scholarly journals Enhancement in Human Insulin Precursor Secretion by Pichia pastoris through Modification of Expression Conditions

2021 ◽  
Vol 29 (1) ◽  
pp. 22-30
Author(s):  
Dini Nurdiani ◽  
Hariyatun Hariyatun ◽  
Nuruliawaty Utami ◽  
Eko Wahyu Putro ◽  
Wien Kusharyoto
Keyword(s):  
Pichia Pastoris ◽  
Cell Density ◽  
Standard Procedure ◽  
Expression System ◽  
Methanol Concentration ◽  
Cell Factory ◽  
Methanol Induction ◽  
Initial Cell ◽  
Initial Cell Density ◽  
Insulin Precursor

Pichia pastoris is an alternative yeast expression system to produce heterologous proteins. It has excellent characteristics for an industrial cell factory, such as its ability to reach high cell densities, high secretory capacity, and a low level of native proteins. In our previous study, we introduced a synthetic insulin precursor (IP)-encoding gene constructed in a pD902 expression vector into P. pastoris. However, the P. pastoris recombinant strains expressed a little amount of IP protein. Here, we modified the expression conditions, including inoculum density, methanol concentration, methanol induction time, pH, and temperature, to obtain a higher amount of secreted IP than our previous result. Protein analysis for studying the five parameters was conducted by SDS-PAGE, and the protein amount was estimated by ImageJ applying lysozyme as standard. We successfully enhanced the IP expression by modifying expression conditions. The highest increased of up to 100 folds was achieved when methanol concentration for induction was arranged at 3% (v/v), and the initial cell density for methanol induction was set at an optical density at 600 nm (OD600) of approximately 10 compared to the standard procedure, where the expression was set at 0.5% (v/v) methanol induction and initial cell density at OD600 = 1.

scholarly journals Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris

2003 ◽  
Vol 69 (10) ◽  
pp. 6064-6072 ◽  
Author(s):  
Mônica C. Triches Damaso ◽  
Marcius S. Almeida ◽  
Eleonora Kurtenbach ◽  
Orlando B. Martins ◽  
Nei Pereira ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Cell Density ◽  
Thermomyces Lanuginosus ◽  
Small Scale ◽  
Efficient Production ◽  
Optimum Temperature ◽  
Nitrogen Base ◽  
Xylanase Production ◽  
Initial Cell ◽  
Initial Cell Density

ABSTRACT Highly efficient production of a Thermomyces lanuginosus IOC-4145 β-1,4-xylanase was achieved in Pichia pastoris under the control of the AOX1 promoter. P. pastoris colonies expressing recombinant xylanase were selected by enzymatic activity plate assay, and their ability to secrete high levels of the enzyme was evaluated in small-scale cultures. Furthermore, an optimization of enzyme production was carried out with a 23 factorial design. The influence of initial cell density, methanol, and yeast nitrogen base concentration was evaluated, and initial cell density was found to be the most important parameter. A time course profile of recombinant xylanase production in 1-liter flasks with the optimized conditions was performed and 148 mg of xylanase per liter was achieved. Native and recombinant xylanases were purified by gel filtration and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, matrix-assisted laser desorption ionization-time of flight-mass spectrometry and physicochemical behavior. Three recombinant protein species of 21.9, 22.1, and 22.3 kDa were detected in the mass spectrum due to variability in the amino terminus. The optimum temperature, thermostability, and circular dichroic spectra of the recombinant and native xylanases were identical. For both enzymes, the optimum temperature was 75°C, and they retained 60% of their original activity after 80 min at 70°C or 40 min at 80°C. The high level of fully active recombinant xylanase obtained in P. pastoris makes this expression system attractive for fermentor growth and industrial applications.

