scholarly journals Heterologous Ectoine Production inEscherichia coli: Optimization Using Response Surface Methodology

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
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.

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

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.   

Optimization of Carotenoid Production by Aspergillus Carbonarius in Submerged Fermentation Using a Response Surface Methodology

2007 ◽  
Vol 3 (5) ◽  
Author(s):  
K. R Sanjay ◽  
N. Kumaresan ◽  
B. Manohar ◽  
S. Umesh Kumar ◽  
G. Vijayalakshmi
Keyword(s):  
Response Surface Methodology ◽  
Response Surface ◽  
Incubation Temperature ◽  
Process Condition ◽  
Dry Weight ◽  
Polynomial Equation ◽  
Aspergillus Carbonarius ◽  
Fermentation Time ◽  
Carotenoid Production ◽  
Initial Medium

Based on a three level Box Behnken design involving the variables pH (X1), incubation temperature (X2) and fermentation time (X3), a response surface methodology for the production of carotenoid by a mutant Aspergillus carbonarius CFTRI-UV10046 was standardized. Data analyzed using a second order polynomial equation resulted in the optimized process condition of 46.7 h growth at 29.30C in a corn flour medium of pH 3.3 for carotenoid production. Under these conditions the model predicted 1283.00 µg of carotenoid /g of dry fungal biomass which is 52.19% higher then the initial medium. With regard to biomass, 2.44 g dry weight/100 ml culture medium was obtained after 56 h of growth at 29.70C in the medium of pH-3.6.

scholarly journals R unning Head: Novel Recombinant Phytate - d egrading Enzyme Production Statistical Optimization of the Induction of Phytase Production by Arabinose in a recombinant E. coli using Response Surface Methodology

2017 ◽  
Vol 26 (1) ◽  
Author(s):  
Abd - ElAziem Farouk ◽  
Anis Shobirin Meor Hussin ◽  
Ralf Greiner ◽  
Shareef Mohideen Ismail ◽  
Mohamed Faizal Batcha ◽  
...  
Keyword(s):  
Response Surface Methodology ◽  
Response Surface ◽  
Incubation Temperature ◽  
Expression System ◽  
Phytase Production ◽  
E Coli ◽  
Significant Parameter ◽  
Central Composite Experimental Design ◽  
Full Factorial ◽  
Central Composite

The production of phytase in a recombinant E.coliusing thepBAD expression system wasoptimized using response surface methodology with full-factorial faced centered central composite design. The ampicilin and arabinose concentrationin the cultivation media and the incubation temperature were optimized in order to maximize phytase productionusing 23 central composite experimental design. With this design the number of actual experiment performed could be reduced while allowing eludidation of possible interactions among thesefactors. The most significant parameter was shown to be the linear and quadratic effect of the incubation temperature. OPtim al conditionsfor phytase production were determinedto be100 μg/ml ampicilin, 0.2 % arabinose and an incubation temperature of37ºC.The production of phytase in the recombinant E. coliwas scaledup to 100ml and 1000ml.

scholarly journals Enzyme optimization to reduce the viscosity of pitanga (Eugenia uniflora L.) juice

2016 ◽  
Vol 19 (0) ◽  
Author(s):  
Ricardo Schmitz Ongaratto ◽  
Luiz Antonio Viotto
Keyword(s):  
Response Surface Methodology ◽  
Response Surface ◽  
Incubation Time ◽  
Regression Models ◽  
Incubation Temperature ◽  
Correlation Coefficients ◽  
Enzyme Concentration ◽  
Optimum Conditions ◽  
Activity Time ◽  
Independent Variables

Summary The aim of this work was to separately evaluate the effects of pectinase and cellulase on the viscosity of pitanga juice, and determine the optimum conditions for their use employing response surface methodology. The independent variables were pectinase concentration (0-2.0 mg.g–1) and cellulase concentration (0-1.0 mg.g–1), activity time (10-110 min) and incubation temperature (23.2-56.8 °C). The use of pectinase and cellulase reduced the viscosity by about 15% and 25%, respectively. The results showed that enzyme concentration was the most important factor followed by activity time, and for the application of cellulase the incubation temperature had a significant effect too. The regression models showed correlation coefficients (R2) near to 0.90. The pectinase application conditions that led to the lowest viscosity were: concentration of 1.7 mg.g–1, incubation temperature of 37.6 °C and incubation time of 80 minutes, while for cellulase the values were: concentration of 1.0 mg.g-1, temperature range of 25 °C to 35 °C and incubation time of 110 minutes.

scholarly journals Design and Optimization of a Process for Sugarcane Molasses Fermentation bySaccharomyces cerevisiaeUsing Response Surface Methodology

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Nour Sh. El-Gendy ◽  
Hekmat R. Madian ◽  
Salem S. Abu Amr
Keyword(s):  
Response Surface Methodology ◽  
Response Surface ◽  
Batch Fermentation ◽  
Fermentation Process ◽  
Incubation Temperature ◽  
Operating Conditions ◽  
Quadratic Model ◽  
Optimum Operating ◽  
Sugarcane Molasses ◽  
Bioethanol Production

A statistical model was developed in this study to describe bioethanol production through a batch fermentation process of sugarcane molasses by locally isolatedSaccharomyces cerevisiaeY-39. Response surface methodology RSM based on central composite face centered design CCFD was employed to statistically evaluate and optimize the conditions for maximum bioethanol production and study the significance and interaction of incubation period, initial pH, incubation temperature, and molasses concentration on bioethanol yield. With the use of the developed quadratic model equation, a maximum ethanol production of 255 g/L was obtained in a batch fermentation process at optimum operating conditions of approximately 71 h, pH 5.6, 38°C, molasses concentration 18% wt.%, and 100 rpm.

