TW201932599A - Method for producing astaxanthin by using Escherichia coli especially transplanting the Escherichia coli expression vector into the Escherichia coli strain - Google Patents

Abstract

The invention provides a method for producing astaxanthin by using Escherichia coli, which comprises: (1) providing an Escherichia coli expression vector pET-30a; (2) providing an Escherichia coli BL-21 (DE3) strain; (3) transplanting the Escherichia coli expression vector pET-30a into the Escherichia coli BL-21 (DE3) strain; (4) culturing the resulting recombinant Escherichia coli strain; and (5) collecting the recombinant Escherichia coli strain, purifying and extracting it to obtain astaxanthin. The method is characterized in that the Escherichia coli expression vector pET-30a is specifically regulated to achieve a free form of astaxanthin with high production and high purity.

Description

Method for producing astaxanthin by using Escherichia coli

The present invention provides a method for producing astaxanthin using Escherichia coli. Further, the present invention provides a method for increasing the yield and purity of free form astaxanthin using Escherichia coli.

Astaxanthin is a red pigment belonging to the natural carotenoids. It has an orange-red color and its chemical name is 3,3'-dihydroxy-β. β'-carotene-4, 4'-dione has optics. A mirror stereoisomer with a molecular formula of C ₄₀ H ₅₂ O ₄ having polar and lipophilic properties. Astaxanthin is widely found in algae, shrimp, crabs and fish. Terrestrial animals cannot synthesize on their own, so they must be taken from food. In nature, only plants, microalgae, mold and red yeast have the ability to synthesize astaxanthin, and there is no ability to synthesize astaxanthin in animals. Therefore, it is necessary to obtain astaxanthin by ingesting microalgae. In addition to being an antioxidant, astaxanthin has other physiological effects, such as: astaxanthin has anti-tumor ability, enhances immunity, anti-inflammatory activity, resists ultraviolet radiation damage and inhibits arterial atherosclerosis. The natural composition of esterified astaxanthin is quite complex. The literature proves that the free form of astaxanthin has higher digestibility and absorption rate in the rainbow trout and trout than the esterified astaxanthin, and adds the free form of astaxanthin to high cholesterol. In the diet, it can effectively increase the antioxidant activity of the liver and reduce the concentration of lipid peroxidase. In addition, the free form of astaxanthin can be linked to a specific fatty acid or a functional peptide to produce a specific esterified astaxanthin or astaxanthin derivative, which can be used for future drug development. Therefore, the free form of astaxanthin is commercially more valuable than esterified astaxanthin.

It has been documented ( Misawa, N. et. al 1995, 177 , 6575-6584.) In E. coli, there are six exogenous genes ( crt E, crt Y, crt I, crt B, which must be synthesized by astaxanthin. Crt Z and crt W) Construction can make E. coli synthesize astaxanthin. Document all past using M13 phage infected E. coli colonize the mode is switched, and the use of all non-photosynthetic soil bacteria (Erwinia uredovora; Pantoea ananatis) genes in the carotenoid biosynthetic enzymes carotene hydroxide (crt E, crt Y , crt I, crt B, crt Z) to form the pACCAR25 ΔcrtX plastid, and in combination with other well-established carotenoid ketolase gene ( crt W) plastids, packaged together in the M13 phage vector Infection is then transmitted to E. coli (Misawa, N. et al. 1995 , 177, 6575-6584; Choi, SK et al.. Mar. Biotechnol. 2005 , 7, 515-522; Ye, RW et al. Appl. Environ .Microbiol. 2006 , 72, 5829-5837 ). However, its disadvantage is a significant decrease in genetic stability and a lower transcriptional efficiency than a high performance performance vector. The literature also confirmed the use of non-photosynthetic soil bacteria ( E.uredovora; P. ananatis ) carotenoid synthesis gene group and carotene hydrolase ( crt E, crt Y, crt I, crt B, crt Z) plus the ocean The carotenoid ketolase ( crt W) of bacteria ( Agrobacterium aurantiacum; Paracoccus sp . strain N81106) produces the free form of levofloxacin (Misawa, N. et al. 1995 , 177, 6575-6584 ; Misawa) , N et al, Bacteriol. 1990 , 172, 6704-6712), because the specific isomer has the highest antioxidant capacity, the specific isomer produced by this strain and the free form of astaxanthin produced by Haematococcus pluvialis Also, the antioxidant capacity is much higher than that of the red yeast.

Previous studies have also indicated that Haematococcus pluvialis and Chlorella are both common sources of esterified astaxanthin production. Saponification can be used to make the esterified astaxanthin a free form of astaxanthin, but it must undergo a complicated extraction process. In addition, it is susceptible to contamination by other compounds during extraction (eg, chlorophyll, glycerides, and other contaminants.) (Yokoyama, A et al, Biosci. Biotech. Biochem. 1994 , 58,1842-1844.) . Due to the difficulty in extracting the free form of astaxanthin and the high cost, the commercially available purity of 95% of the free form of astaxanthin is 300,000 yuan per gram. Therefore, mass production of free form of astaxanthin and simplification of the extraction process and reduction of contamination of other compounds are an important subject to be solved by those skilled in the art to which the present invention pertains.

