Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli

Olaofe, Oluwafemi A.; Burton, Stephanie G.; Cowan, Don A.; Harrison, Susan T.L.

doi:10.1016/j.bej.2010.06.013

Cited by 29 publications

(14 citation statements)

References 38 publications

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“…To further improve protein concentration and possibly improve gene expression, a glucose-based defined medium was investigated due to an expected increase in biomass concentration [24]. This necessitated varying inducer concentration (IPTG), as previous investigations have suggested that inducer concentration should be determined on a basis of biomass concentration [25]. The concentration of δ-ALA which is required for heme synthesis was varied simultaneously across the same range.…”

Section: Discussionmentioning

confidence: 99%

The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6

et al. 2013

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BackgroundBiocatalyst improvement through molecular and recombinant means should be complemented with efficient process design to facilitate process feasibility and improve process economics. This study focused on understanding the bioprocess limitations to identify factors that impact the expression of the terminal hydroxylase CYP153A6 and also influence the biocatalytic transformation of n–octane to 1-octanol using resting whole cells of recombinant E. coli expressing the CYP153A6 operon which includes the ferredoxin (Fdx) and the ferredoxin reductase (FdR).ResultsSpecific hydroxylation activity decreased with increasing protein expression showing that the concentration of active biocatalyst is not the sole determinant of optimum process efficiency. Process physiological conditions including the medium composition, temperature, glucose metabolism and product toxicity were investigated. A fed-batch system with intermittent glucose feeding was necessary to ease overflow metabolism and improve process efficiency while the introduction of a product sink (BEHP) was required to alleviate octanol toxicity. Resting cells cultivated on complex LB and glucose-based defined medium with similar CYP level (0.20 μmol gDCW-1) showed different biocatalyst activity and efficiency in the hydroxylation of octane over a period of 120 h. This was influenced by differing glucose uptake rate which is directly coupled to cofactor regeneration and cell energy in whole cell biocatalysis. The maximum activity and biocatalyst efficiency achieved presents a significant improvement in the use of CYP153A6 for alkane activation. This biocatalyst system shows potential to improve productivity if substrate transfer limitation across the cell membrane and enzyme stability can be addressed especially at higher temperature.ConclusionThis study emphasises that the overall process efficiency is primarily dependent on the interaction between the whole cell biocatalyst and bioprocess conditions.

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Section: Discussionmentioning

confidence: 99%

The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6

et al. 2013

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“…This allows to increase biomass concentration within particles and therefore increase reaction rate (Olaofe et al . ).…”

Section: Resultsmentioning

confidence: 97%

“…Biomass was propagated over a series of seven repeated batches with 50 mg l À1 of ampicillin. This allows to increase biomass concentration within particles and therefore increase reaction rate (Olaofe et al 2010).…”

Section: Propagation Of Biomass and Induction Of Recombinant Enzymementioning

confidence: 99%

Biocatalysis with Escherichia coli -overexpressing cyclopentanone monooxygenase immobilized in polyvinyl alcohol gel

Rebroš

Lipták

Rosenberg

et al. 2014

Lett Appl Microbiol

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Significance and Impact of the Study: Immobilization in polyvinylalcohol gel particles is desirable technique with presumptive impact on industrial applications of recombinant whole-cell Baeyer-Villiger monooxygenases as biocatalysts for production of bioactive compounds and precursors of potentially new drugs. An original immobilization of cells E. coli with overproduced Baeyer-Villiger monooxygenase improved their stability in repetitive batch biooxidations as compared to free cells. Detected autoinduction of recombinant enzyme in pET22b+ plays significant role in application of immobilized cells as it may increase specific activity of cells in repetitive use under growing reaction conditions. Original technique for qualitative analysis of enzyme expression within immobilized cells was developed. was developed and used for verification of optimal conditions for the induction of cyclopentanone monooxygenase. Here, we successfully performed six repeated batch Baeyer-Villiger biooxidations utilizing entrapped cells using 40% (w/v) polyvinyl alcohol gel particles in flasks with baffles. The latter conditions have been found to be the most appropriate achieving optimal oxygen transfer within LentiKats â . Moreover, immobilized cells retained their catalytic efficiency over six reaction cycles, while the catalytic efficiency of free cells decreased after three reaction cycles.

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“…Tabela 5 -Parâmetros cinéticos obtidos a partir de cultivo em reator de 3 L. foram plaqueadas em meio LB solidificado (não seletivo sobrecarga metabólica do crescimento celular e expressão da proteína (OLAOFE et al, 2010 Com relação à utilização do IPTG como indutor, apesar de ampla extensão de concentrações ter sido reportada, variando-se de 0,005 a 5 mM, a expressão é geralmente realizada com uma concentração final a 1 mM, independente da densidade celular da cultura, haja visto que esta molécula não é metabolizada pela célula (DONOVAN et al, 1996). Tal concentração, considerada em excesso, tem sido utilizada no intuito de induzir completamente o operador lac, resultando numa maior expressão do gene.…”

Section: Parâmetros Cinéticosunclassified

Avaliação do sistema de estabilização plasmidial toxina-antitoxina para a produção de proteínas recombinantes em <i>Escherichia coli</i>.

Katayama¹

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Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli

Cited by 29 publications

References 38 publications

The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6

The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6

Biocatalysis with Escherichia coli -overexpressing cyclopentanone monooxygenase immobilized in polyvinyl alcohol gel

Avaliação do sistema de estabilização plasmidial toxina-antitoxina para a produção de proteínas recombinantes em <i>Escherichia coli</i>.

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