Development of fed-batch strategies for the production of streptavidin by Streptomyces avidinii based on power input and oxygen supply studies

Müller, Jakob; Risse, Joe Max; Jussen, Daniel; Flaschel, Erwin

doi:10.1016/j.jbiotec.2012.10.021

Cited by 17 publications

(11 citation statements)

References 28 publications

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Section: Resultsmentioning

confidence: 99%

“…Despite lower product concentrations and productivities, the process offers advantages compared to cultivation of the natural producer S. avidinii, since this organism is much more sensitive towards environmental factors, shows a complex morphology, slow growth and resulting long process times in an un-optimized environment, and imposes higher requirements for biochemical engineering. 26 The new process strategy necessitates complex media compounds, which may result in batch-to-batch variability and the need for diafiltration during downstream processing. However, it is methanol-independent, thus eliminating central disadvantages of the application of P AOX (see Introduction).…”

Section: Bioreactor Experimentsmentioning

confidence: 99%

“…Publications on the expression of both core and full‐length SAV in P. pastoris resulted in remarkable concentrations of up to 4 g L −1 (71 µM) and 0.65 g L −1 (11 µM) of SAV, respectively. Comparable product concentrations have so far only been reported for the secretion of the protein by the natural producer S. avidinii , documenting the outstanding properties of P. pastoris as a host for heterologous protein production. However, both studies were based on the methanol‐inducible alcohol oxidase 1 ( AOX 1, abbreviated AOX hereinafter) promoter.…”

Section: Introductionmentioning

confidence: 99%

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GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Müller

Bruhn

Flaschel

et al. 2016

Biotechnology Progress

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Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin-auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol-based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol-based processes lead to large proportions of biotin-blocked binding sites of streptavidin due to biotin-supplemented media. Targeting these problems, this paper provides strategies for the methanol-free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin-blocked streptavidin. The optimized, easily scalable fed-batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin-free streptavidin and a productivity of 57.8 nM h(-1) based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un-optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h(-1) ), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855-864, 2016.

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Section: Resultsmentioning

confidence: 99%

Section: Bioreactor Experimentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Müller

Bruhn

Flaschel

et al. 2016

Biotechnology Progress

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“…Biakan dengan nutrien yang kompleks, dimana sumber nitrogen berasal dari casein hydrolysate dan soy peptone dengan kecepatan goyangan yang sama menghasilkan pertumbuhan bakteri yang lebat tetapi produksi streptavidin sangat rendah (2,5 µg/ml). Biakan dalam LAB bioreactor dengan media kompleks yang sama tetapi dengan stirrer kecepatan tinggi (400-700 rpm) dan pH yang terkontrol dilaporkan menghasilkan streptavidin dengan konsentrasi sekitar 450 µg/ml (Muller et al 2013). Berdasarkan fakta diatas dapat disimpulkan bahwa goyangan atau shaking yang cepat mutlak diperlukan untuk produksi streptavidin, makin cepat pertumuhan bakteri makin cepat goyangan yang dibutuhkan.…”

Section: Produksi Streptavidinunclassified

“…Pada awalnya, streptavidin diproduksi dengan media yang mengandung senyawa kompleks seperti protein (Stapley et al 1963;Chaiet & Wolf 1964). Produksi streptavidin yang tinggi dengan menggunakan media kompleks dapat dihasilkan menggunakan bioreaktor dimana aerasi dan shearing dapat terkontrol (Aldwin et al 1990;Muller et al 2013). Media sintetik yang tidak mengandung senyawa kompleks untuk produksi streptavidin sehingga memudahkan purifikasi streptavidin telah dilaporkan .…”

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Production and purification of streptavidin with higher biotin-binding activity

Tarigan¹,

Sumarningsih²

2015

JITV

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Tarigan S, Sumarningsih. 2014. Production and purification of streptavidin with higher biotin-binding activity. JITV 19(3): 231-238. DOI: http://dx.doi.org/10.14334/jitv.v19i3.1086The objective of this study was to develop practical, efficient method for production, purification and assay of binding activity of streptavidin. Streptomyces avidinii was first propagated on agar plates, the bacterial cells on the agar were scrapped and suspended in a defined synthetic media (4.4 ml/cm 2 ). After 7 days agitation on a rotary shaker (200 rpm/min) at room temprature (≈28°C), the bacterial cells in the culture were pelleted. The culture supernatant was concentrated to 1/62 original volume with 75% saturation ammonium sulphate. After intensive dialysis against ammonium carbonate buffer pH 11, the suspension was loaded into an iminobiotin agarose column chromatography. The adsorbed protein (streptavidin) was eluted with sodium acetate buffer, pH 4, and the eluate was concentrated with an ultrafiltration divice and suspended in PBS. The strepatavidin-binding activity was assayed by a competitive ELISA, a competition between streptavidin in the sample and the HRP-streptavidin conjugate for the biotin (biotinyl IgG) immobilised on wells of a microtitre plate. The detection limit of this assay measured 0.16 µg/ml streptavidin. The method developed in this study produced 160 µg/ml streptavidin in the culture supernatant. After concentration with the ammonium sulphate, the streptavidin concentration increased to 4 mg/ml (69% recovery). At the final step of purification, streptavidin with 10 mg/ml concentration was obtained. The purity of the streptavidin was higher (95%) with a recovary of 19%. The purified streptavidin in this study appeared as a dimer core streptavidin on SDS PAGE and its binding activity was twice as high as that of a commercial one. Penelitian ini bertujuan mengembangkan teknik produksi, purifikasi dan pengukuran streptavidin yang efisien dan praktis. Streptomyces avidinii mula-mula dipropagasi dalam lempeng agar kemudian sel bakteri dari agar dipindahkan kedalam media cair sintetik (4,4 ml/cm 2 ). Setelah 7 hari diagitasi diatas rotary shaker 200 rpm/min pada suhu ruangan (≈28°C), sel bakteri dipeletkan, supernatan dikonsentrasikan menjadi 1/62 volume awal dengan amonium sulfat saturasi 75%. Setelah didialisis dalam larutan amonium carbonat pH 11, suspensi protein diadsorbsikan kedalam kolom iminobiotin agarose. Protein (streptavidin) yang teradsorbsi dielusi dengan larutan sodium asetat pH 4, eluat dikonsentrasikan dengan ultrafiltrasi dan disuspensikan dalam PBS. Pengukuran aktivitas binding streptavidin dilakukan dengan ELISA kompetitif, kompetisi antara streptavidin dalam sampel yang diukur dengan konjugat HRP-streptavidin memperebutkan biotin (IgG biotinil) yang diimobilisasi pada microtitre plate. ELISA ini mempunyai limit deteksi 0.16 µg/ml streptavidin. Metode produksi dan purifikasi yang dikembangkan dalam penelitian ini menghasilkan 160 µg/ml pada biakan supernatan. Setelah dikonsentrasik...

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