of aeration of 0á25 vvm. It was found out that while impeller speed had a direct effect on the production of enzyme, a correlation between k L a and speci®c GOD production could not be established. Conclusions: At the agitation speed of 420 rev min ±1 and at 0á25 vvm aeration rate, the degree of turbulence and the dissolved oxygen concentration were thought to be optimal both for cellular growth and production of enzyme. Signi®cance and Impact of the Study: The combined effect of agitation and aeration on recombinant glucose oxidase production in batch cultivation has not yet been reported in the literature. Therefore, this study gives an insight into the effect of these two important physical parameters on recombinant protein production. It also suggests that since there is no correlation between k L a and speci®c production of GOD, k L a should not be used as one of the scale-up parameters.

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Kinetics of glucose oxidase excretion by recombinantAspergillus niger

Pluschkell¹,

Hellmuth²,

Rinas³

1996

Biotechnol. Bioeng.

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The kinetics of glucose oxidase (GOD) excretion by recombinant Aspergillus niger NRRL‐3 (GOD3‐18) were investigated using enzymatic activity measurements as well as gel electrophoresis techniques. The majority of GOD was produced during rapid growth in the first phase of the cultivation. The high excretion rate during this phase did not prevent the endocellular accumulation of GOD up to 40% of the total soluble cell protein demonstrating that the production rate exceeded the excretion rate of the enzyme into the culture medium. During the second phase of the cultivation, excretion of GOD occurred at a slower rate, although the majority of GOD produced during the first phase was excreted during the second phase of the cultivation. At the end, about 90% of the total GOD produced was recovered from the culture medium. Two‐dimensional gel electrophoresis provided evidence that endo‐ and exocellular GOD were indistinguishable, revealing identical posttranslational modifications (e.g., signal sequence cleavage, glycosylation pattern). The results demonstrate that the initial steps of the secretory pathway are fast and that the excretion of the enzyme into the culture fluid was most likely delayed due to retention by the cell wall. © 1996 John Wiley & Sons, Inc.

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Optimization of glucose oxidase production by Aspergillus niger using genetic-and process-engineering techniques

Cited by 34 publications

References 22 publications

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

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

Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition

Kinetics of glucose oxidase excretion by recombinantAspergillus niger

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