CQ10 Coenzyme Production Bacteria Screening
Coenzyme Q10, chemical formula C₉HoO₄, also known as ubidecarenone and ubiquinone, is a fat-soluble quinone compound containing a side chain consisting of multiple isoprenoid units connected to the parent nucleus of p-benzoquinone. The molecule contains a side chain of multiple isoprene units connected to the p-benzoquinone nucleus, which has 10 isoprene units. Ubiquinone is involved in energy production and activation in human cells.
Coenzyme Q10 is a natural antioxidant and activator of cellular metabolism. It prevents the oxidation of LDL and cholesterol and reduces the incidence of coronary atherosclerosis. It is used clinically for heart disease, diabetes, cancer, acute and chronic hepatitis, Parkinson's disease, and other diseases, to improve immunity and treat immune system disorders.2 Recent studies have found that Coenzyme Q10 has anti-aging effects. Coenzyme Q10 is produced by plant and animal tissue extraction, chemical synthesis, plant cell culture, and microbial fermentation3 .
Coenzyme Q10 is characterized by the Craven's color test, the Wiss-Brubacher method and thin-layer chromatography. For quantitative analysis, visible spectrophotometry, mass spectrometry (MS), infrared (IR) and ultraviolet (UV) absorption spectrometry (UVAS), optical density analysis (ODA), spectrofluorimetry (SF), and high-performance liquid chromatography (HPLC)l- were used. In this experiment, the content of coenzyme Q10 in Saccharomyces cerevisiae was determined by visible spectrophotometry and inverse high-pressure liquid chromatography (HPLC), and the coenzyme Q10-producing strains of Saccharomyces cerevisiae were screened.
1 Materials and Methods
1.1 Test Materials
1.1.1 Test Strains
Yeast strains from the library were selected as the starting strains for coenzyme Q10 production, as shown in Table 1.
1.1.2 Test medium. Wort 100mL, glucose 10 g, peptone 5g, yeast paste 3g, K₂HPO₄0.5 g, KH₂PO₄0.5 g, MgSO₄-7H₂O 0.3g, pH value 6.0, fixed 1000 mL.
1.1.3 Instruments and Reagents
721 spectrophotometer, ethyl cyanoacetate (analytically pure), anhydrous ethanol (analytically pure), 0.5% potassium hydroxide ethanol solution (weigh 1g of potassium hydroxide (analytically pure) dissolved in 200 mL of ethanol (95%)), coenzyme Q10 standard (Sigma C9538-coenzymeQ10 minium98%HPLC). ).
Table 1 Coenzyme Q10-producing Departure Strains
1.2 Test Methods
1.2.1 Shake Flask Culture, Isolation of Bacteria and CQ10 Coenzyme Extraction
Take 500 mL triangular flask, pour 100 mL of medium, sterilized and inoculated according to 2% of the volume, 200 r/min shaking bed, 30 ℃ shaking culture for 72h, fermentation liquid 3000 r/min centrifugation for 20 min, collect the bacterial body, washed with sterile water twice, centrifugation, to get the yeast bacterial body, weighed. Take 5g of fermented bacteria, transfer to 500 mL round bottom flask, add antioxidant, methanol, sodium hydroxide, distilled water and mix well, reflux at 90 ℃ for 45 min, cool down and add 40 mL of petroleum ether, extract in 2 times, refrigerate at 4 ℃ overnight, remove precipitated proteins and lipids, use rotary evaporator to evaporate out the petroleum ether, and dissolve the yellow oily substance at the bottom of the flask in anhydrous ethanol to be determined.
1.2.2 Visible Colorimetric Standard Curves
Prepare 0.2 mg/mL anhydrous ethanol solution of CQ10 Coenzyme standard, according to the data in Table 2, take different volumes of anhydrous ethanol solution of Coenzyme Q10 standard, add ethyl cyanoacetate and 0.5% potassium hydroxide ethanol solution of 1 mL each, and make a volume of 25 mL, stoppered, shaken, and placed in a dark place at room temperature of 20~30 ℃ for 35 min, and then determine the absorbance at 620 nm (OD) by 721 spectrophotometer, and plot the standard curve with the absorbance and the concentration of Coenzyme Q10. The absorbance at 620 nm was measured by 721 spectrophotometer, and the standard curve was plotted with this absorbance and the concentration of coenzyme Q10.
Table 2 Data related to standard curve of visible colorimetric method
1.2.3 Determination of CQ10 Coenzyme in Strains by Colorimetric Method
Take 3mL of anhydrous ethanol solution and add 1mL each of ethyl cyanoacetate and 0.5% potassium hydroxide ethanol solution respectively, stopper tightly, shake well, and put it in a dark place at 20~30℃ for 35 min, then measure the OD of the samples at 620 nm by using a 721 spectrophotometer, and then calculate the content of CoQ10 in the samples according to the standard curve plotted by the determination.
1.2.4 Identification of Coenzyme Q10 content by HPLC
Chromatographic conditions: the packing material was octadecylsilane-bonded silica gel, the mobile phase was methanol: anhydrous ethanol (1:1), the flow rate was 1 mL/min, the detection wavelength was 275 nm, the temperature was 35 ℃, and the mass concentration of coenzyme Q10 standard (Sigma C9538-coenzymeQ10 minium 98% HPLC) was 0.2 mg/mL, and the injection volume was 10 μL.
