Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli

Sohoni, Sujata Vijay; Nelapati, Dhanaraj; Sathe, Sneha S.; Javadekar-Subhedar, Vaishali; Gaikaiwari, Raghavendra P.; Wangikar, Pramod P.

doi:10.1016/j.biortech.2015.02.038

Cited by 45 publications

(19 citation statements)

References 27 publications

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“…For the chosen shake flask geometry, liquid levels, and the agitation conditions, the k L a values were 4.2 and 8.9 hours −1 for 60 and 80 rpm, respectively, for a pitch of 5 cm. The k L a was measured by the gassing out‐gassing in method as described previously …”

Section: Resultsmentioning

confidence: 99%

“…This work presents a novel method to estimate instantaneous OER for cyanobacterial cultures grown in shake flasks. The method requires the knowledge of k L a for oxygen under the pertinent growth conditions, which can be readily measured experimentally by the gassing‐out, gassing‐in method, as described previously . The k L a values from the published literature can be used when available for the matching conditions including agitation speed and pitch, flask geometry, and liquid levels in flask .…”

Section: Resultsmentioning

confidence: 99%

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A method to compute instantaneous oxygen evolution rates in cyanobacterial cultures grown in shake flasks

Sengupta

Wangikar

2020

Engineering Reports

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Cyanobacteria have been attracting attention for various biotechnological applications due to their ability to carry out oxygenic photosynthesis. The oxygen evolution rate (OER) of cyanobacterial cultures is a key physiological parameter, which is typically measured offline by using oxygen electrodes. In situ measurement of OER has been reported in a photobioreactor fitted with a dissolved oxygen (DO) probe. Here, we show that the OER can be estimated from online measurements of the DO levels by using sensors fitted in shake flasks, which are widely used for preliminary characterization of microbial strains. We study how the DO levels change under diurnal light regime and different growth conditions in two strains of Synechococcus elongatus, namely, PCC 7942 and PCC 11801, in shake flasks. We use the DO levels coupled with the knowledge of the overall gas-liquid mass transfer coefficient for oxygen (k L a) to compute the OER.For the growth conditions tested, the cumulative oxygen evolved in a day correlated with the increase in biomass suggesting that the calculated OER, in turn, might provide an estimate of the organism's growth rate. We believe that the method may be widely applicable for the growth characterization of organisms that perform oxygenic photosynthesis. K E Y W O R D Sabiotic factors, cyanobacteria, dissolved oxygen profile, oxygen evolution rates, PreSens technology, shake flaskThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

A method to compute instantaneous oxygen evolution rates in cyanobacterial cultures grown in shake flasks

Sengupta

Wangikar

2020

Engineering Reports

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“…Nitrilase activity was determined in whole cells as described by Sohoni et al (2015). Cells from 1ml culture were resuspended in 0.1 M sodium phosphate buffer (pH 7.5).…”

Section: Experimental Methodsmentioning

confidence: 99%

A plug-and-play system for enzyme production at commercially viable levels in fed-batch cultures of Escherichia coli BL21 (DE3)

et al. 2018

Preprint

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Commercial exploitation of enzymes in biotransformation necessitates a robust method for enzyme production that yields high enzyme titer. Nitrilases are a family of hydrolases that can transform nitriles to enantiopure carboxylic acids, which are important pharmaceutical intermediates. Here, we report a fed-batch method that uses a defined medium and involves growth under carbon limiting conditions using DO-stat feeding approach combined with an optimized post-induction strategy, yielding high cell densities and maximum levels of active and soluble enzyme. This strategy affords strict control of nutrient feeding and growth rates, and ensures sustained protein synthesis over a longer period. The method was optimized for highest titer of nitrilase reported so far (247 kU/l) using recombinant E. coli expressing the Alcaligenes sp. ECU0401 nitrilase. The fed-batch protocol presented here can also be employed as template to produce a wide variety of enzymes with minimal modification, as demonstrated for alcohol dehydrogenase and formate dehydrogenase.

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“…It has been reported that the expression of recombinant proteins under the control of lac operon induced by IPTG leads to low expression of recombinant proteins. According to the previous studies, due to the high cost and toxicity of IPTG, complex operating procedure, and nonuniform protein expression pattern, the use of lactose as an inducer for expression of recombinant proteins is preferable for overproduction of recombinant proteins [3]. By using lactose as inducer more soluble protein has been produced.…”

Section: Introductionmentioning

confidence: 99%

Enhancement of extracellular bispecific anti‐MUC1 nanobody expression in E. coli BL21 (DE3) by optimization of temperature and carbon sources through an autoinduction condition

Rezaei

Shojaosadati

Farahmand

et al. 2020

Engineering in Life Sciences

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Escherichia coli is one of the most suitable hosts for production of antibodies and antibody fragments. Antibody fragment secretion to the culture medium improves product purity in cell culture and diminishes downstream costs. In this study, E. coli strain BL21 (DE3) harboring gene encoding bispecific anti‐MUC1 nanobody was selected, and the autoinduction methodology for expression of bispecific anti‐MUC1 nanobody was investigated. Due to the replacement of IPTG by lactose as inducer, less impurity and toxicity in the final product were observed. To increase both intracellular and extracellular nanobody production, initially, the experiments were performed for the key factors including temperature and duration of protein expression. The highest amount of nanobody was produced after 21 h at 33°C. The effect of different carbon sources, glycerol, glucose, lactose, and glycine as a medium additive at optimum temperature and time were also assessed by using response surface methodology. The optimized concentrations of carbon sources were obtained as 0.75% (w/v), 0.03% (w/v), 0.1% (w/v), and 0.75% (w/v) for glycerol, glucose, lactose, and glycine, respectively. Finally, the production of nanobody in 2 L fermenter under the optimized autoinduction conditions was evaluated. The results show that the total titer of 87.66 µg/mL anti‐MUC1 nanobody, which is approximately seven times more than the total titer of nanobody produced in LB culture medium, is 12.23 µg/L .

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Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli

Cited by 45 publications

References 27 publications

A method to compute instantaneous oxygen evolution rates in cyanobacterial cultures grown in shake flasks

A method to compute instantaneous oxygen evolution rates in cyanobacterial cultures grown in shake flasks

A plug-and-play system for enzyme production at commercially viable levels in fed-batch cultures of Escherichia coli BL21 (DE3)

Enhancement of extracellular bispecific anti‐MUC1 nanobody expression in E. coli BL21 (DE3) by optimization of temperature and carbon sources through an autoinduction condition

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