Enhanced Production of Mycelia by the Medicinal Mushroom Cordyceps militaris Using Plackett-Burman Design and Response Surface Methodology

2011 ◽  
Vol 138-139 ◽  
pp. 1209-1214
Author(s):  
Xiao Yu Liu ◽  
Fan Xing Meng ◽  
Yi Bo Zhang ◽  
Huan He ◽  
Wei Han ◽  
...  
Keyword(s):  
Response Surface Methodology ◽  
Response Surface ◽  
Yeast Extract ◽  
Dry Weight ◽  
Fermentation Medium ◽  
Cordyceps Militaris ◽  
Enhanced Production ◽  
Burman Design ◽  
Optimization Of Fermentation ◽  
Plackett Burman Design

Response surface methodology (RSM) was used for statistical optimization of fermentation medium that influenced the yield of endo-polysaccharide from cultivated mycelia of Cordyceps militaris. First, the Plackett-Burman design was used to evaluate the effects of ten variables including glucose, maltose, peptone, yeast extract, KH2PO4, MgSO4, CaCl2, VB1, inoculum density and medium capacity. Among these variables, glucose, peptone and yeast extract were identified to have the significant effects. Subsequently, response surface methodology based on a five-level three-factor central composite design was employed to determine the maximum dry weight (DW) of mycelial biomass at optimum concentration of glucose, peptone and yeast extract. The mycelia growth was found to correlate to the three parameters that could be represented by second-order polynomial models. The optimal values of the three parameters were determined as 4.62% glucose, 3.36% peptone and 0.43% yeast extract. The prediction DW was 23.727g/L. The actual experimental results were in agreement with the prediction.

scholarly journals Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C50 Carotenoid Cyclases

2010 ◽  
Vol 192 (21) ◽  
pp. 5688-5699 ◽  
Author(s):  
Roman Netzer ◽  
Marit H. Stafsnes ◽  
Trygve Andreassen ◽  
Audun Goksøyr ◽  
Per Bruheim ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Micrococcus Luteus ◽  
Dry Weight ◽  
Biosynthetic Gene Cluster ◽  
Hybrid Gene ◽  
E Coli ◽  
Shake Flasks ◽  
Catalytic Functions ◽  
First Time

ABSTRACT We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C50 carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C40 lycopene, C45 nonaflavuxanthin, C50 flavuxanthin, and C50 sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C50 carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C50 carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ε-cyclic C50 carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C50 carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C50 carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.

scholarly journals Optimum Parameters for Extracting Three Kinds of Carotenoids from Pepper Leaves by Response Surface Methodology

Separations ◽  
2021 ◽  
Vol 8 (9) ◽  
pp. 134
Author(s):  
Nenghui Li ◽  
Jing Li ◽  
Dongxia Ding ◽  
Jianming Xie ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Response Surface Methodology ◽  
Response Surface ◽  
Dry Weight ◽  
Optimum Parameters ◽  
Order Polynomial ◽  
Pepper Leaves ◽  
New Varieties ◽  
Weak Light ◽  
Experimental Values ◽  
Optimum Extraction

To determine the optimum parameters for extracting three carotenoids including zeaxanthin, lutein epoxide, and violaxanthin from pepper leaves by response surface methodology (RSM), a solvent of acetone and ethyl acetate (1:2) was used to extract carotenoids with four independent factors: ultrasound time (20–60 min); ratio of sample to solvent (1:12–1:4); saponification time (10–50 min); and concentration of saponification solution (KOH–methanol) (10–30%). A second-order polynomial model produced a satisfactory fitting of the experimental data with regard to zeaxanthin (R2 = 75.95%, p < 0.0197), lutein epoxide (R2 = 90.24%, p < 0.0001), and violaxanthin (R2 = 73.84%, p < 0.0809) content. The optimum joint extraction conditions of zeaxanthin, lutein epoxide, and violaxanthin were 40 min, 1:8, 32 min, and 20%, respectively. The optimal predicted contents for zeaxanthin (0.823022 µg/g DW), lutein epoxide (4.03684 µg/g dry; DW—dry weight), and violaxanthin (16.1972 µg/g DW) in extraction had little difference with the actual experimental values obtained under the optimum extraction conditions for each response: zeaxanthin (0.8118 µg/g DW), lutein epoxide (3.9497 µg/g DW), and violaxanthin (16.1590 µg/g DW), which provides a theoretical basis and method for cultivating new varieties at low temperatures and weak light resistance.

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