1.2.5 Concentration of Samples by HPLC Method
CQ10 Coenzyme samples with concentration differences were measured by visible spectrophotometry and HPLC, respectively, and the correlation between the OD values obtained by visible spectrophotometry and the values determined by HPLC (peak area of the sample/peak area of the standard) were compared to determine the correlation between the data. The correlation between the OD values obtained by VIS and HPLC (peak area of sample/peak area of standard) was determined and the correlation between the data could be estimated.
2 Results and Analysis
2.1 Visible Colorimetric Method for the Determination of CQ10 Coenzyme
2.1.1 Standard curve
The linearity of the standard curve was good, R²=0.997, which can be used to measure the content of coenzyme Q10 by the visible light colorimetric method (Fig. 1); the regression equation of the standard curve of coenzyme Q10: y=3.9268xox is the mass concentration of coenzyme Q10, and y is the OD value.
Figure 1 Coenzyme Q10 standard curve
2.1.2 Determination of CQ10 Coenzyme in Strains by Visible Light Method
The saponification extraction method was used to isolate coenzyme Q10 from the fermenting organisms, and the results of colorimetric detection of coenzyme Q10 in the samples are shown in Table 3, which showed that the highest coenzyme Q10 yields were obtained from C. tropicalis (C. tropi-calis(cast).Berkhout) B021 and Schizosaccheromyces pombe Lindner B147 with reference to the yield of the organisms and the yield of coenzyme Q10 extracted. Berkhout) B021 and Schizosaccharomyces pombe Lindner (B147) had the highest coenzyme Q10 yields.
Table 3 Content of coenzyme Q10 in different strains of bacteria
2.2 Validation of the HPLC Method for the Detection of Coenzyme Q10
The samples with higher content were verified by HPLC assay. Comparison with the standard profiles (Fig. 2) showed that CQ10 Coenzyme was produced by both Saccharomyces cerevisiae B147 (Fig. 3) and Pseudohyphae tropicalis B021 (Fig. 4).
Peak time//min
Fig. 2 Graphical representation of standardized products
Figure 4 HPLC profile of B021 sample
The CQ10 Coenzyme content in the samples was determined by HPLC using standard concentration and peak area conversion, and the results are shown in Table 4. The results are shown in Table 4. Like the visible light colorimetric method, high levels of coenzyme Q10 were found in both Saccharomyces cerevisiae B147 and Pseudohyphae tropicalis B021, which were identified as the starting strains for the metabolic control breeding technique.
Table 4 Determination of coenzyme Q10 by HPLC method
2.3 Linearity Between Visible Spectrophotometry and HPLC method
There was a correlation between the values obtained by 721 VIS and the HPLC values with a linear equation: y=0.0675x+0.0187 and a correlation coefficient R²= 0.986 (Figure 5).
3 Conclusion and Discussion
In this experiment, the visible spectrophotometric method was chosen to measure the content of CQ10 Coenzyme. According to the principle that ethyl cyanoacetate can replace the methoxy group on the coenzyme Q10 molecule to produce blue color under alkaline condition, we established a colorimetric detection method for coenzyme Q10 with 721 spectrophotometer. This method has two advantages: firstly, it can qualitatively detect whether the strain contains coenzyme Q10 through the color reaction, and qualitatively screen the strains; secondly, it can determine the OD value according to the degree of color development by 721 spectrophotometer, and then comparing with the standard curve to calculate the content of coenzyme Q10, which can be used to quantitatively analyze the yield of the strains under certain conditions. This method can be used to screen the starting strains of metabolic control breeding, both qualitative and quantitative, is a simple and fast means of coenzyme Q10 detection.
This test used alkaline alcohol saponification for the extraction of coenzyme Q10. Organic solvents have also been used in the literature. Due to the cellular structure of Saccharomyces cerevisiae, organic solvent extraction requires wall-breaking. However, it was found that the alkanol saponification method was relatively more effective. The saponification time and temperature affect the yield of coenzyme Q10, and there is a certain loss of coenzyme Q10 during the transfer of the extract, protein and fat removal, and evaporation of the extract. Therefore, the actual coenzyme Q10 content was higher than the measured value. The slightly higher values of coenzyme Q10 determined by the visible colorimetric method for strain screening may be due to the presence of some impurities in the yeast cell extracts, which may have interfered with the results of the assay.
In this experiment, two strains, C. tropical- alis (cast) Berkhout B021 and Schizosaccharomyces pombe Lindner B147, were screened for the production of coenzyme Q10. According to the literature7 , coenzyme Q10 production may be further increased if the cultivation conditions are improved with the addition of suitable precursors for coenzyme Q10 biosynthesis. In addition, it is expected that the yield of coenzyme Q10 can be increased by optimizing the nutrient conditions of the fermentation culture of the strains and using metabolic control breeding techniques, such as I3-9: nutrient-deficient mutation, anti-feedback-regulated mutant strains, selecting nutrient-deficient revertant or conditional mutant strains, to deregulate the key enzymes of the end-products, or constructing engineered strains to select high-yielding strains.
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