The Effects of Nutritional Media and Initial Cell Density on the Growth and Development of Spirulina platensis

2017 ◽  
Vol 7 (10) ◽  
Author(s):  
Nguyen Van Khanh ◽  
Nguyen Thi Diem ◽  
Le Thi Tuyet Nhan ◽  
Phan Van Cu ◽  
Tran Quang Khanh Van ◽  
...  
Keyword(s):  
Cell Density ◽  
Spirulina Platensis ◽  
Growth And Development ◽  
Initial Cell ◽  
Initial Cell Density

scholarly journals Cultivation of Chlorella sorokiniana in a bubble-column bioreactor coupled with cooking cocoon wastewater treatment: effects of initial cell density and aeration rate

2021 ◽  
Author(s):  
Chunye Xue ◽  
Kun Gao ◽  
Pingkang Qian ◽  
Jingwei Dong ◽  
Zheng Gao ◽  
...  
Keyword(s):  
Cell Density ◽  
Biomass Production ◽  
Bubble Column ◽  
Chlorella Sorokiniana ◽  
Aeration Rate ◽  
Chemical Compositions ◽  
Initial Cell ◽  
Initial Cell Density ◽  
Bubble Column Bioreactor ◽  
Column Bioreactor

Abstract Previous studies documented that Chlorella sorokiniana could grow well on cooking cocoon wastewater (CCW) with a maximum biomass of 0.49 g/L. In order to further enhance the biomass production and nutrient removals, a bubble-column bioreactor was designed and performed to cultivate C. sorokiniana in CCW, and two main cultivation parameters were investigated in this work. Results showed that a maximum algal biomass, specific growth rate, and biomass productivity of 2.83 g/L, 0.854 d−1, and 476.25 g/L/d, respectively, were achieved when this alga was cultivated in the bioreactor with an initial cell density of 0.8 g/L and an aeration rate of 3.34 L air/L culture/min; meanwhile, removal efficiencies of ammonium, total nitrogen, total phosphorus, and chemical oxygen demand reached 97.96, 85.66, 97.96, and 86.43%, respectively, which were significantly higher than that obtained in our previous studies. Moreover, chemical compositions in the algal cells varied with the changes of cultivation conditions (i.e., initial cell density and aeration rate). Thus, it is concluded that (1) the bubble-column bioreactor was suitable for cultivation of C. sorokiniana coupled with the CCW treatment; and (2) initial cell density and aeration rate affected the biomass production, nutrient removals and chemical compositions of this alga.

scholarly journals Study on the biosynthesis and structure characterization of exopolysaccharide from Lactobacillus fermentum MC3

2018 ◽  
Vol 127 (1D) ◽  
pp. 13
Author(s):  
Trần Thị Ái Luyến ◽  
Đỗ Thị Bích Thủy ◽  
Trần Thị Văn Thi ◽  
Phan Thị Thu Huyền
Keyword(s):  
Cell Density ◽  
Culture Medium ◽  
Structure Characterization ◽  
Lactobacillus Fermentum ◽  
Molar Ratio ◽  
Gas Liquid Chromatography ◽  
Liquid Chromatography Mass Spectrometry ◽  
Initial Cell ◽  
Initial Cell Density

<p><strong>Abstract: </strong>The effects of carbohydrate sources in various concentration (2%, 3%, 4%, 5%, 6%) and fermentation conditions (such as initial cell density, temperature, pH and incubation time) on EPS synthesis of <em>Lactobacillus fermentum </em>MC3 were also studied. The results showed that adding different sugars (including glucose, lactose and sucrose) to culture medium significantly increased the EPS production. In comparison with other concentrations, EPS amounts were maximized in the medium supplemented with 4% (w/v) of sugars. The outcome was the highest for glucose, which was 178.207 mg/L, the obtained figures for lactose and for sucrose were 148.614 mg/L and 152.272 mg/L respectively. The results indicated that EPS production by <em>L. fermentum </em>MC3 reached the maximum values in the medium supplemented with 4% (w/v) glucose at 40<sup>0</sup>C, pH 6.0, initial cell density of 10<sup>6</sup>CFU/ml for 48 h cultivation with amount of 200.728 mg/L. By methylation analysis and gas–liquid chromatography–mass spectrometry (GLC–MS), the exopolysaccharide was found to be composed of D-mannose: D-glucose: D-galactose in a molar ratio of 1 : 0.74 : 0.09.</p>

scholarly journals Optimal initial cell density that yields the highest number of primary synovial mesenchymal stem cells in a clinical setting

2020 ◽  
Author(s):  
Kiyotaka Horiuchi ◽  
Mitsuru Mizuno ◽  
Hisako Katano ◽  
Kentaro Endo ◽  
Nobutale Ozeki ◽  
...  
Keyword(s):  
Human Serum ◽  
Cell Density ◽  
Clinical Setting ◽  
Clinical Studies ◽  
Initial Density ◽  
Fold Increase ◽  
Cell Number ◽  
Autologous Serum ◽  
Initial Cell ◽  
Initial Cell Density

