Correction: Exploring Codon Optimization and Response Surface Methodology to Express Biologically Active Transmembrane RANKL in E. coli

Abstract Background: Pseudomonas lipases are widely used in industrial applications due to its unique biochemical properties, but one of the biggest limitations are the low yields obtained in native strains therefore, organisms as E. coli are used for the recombinant lipase overexpression. However, the recombinant lipase is accumulated as inclusion bodies and it affects biological activity, making that researchers evaluate different fermentation conditions to improve the activity of recombinant enzymes. In this study, a statistical experimental design was implemented to evaluate the effects of temperature, agitation rate and osmolyte concentration on the recombinant lipase activity produced in E. coli BL21 (DE3). Once the significant variables were identified, an optimization by a Response Surface Methodology was applied to maximize the lipase production. Results: The Box-Behnken designs revealed different optimal fermentation conditions for each osmolyte experiment. The glycerol showed the highest specific lipase activity compared to the other osmolytes and 0.1 M of osmolyte glycerol,5°C and 110 rpm showed the highest significant increase on the specific lipase activity and the data fitted the model very well. The validation showed that 452.01 U/mg of specific lipase activity was obtained which was significantly higher compared to the group where no glycerol was added (271.38 U/mg). The relative recombinant lipase expression was 2.7-fold lower at 5°C compared to 25 °C, but at 5°C the lipase activity was significantly higher. In addition, when the 3 L shaken Erlenmeyer Bioreactor was used to produce the recombinant lipase based on the power input parameter, the specific lipase activity was not significantly different from that found in Schott (408,4 U/mg and 452 U/mg, respectively), which means that this Bioreactor platform should be used for future scale-up processes.Conclusion: Low temperatures, low agitation rates and 0.1 M of glycerol in the autoinduction media enhanced the activity of the recombinant lipase produced in E. coli BL21(DE3). The optimized conditions and the 3 L shaken Erlenmeyer Bioreactor can be used to produce the recombinant enzyme in a higher volume based on the power input parameter. Further studies using this strategy may lead to the identification of optimal culture conditions for a given recombinant enzyme facilitating the large-scale bioprocess implementation.

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Optimization of ultrasound-assisted extraction of polyphenolic compounds from coriander seeds using response surface methodology

Acta periodica technologica ◽

10.2298/apt1647249z ◽

2016 ◽

pp. 249-263 ◽

Cited By ~ 1

Author(s):

Zoran Zekovic ◽

Sasa Djurovic ◽

Branimir Pavlic

Keyword(s):

Response Surface Methodology ◽

Response Surface ◽

Three Dimensional ◽

Extraction Process ◽

Biologically Active ◽

Ultrasonic Power ◽

Ultrasound Assisted Extraction ◽

Assisted Extraction ◽

Order Polynomial ◽

Predicted Values

Coriandrum sativum L. (coriander) seeds (CS) were used for preparation of extracts with high content of biologically active compounds. In order to optimize ultrasoundassisted extraction process, three levels and three variables of Box-Behnken experimental design (BBD) in combination with response surface methodology (RSM) were applied, yielding maximized total phenolics (TP) and flavonoids (TF) content and antioxidant activity (IC50 and EC50 values). Independent variables were temperature (40-80oC), extraction time (40-80 min) and ultrasonic power (96-216 W). Experimental results were fitted to a second-order polynomial model with multiple regression, while the analysis of variance (ANOVA) was employed to assess the model fitness and determine optimal conditions for TP (79.60oC, 49.20 min, 96.69 W), TF (79.40oC, 43.60 min, 216.00 W), IC50 (80.00oC, 60.40 min, 216.00 W) and EC50 (78.40oC, 68.60 min, 214.80 W). On the basis of the obtained mathematical models, three-dimensional surface plots were generated. The predicted values for TP, TF, IC50 and EC50 were: 382.68 mg GAE/100 g CS, 216 mg CE/100 g CS, 0.03764 mg/mL and 0.1425 mg/mL, respectively.

