A fermentation strategy for production of recombinant protein subjected to plasmid instability

Chen, Shu‐Jen; Ke, Bo-Shun; Chiu, I-Chung

doi:10.1007/s11814-008-0181-4

Cited by 5 publications

(3 citation statements)

References 19 publications

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“…The behaviour of BK4063 was also analysed in batch experiments revealing a slower growth phenotype and a lower oxygen request compared to the wild type strain and the complete loss of recombinant plasmid after 24 h of growth. Chen et al (2008) illustrated a method to perform recombinant E. coli cultures in the absence of selective marker, designating the fast degradation of ampicillin, and therefore loss of selective pressure, as the main cause of reduced yields. In order to check whether selective marker degradation affected plasmid stability also in the present case, a batch experiment in which ampicillin was repeatedly added to the broth was performed with strain BK4063.…”

Section: Discussionmentioning

confidence: 99%

Improved fructosylated chondroitin production by kfoC overexpression in E. coli K4

Cimini

Rosa

Viggiani

et al. 2010

Journal of Biotechnology

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Section: Discussionmentioning

confidence: 99%

Improved fructosylated chondroitin production by kfoC overexpression in E. coli K4

Cimini

Rosa

Viggiani

et al. 2010

Journal of Biotechnology

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“…Two-stage fermentation strategy has been used for production of recombinant protein and other metabolites [24][25][26][27]. For FB A, we proposed a two-stage fermentation strategy and expected to get a high production.…”

Section: Nk Expression By Ask Culture and Fed-batch Experimentsmentioning

confidence: 99%

High cell density cultivation of a recombinant Bacillus subtilis for nattokinase production

Cui

Bian

Sun

et al. 2020

Preprint

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Background: Nattokinase (NK), a brinolytic enzyme, can be produced by culturing recombinant Bacillus subtilis in Luria-Bertani broth in a shaking ask. For use as a nutraceutical, however, a large-scale preparation and a simple puri cation process are required. Results: The present study utilized a fed-batch process to cultivate a B. subtilis strain carrying a pHT01 plasmid with an NK-encoding gene (B. subtilis/pHT01-aprN1). For batch A (FB A), with a pH-stat twostage fermentation strategy, we achieved an activity of 2910.5 ± 21.6 U mL-1 and a speci c activity of 30.32 U ml-1 OD 600-1. Then, we changed the strategy with a later induction and lower feeding rate to pursue higher cell density and thus higher enzyme activity, a 11.9-fold activity of 4521.8 ± 23.8 U mL-1 was acquired, however, the speci c activity was lower than FB A. For the third batch, low-glycerol-levelmaintain feeding strategy was followed, and nally, a NK activity of 7778 ±17.28 U mL-1 was obtained, according to our knowledge, it was the highest activity assayed by the brin plate method ever reported. Furthermore, fermentation supernatant was successively puri ed by ammonium sulfate precipitation and nickel column a nity chromatography with a total NK recovery rate of 65.2%. Conclusions: Our results indicate that there is a balance between the cell growth rate and NK expression when recombinant Bacillus subtilis is cultured with a fed-batch process. The equilibrium state can be attained by optimizing the induction and feeding strategy, and thus a high cell density and enzyme activity can be achieved.

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“…The combination of recombinant proteins and large-scale processes make it possible to produce these proteins in amounts exceeding greatly the availability in natural resources [1][2][3][4]. The most commonly used host organism for recombinant protein production is still Escherichia coli because its genetic properties and physiological behaviour are well-known [1][2][3][5][6][7][8][9]. Within this study, High-Cell-Density Cultivation (HCDC) using E. coli as host organism was evaluated in view of a good expression and production of the L-arabinose isomerase (L-AI) from Geobacillus stearothermophilus.…”

Section: Introductionmentioning

confidence: 99%

Overexpression of L-Arabinose Isomerase for Production of the Low-Calorie Bulk Sweetener D-Tagatose

Holsbeeck¹

2014

Enz Eng

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High-Cell-Density Cultivations show a huge potential to produce recombinant proteins to amounts greatly exceeding the availability in natural resources. An interesting example of a recombinant protein is an L-arabinose isomerase, which is able to convert D-galactose to the low-caloric and low-glycaemic bulk sweetener D-tagatose. Within this study, the L-arabinose isomerase from Geobacillus stearothermophilus was expressed intracellularly in Escherichia coli. The cultivation medium contained glucose, yeast extract and various macro-and micronutrients. The effect of air flow rate on E. coli growth and expression of L-arabinose isomerase was studied. After 52 hours, an Optical Density and Dry Cell Weight of 154 ± 4 and 54.8 ± 1.3 g L-1 were reached, respectively by regulating the air flow rate between 0.2 and 30 L min-1. A corresponding L-arabinose isomerase activity of 6.99 ± 0.46 U mL-1 was reached. A drawback of High-Cell-Density Cultivation is the production of the by-product acetic acid which may inhibit growth. However, the acetic acid concentration was maintained as low as possible during fermentation to avoid inhibitory effects inherent to this compound. With the L-arabinose isomerase produced, a conversion percentage of 37.1 ± 1.5% was achieved, corresponding to 94.9 ± 3.7 g L-1 D-tagatose. Thus, the implementation of a High-Cell-Density Cultivation led to an efficient expression of the L-arabinose isomerase enzyme and D-tagatose production. Also the storage stability of the cells was investigated during several months at 4°C. A stable L-arabinose isomerase enzyme was noticed during at least 8 months storage at 4°C.

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A fermentation strategy for production of recombinant protein subjected to plasmid instability

Cited by 5 publications

References 19 publications

Improved fructosylated chondroitin production by kfoC overexpression in E. coli K4

Improved fructosylated chondroitin production by kfoC overexpression in E. coli K4

High cell density cultivation of a recombinant Bacillus subtilis for nattokinase production

Overexpression of L-Arabinose Isomerase for Production of the Low-Calorie Bulk Sweetener D-Tagatose

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