Taiwan Patent No. TW200934872 discloses a method for commercial production of natural carotenoids, which is transformed with a double vector (mp25yz/pwizI6) into an E. coli M2H host (this strain has been subjected to UV screening of a mutant strain). It can produce astaxanthin 3.7mg/g DCW (dry cell weight) with a purity of 59%. Using high-density fermentation, 1100 mg/L of astaxanthin can be produced with a purity of 40%. Using solvent extraction and purification, it can obtain 5.24mg/g DCW of astaxanthin with a purity of 85% or more. However, the method is complicated and the purity of the extracted astaxanthin is still less than 95%.

Taiwan Patent No. TW201544593 discloses a recombinant polynucleotide sequence for preparing astaxanthin and a use thereof, wherein a plurality of promoters regulate different genes and modify the original genes of host cells by synthetic biology, and produce esters. Astaxanthin. However, it produces esterified astaxanthin, a non-free form of astaxanthin, and the original E. coli gene must be changed. The method is complicated.

In view of this, the inventor of this case has a deep understanding of the shortcomings and shortcomings of the previous case, but he has made improvements and innovations, and after years of painstaking research, he finally succeeded in research and development of this method to produce high purity free form of astaxanthin using E. coli. .

It is an object of the present invention to provide a method for producing a high purity free form of astaxanthin using Escherichia coli.

For the purpose of the foregoing invention, the method comprises the following steps: (1) providing an E. coli expression vector pET-30a; (2) providing an Escherichia coli BL-21 (DE3) strain; (3) expressing the Escherichia coli The vector pET-30a is transfected into the E. coli BL-21 (DE3) strain; (4) the recombinant E. coli strain obtained is cultured; and (5) the recombinant E. coli strain is collected, purified and extracted to obtain astaxanthin .

To achieve the aforementioned object, the E. coli expression vector pET-30a consists of the crtE, crtY, crtI, crtB, crtZ and crtW genes and four promoters.

For the purposes of the foregoing invention, wherein the promoters are selected from three constitutive promoters, and an inducible promoter on the vector, the promoter exhibiting the persistence Different from the start-up time of the inducible promoter, the purity of the initially extracted astaxant was 85%.

For the purposes of the foregoing invention, wherein the inducible promoter is selected from, but not limited to, the T7 promotor.

To achieve the aforementioned object, wherein the inducible promoter controls the activation of the crtW gene.

For the purpose of the foregoing invention, the promoter of the persistent expression can simultaneously activate the crtE gene, the crtZ gene, and the crtY, crtI, crtB genes, respectively.

For the purpose of the foregoing invention, the regulation of the E. coli expression vector pET-30a is specifically regulated.

For the purpose of the foregoing invention, wherein the specific regulatory system activates the crtE, crtY, crtI, crtB and crtZ genes by the promoter of the persistent expression, and then activates the crtW gene by the inducible promoter.

For the purpose of the foregoing invention, the Escherichia coli clones a carotenoid biosynthetic gene group and a carotenoid hydroxylase gene (crtE, crtY, crtI, crtB and crtZ, GenBank: D90087.2) from E. uredorora, And the carotenoid ketolase gene (crtW, PRF: 1096128) cloned from A. aurantiacum was constructed on the pET-30a expression vector.

For the purpose of the foregoing invention, the astaxanthin is a free form of astaxanthin.

For the purpose of the foregoing invention, the purification and extraction of astaxanthin is recovered and purified by high-density fermentation and food grade solvent extraction to obtain more than 95% of astaxanthin.

Figure 1 The composition of the single E. coli expression vector pET-30a and the promoter; Figure 2 The general regulation of the free form of astaxanthin production; Figure 3 The specific regulation of the free form of astaxanthin production; Figure 4A, 4B free form shrimp LC-MS/MS diagram of erythromycin standard; LC-MS/MS diagram of free form astaxanthin produced by strains of Figs. 5A and 5B; Fig. 6 Experimental results of purity >95% astaxanthin; Fig. 7A HPLC spectrum of the free form of astaxanthin in the form; Figure 7B Storage of free form astaxanthin in different forms of preservation; Figure 8. Stability of free form astaxanthin dissolved in soybean oil (A) and dissolved in alcohol (B) for temperature; Figure 9 Antioxidant activity of free form astaxanthin in soybean oil.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples. The materials used in the present invention are commercially available materials, unless otherwise specified.