Abstract Background Synovial mesenchymal stem cell (MSC) transplantation improved clinical outcomes in knees with cartilage defects and after repairs of degenerative torn menisci. For these, nucleated cells were cultured with 10% autologous serum for 14 days. Importantly, nucleated cell number and serum volume practically varied by patient, and initial cell density and dish number affected yield/donor. Our purpose was to examine the effect of plating density within range in clinical studies on fold increase and to estimate the MSC yield/donor, which are affected by the nucleated cell number and serum volume. Methods We obtained the data of the autologous serum volume, nucleated cell number, and initial plating cell density from 10 patients who participated in our previous clinical studies. Experimentally, synovial nucleated cells harvested from three patients with osteoarthritis during total knee arthroplasty were cultured with 10% mixed allogenic human serum for 14 days to examine the effect of initial cell density on fold increase. We used both our previous clinical data and our current experimental data to predict the yields of synovial MSCs in nine cases within the range of clinical studies. Results In the clinical studies, collected serum ranged between 87 and 186 ml, harvested nucleated cells ranged between 6.2 and 23 million, and the top five total MSC yields were obtained from donors when the initial plating cell density ranged from 919 to 1905 cells/cm 2 . Experimentally, fold increase significantly decreased between 700 and 1,900 cells/cm 2 . In cases where the cell number was low and the serum volume was high, the estimated yield/donor decreased along with the initial density, while in cases where the cell number was high and the serum volume was low, the estimated yield/donor increased along with the initial density. In cases both where some nucleated cells were discarded at low density and no cells were discarded at high density, the maximum yield/donor were obtained when the initial density was the lowest density that does not discard cells. Conclusions There was an optimal initial density that yields the highest number of primary MSCs in a clinical setting when cell numbers and human serum volumes are limited.

scholarly journals Distribution and Physiological Characteristics of Hyperthermophiles in the Kubiki Oil Reservoir in Niigata, Japan

2000 ◽  
Vol 66 (1) ◽  
pp. 73-79 ◽  
Author(s):  
Yoh Takahata ◽  
Miyuki Nishijima ◽  
Toshihiro Hoaki ◽  
Tadashi Maruyama
Keyword(s):  
Cell Density ◽  
Carbon Sources ◽  
Most Probable Number ◽  
Oil Reservoir ◽  
Formation Water ◽  
Probable Number ◽  
Initial Cell ◽  
Initial Cell Density ◽  
Inorganic Substances ◽  
Subterranean Environment

ABSTRACT The distribution of culturable hyperthermophiles was studied in relation to environmental conditions in the Kubiki oil reservoir in Japan, where the temperature was between 50 and 58°C. Dominant hyperthermophilic cocci and rods were isolated and shown to belong to the genera Thermococcus and Thermotoga, respectively, by 16S rDNA analyses. Using the most-probable-number method, we found that hyperthermophilic cocci were widely distributed in several unconnected fault blocks in the Kubiki oil reservoir. In 1996 to 1997, their populations in the production waters from oil wells were 9.2 × 103 to 4.6 × 104cells/ml, or 10 to 42% of total cocci. On the other hand, hyperthermophilic rods were found in only one fault block of the reservoir with populations less than 10 cells/ml. DominantThermococcus and Thermotoga spp. grew at reservoir temperatures and utilized amino acids and sugars, respectively, as sole carbon sources. While organic carbon was plentiful in the environment, these hyperthermophiles were unable to grow in the formation water due to lack of essential nutrients. Concentrations of some organic and inorganic substances differed among fault blocks, indicating that the movement of formation water between fault blocks was restricted. This finding suggests that the supply of nutrients via fluid current is limited in this subterranean environment and that the organisms are starved in the oil reservoir. Under starved conditions at 50°C, culturable cells of Thermococcus sp. remained around the initial cell density for about 200 days, while those of Thermotoga sp. decreased exponentially to 0.01% of the initial cell density after incubation for the same period. The difference in survivability between these two hyperthermophiles seems to reflect their populations in the fault blocks. These results indicate that hyperthermophilic cocci and rods adapt to the subterranean environment of the Kubiki oil reservoir by developing an ability to survive under starved conditions.

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