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Comparative Study of VariousE. coliStrains for Biohydrogen Production Applying Response Surface Methodology

The Scientific World JOURNAL ◽

10.1100/2012/819793 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 4

Author(s):

Péter Bakonyi ◽

Nándor Nemestóthy ◽

Katalin Bélafi-Bakó

Keyword(s):

Response Surface Methodology ◽

Comparative Study ◽

Response Surface ◽

Strain Engineering ◽

Genetically Engineered ◽

Wild Type ◽

E Coli ◽

Physiological Characterization ◽

Optimized Conditions

The proper strategy to establish efficient hydrogen-producing biosystems is the biochemical, physiological characterization of hydrogen-producing microbes followed by metabolic engineering in order to give extraordinary properties to the strains and, finally, bioprocess optimization to realize enhanced hydrogen fermentation capability. In present paper, it was aimed to show the utility both of strain engineering and process optimization through a comparative study of wild-type and genetically modifiedE. colistrains, where the effect of two major operational factors (substrate concentration and pH) on bioH2production was investigated by experimental design and response surface methodology (RSM) was used to determine the suitable conditions in order to obtain maximum yields. The results revealed that by employing the genetically engineeredE. coli(DJT 135) strain under optimized conditions (pH: 6.5; Formate conc.: 1.25 g/L), 0.63 mol H2/mol formate could be attained, which was 1.5 times higher compared to the wild-typeE. coli(XL1-BLUE) that produced 0.42 mol H2/mol formate (pH: 6.4; Formate conc.: 1.3 g/L).

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Heterologous Ectoine Production inEscherichia coli: Optimization Using Response Surface Methodology

International Journal of Microbiology ◽

10.1155/2019/5475361 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13 ◽

Cited By ~ 4

Author(s):

I Putu Parwata ◽

Deana Wahyuningrum ◽

Sony Suhandono ◽

Rukman Hertadi

Keyword(s):

Response Surface Methodology ◽

Response Surface ◽

Gene Cluster ◽

Incubation Temperature ◽

Halophilic Bacteria ◽

Dry Weight ◽

Halophilic Bacterium ◽

E Coli ◽

Halomonas Elongata ◽

Recombinant Cell

Introduction. A halophilic bacterium of theHalomonas elongataBK-AG25 has successfully produced ectoine with high productivity. To overcome the drawbacks of high levels of salt in the production process, a nonhalophilic bacteria ofEscherichia coli(E. coli) was used to express the ectoine gene cluster of the halophilic bacteria, and the production of ectoine by the recombinant cell was optimized.Methods. The ectoine gene cluster from the halophilic bacterium was isolated and inserted into an expression plasmid of pET30(a) and subsequently transformed intoE. coliBL21 (DE3). Production of ectoine from the recombinantE. coliwas investigated and then maximized by optimizing the level of nutrients in the medium, as well as the bioprocess conditions using response surface methodology. The experimental designs were performed using a central composite design.Results. The recombinantE. colisuccessfully expressed the ectoine gene cluster ofHalomonas elongataBK-AG25 under the control of theT7promoter. The recombinant cell was able to produce ectoine, of which most were excreted into the medium. The optimization of ectoine production with the response surface methodology showed that the level of salt in the medium, the incubation temperature, the optical density of the bacteria before induction, and the final concentration of the inducer gave a significant effect on ectoine production by the recombinantE. coli. Interestingly, the level of salt in the medium and the incubation temperature showed an inverse effect on the production of intracellular and extracellular ectoine by the recombinant cell. At the optimum conditions, the production yield was about 418 mg ectoine/g cdw (cell dry weight) after 12 hours of incubation.Conclusion. This study is the first report on the expression of an ectoine gene cluster ofHalomonas elongataBK-AG25 inE. coliBL21, under the control of theT7promoter. Optimization of the level of nutrients in the medium, as well as the bioprocess condition using response surface methodology, has successfully increased the production of ectoine by the recombinant bacteria.