The present invention utilizes a single E. coli expression vector: pET-30a, and 6 co-clones in the vector can synthesize the free form of the astaxanthin gene and four promoters, as shown in FIG. The crtE, crtY, crtI, crtB, and crtZ genes are from Erwinia uredovora (alias: Pantoea ananatis ) and the crtW gene is from Agrobacterium aurantiacum (alias: Paracoccus sp. N81106). The T7 promoter (T7 promoter) serves as an inducible promoter, which controls the activation of the crtW gene. The constitutive promoter (P, a promoter that exhibits sustained expression) has three genes, which can simultaneously activate the crtE gene. Activation of the crtZ gene and crtY , crtI , crtB genes. Therefore, the regulation of the promoter is divided into two directions, one of which is the normal condition: the activation of the 6 genes together with the promoter of the inducible promoter and the persistent expression will result in a very low free form of astaxanthin. Production (Figure 2), the second is the specific condition: the promoter of the persistent expression first activates 5 genes ( crtE , crtY , crtI , crtB , and crtZ ) and then adds 0.1 mM IPTG (isopropyl -β-D-thiogalactopyranoside) enables the inducible promoter to regulate the activation of the crtW gene, resulting in a substantial increase in free form of astaxanthin production (Figure 2).

Example 1 Experimental materials

Carotenoid synthesis gene group and the hydroxyl group E.uredorora gene cloned from biological carotenoids (crtE, crtY, crtI, crtB and crtZ, GenBank: D90087.2) and cloned enzyme-keto-carotene from A.aurantiacum The gene ( crtW , PRF: 1096128) was co-constructed on the pET-30a expression vector. The promoter size of these six genes plus three persistent expressions was 6439 bp. Escherichia coli BL-21 (DE3) strain was used as an expression host and cultured in LB medium containing kanamycin. The treatment of the specific state and the normal state are as follows: the specific condition: by culturing the expression host to 18 hours at 37 ° C, followed by the addition of 1% fresh LB medium and kanamycin, and adding the final concentration Incubation at 0.1 °C for 20 hours after 0.1 mM IPTG; general condition: by incubating the expression host to 18 hours at 37 ° C, without adding 1% fresh LB medium with kanamycin and IPTG Incubate at 16 ° C for 20 hours.

Example 2 Extraction and purification of free form astaxanthin

After the bacteria were centrifuged at 4 ° C, 5000 rpm for 10 minutes, the supernatant was decanted, and the strain was washed with 0.1 M Tris buffer pH 7, followed by centrifugation at 5000 rpm for 10 minutes, and then 99.5% anhydrous alcohol was added to extract the free form of shrimp red. Prime, violent shock for 20 minutes. After centrifugation for 15 minutes at 4 ° C, 13,000 rpm, the supernatant was collected and placed in a vacuum concentrator for 30 minutes, 40 ° C and 80 MPa. Thereafter, the concentrated precipitate is dissolved in a volume ratio of 3:2 by using n-hexane and sterile secondary water, and the two layers of the liquid phase can be separated, and then the precipitate is collected, and the dried form of the free form of astaxanthin is obtained by using a vacuum freeze dryer, and stored. Store at minus 20 °C. All of the extraction and purification processes must be carried out in the dark. All of the above solvents are food grade usable solvents.

Example 3 Astaxanthin Form and Purity Analysis

The free form of commercially available free form of astaxanthin is 97%. The astaxanthin produced by the strain was measured by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) before purification. Compared with the standard (Fig. 4), the astaxanthin produced by the strain is free form astaxanthin (Fig. 5), and the purity is 85%. Purified free form of astaxanthin 95% (as shown in Figure 6). Table 1 below compares the content of free form astaxanthin and carotenoids.

The purity of the free form of astaxanthin, which is first extracted from E. coli, can be as high as 85%, thus simplifying the subsequent purification step. Further, HPLC can be used for small-scale or preparative liquid chromatography MPLC for industrial grade purification of the free form of astaxanthin, which can increase the purity of 85% to over 95%, thereby effectively reducing the purification cost.

^a percentage of free form astaxanthin.

^b carotenoid content percentage.

^c can't detect.

Example 4 Free form astaxanthin stability test

Free form astaxanthin is sensitive to oxidation, heat and light and can be easily lost Its biological function. Therefore, the storage of free form astaxanthin is a key issue. The free form of astaxanthin was dissolved in alcohol and stored in the dark at -20 ° C (indicated by SS). Another free form of astaxanthin was formed into a powder sample (represented by PS) by using a vacuum freeze dryer and stored at -20 ° C in the dark. After 3 months, the astaxanthin solution and powder were compared to astaxanthin (represented by Control) which was extracted just 3 months ago. The HPLC spectra of the three samples showed similar curves (Fig. 7A). Figure 7B. Quantification of experimental data showed that the free form of astaxanthin was reduced by 6% in the solution sample, while the free form of astaxanthin was only reduced by 1% in the powder sample. This result indicates that the free form of astaxanthin powder stored for 3 months is more stable than the dissolved solution.

The free form of astaxanthin is as critical as the temperature stability during processing of food additives. The free form of astaxanthin is lipophilic and polarophilic. Therefore, the stability of the free form of astaxanthin dissolved in the oil phase or the aqueous phase at a high temperature can be compared. When the free form of astaxanthin is dissolved in soybean oil, it is very stable at room temperature, with 97.3% retention of astaxanthin, and 90.4%-71.1% of shrimp after 4 hours of maintenance at 70-90 ° C, respectively. The erythroside content is retained (see Figure 8A). However, when the temperature was raised to 100 ° C for 4 hours, the stability of astaxanthin in oil dropped to 66.5%.

When the free form of astaxanthin is dissolved in alcohol, it is stable at room temperature, with 97.5% retained astaxanthin content, and after 8 hours at 70-90 ° C, it has 86.1%-50.5% astaxanthin content. Hold (as shown in Figure 8B). However, when the temperature was raised to 100 ° C for 4 hours, the stability of astaxanthin in alcohol was significantly reduced to 20.1%. This result indicates that the free form of astaxanthin dissolved in oil has higher temperature stability than dissolution in alcohol. Therefore, the free form of astaxanthin has potential applications in the processed food industry and can be combined with edible oils. Capsules are used in nutritional medicine and pharmaceutical applications.

Astaxanthin is an effective antioxidant because it reacts with free radicals to convert it into a more stable product. As can be seen from the results of Fig. 9, the free form of astaxanthin in soybean oil has higher temperature stability than alcohol. Therefore, the free form of astaxanthin in soybean oil is tested for antioxidant capacity under various temperature environments. The initial sample indicates that the free form of astaxanthin powder is dissolved in the oil, and then the test sample is directly analyzed. The antioxidant activity of the free form astaxanthin powder dissolved in oil was 80.9% at room temperature, and was 77.4%-64.7% at 70-100 ° C for 4 hours, respectively. This result indicates that the antioxidant capacity of the free form of astaxanthin in oil is stable whether it is at room temperature or at 70-100 ° C for 4 hours, which is consistent with the results of previous studies. Therefore, in a high temperature processing environment, the antioxidant activity of the free form of astaxanthin dissolved in oil can be applied to the food, pharmaceutical and nutraceutical industries.

In summary, the method of the present invention can produce high-purity and high-purity astaxanthin, and can be applied to food or medicine, so this case is not only innovative in terms of technical thinking, but also has a conventional method of conventional use. Less than the above-mentioned multiple functions, it has fully complied with the statutory invention patent requirements of novelty and progressiveness. If you apply in accordance with the law, you are requested to approve the application for this invention patent to encourage invention.

Claims (10)

A method for producing astaxanthin by using Escherichia coli, the method comprising: (1) providing an E. coli expression vector pET-30a; (2) providing an Escherichia coli BL-21 (DE3) strain; (3) Transplanting the E. coli expression vector pET-30a into the E. coli BL-21 (DE3) strain; (4) culturing the resulting recombinant E. coli strain; and (5) collecting the recombinant E. coli strain, purifying and extracting To obtain astaxanthin. The method of claim 1, wherein the E. coli expression vector pET-30a is composed of the crtE , crtY , crtI , crtB , crtZ and crtW genes and four promoters. The method of claim 2, wherein the promoter is selected from three constitutive promoters, and an inducible promoter on the vector, The sustained expression of the promoter differs from the initiation time of the inducible promoter, resulting in a purity of 85% of the initially extracted astaxanthin. The method of claim 3, wherein the inducible promoter is selected from the group consisting of, but not limited to, a T7 promoter. The method of claim 3, wherein the inducible promoter controls the activation of the crtW gene. The method of claim 3, wherein the promoter of the persistent expression simultaneously activates the crtE gene, the crtZ gene, and the crtY , crtI , crtB genes, respectively. The method of claim 1, wherein the regulation of the E. coli expression vector pET-30a is specifically regulated. The method of claim 6, wherein the specific regulatory system activates crtE , crtY , crtI , crtB and crtZ genes by the promoter of the persistent expression, and then activates the crtW gene by the inducible promoter. . The application method of claim 1 patentable scope clause, wherein the synthetic gene of E. coli-based group and the hydroxyl group carotenoid genes (crtE, crtY, crtI, crtB and crtZ, GenBank E.uredorora cloned from biological carotenoids :D90087.2), and the carotenoid ketolase gene ( crtW , PRF: 1096128) cloned from A. aurantiacum was constructed on pET-30a expression vector. The method of claim 1 or 3, wherein the astaxanthin is a free form of astaxanthin.