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Statistical Optimization of the Induction of Phytase Production by Arabinose in a recombinant E. coli using Response Surface Methodology

ASEAN Journal on Science and Technology for Development ◽

10.29037/ajstd.304 ◽

2017 ◽

Vol 26 (1) ◽

pp. 57-64

Author(s):

Abd-El Aziem Farouk ◽

Anis Shobirin Meor Hussin ◽

Ralf Greiner ◽

Shareef Mohideen Ismail ◽

Hamadah Mohd Nur Lubis

Keyword(s):

Response Surface Methodology ◽

Response Surface ◽

Incubation Temperature ◽

Expression System ◽

Optimal Conditions ◽

Phytase Production ◽

E Coli ◽

Central Composite Experimental Design ◽

Full Factorial ◽

Central Composite

The production of phytase in a recombinant E.coli using the pBAD expression system was optimized using response surface methodology with full-factorial faced centered central composite design. The ampicilin and arabinose concentration in the cultivation media and the incubation temperature were optimized in order to maximize phytase production using 2 3 central composite experimental design. With this design the number of actual experiment performed could be reduced while allowing eludidation of possible interactions among these factors. The most significant parameter was shown to be the linear and quadratic effect of the incubation temperature. Optimal conditions for phytase production were determined to be 100 µg/ml ampicilin, 0.2 % arabinose and an incubation temperature of 37ºC. The production of phytase in the recombinant E. coli was scaled up to 100 ml and 1000 ml.

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Rana chensinensis Ovum Oil Based on CO2 Supercritical Fluid Extraction: Response Surface Methodology Optimization and Unsaturated Fatty Acid Ingredient Analysis

Molecules ◽

10.3390/molecules25184170 ◽

2020 ◽

Vol 25 (18) ◽

pp. 4170

Author(s):

Yuanshuai Gan ◽

Dongliang Xu ◽

Jianqiu Zhang ◽

Zhongyao Wang ◽

Shihan Wang ◽

...

Keyword(s):

Response Surface Methodology ◽

Response Surface ◽

Supercritical Fluid Extraction ◽

Supercritical Fluid ◽

Unsaturated Fatty Acids ◽

Model Verification ◽

Biologically Active ◽

Surface Model ◽

Fluid Extraction ◽

Rana Chensinensis

Rana chensinensis ovum oil (RCOO) is an emerging source of unsaturated fatty acids (UFAs), but it is lacking in green and efficient extraction methods. In this work, using the response surface strategy, we developed a green and efficient CO2 supercritical fluid extraction (CO2-SFE) technology for RCOO. The response surface methodology (RSM), based on the Box–Behnken Design (BBD), was used to investigate the influence of four independent factors (pressure, flow, temperature, and time) on the yield of RCOO in the CO2-SFE process, and UPLC-ESI-Q-TOP-MS and HPLC were used to identify and analyze the principal UFA components of RCOO. According to the BBD response surface model, the optimal CO2-SFE condition of RCOO was pressure 29 MPa, flow 82 L/h, temperature 50 °C, and time 132 min, and the corresponding predicted optimal yield was 13.61%. The actual optimal yield obtained from the model verification was 13.29 ± 0.37%, and the average error with the predicted value was 0.38 ± 0.27%. The six principal UFAs identified in RCOO included eicosapentaenoic acid (EPA), α-linolenic acid (ALA), docosahexaenoic acid (DHA), arachidonic acid (ARA), linoleic acid (LA), and oleic acid (OA), which were important biologically active ingredients in RCOO. Pearson correlation analysis showed that the yield of these UFAs was closely related to the yield of RCOO (the correlation coefficients were greater than 0.9). Therefore, under optimal conditions, the yield of RCOO and principal UFAs always reached the optimal value at the same time. Based on the above results, this work realized the optimization of CO2-SFE green extraction process and the confirmation of principal bioactive ingredients of the extract, which laid a foundation for the green production of RCOO.

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