Method for producing astaxanthin by immobilized enzyme technology and application thereof

Abstract

The invention discloses a method for producing astaxanthin by utilizing an immobilized enzyme technology and application of the astaxanthin, the method comprises the following steps: S1, uniformly mixing macroporous resin and nickel chloride for chelating to obtain pretreated macroporous resin; the final concentration of the nickel chloride in the chelating system is 0.15-0.3 mol / L; s2, mixing an enzyme solution containing beta-carotene hydroxylase and beta-carotene ketolase with the pretreated macroporous resin obtained in the step S1 according to a volume-mass ratio of (5-8): 1, and immobilizing to obtain immobilized resin; and S3, uniformly mixing a beta-carotene substrate solution added with metal ions with the immobilized resin obtained in the step S2, carrying out a catalytic reaction, and producing an astaxanthin solution after the reaction is finished. According to the method, the conversion efficiency of beta-carotene is improved by optimizing a catalytic system of the immobilized enzyme, so that the yield of astaxanthin is improved, and the method is efficient and low in cost and has a certain industrial prospect.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a method for producing astaxanthin using immobilized enzyme technology and its application. Background Technology

[0002] Astaxanthin is a potent natural antioxidant, belonging to the ketocarotenoid class of compounds, with the chemical name 3,3′-dihydroxy-4,4′-diketo-β,β′-carotene. Astaxanthin is widely found in the biological world, especially in aquatic animals and plants, such as shrimp, crabs, fish, algae, and bird feathers, where it plays a role in color development. Due to its outstanding antioxidant capacity (its antioxidant activity is 600-1000 times that of vitamin E) and wide range of health benefits, astaxanthin is hailed as "super vitamin E" or "red miracle." Its main functions include antioxidation, anti-inflammation, boosting immunity, protecting eyesight, improving fatigue, and enhancing pigmentation. It has broad application prospects in health products, pharmaceuticals, cosmetics, food additives, and animal husbandry and aquaculture.

[0003] Astaxanthin production methods include natural extraction, biosynthesis, and chemical synthesis. Haematococcus pluvialis is widely recognized as the best organism in nature for producing natural astaxanthin, with its astaxanthin having a 100% levorotatory structure, consistent with the structure required by humans and animals, possessing advantages such as high antioxidant properties and high safety. However, its extraction process is costly and expensive. Furthermore, Haematococcus pluvialis-derived astaxanthin has an off-flavor, fishy, ​​and bitter taste, making it unsuitable for animal consumption, requiring the addition of other flavoring agents or special treatment in feed. Chemically synthesized astaxanthin has a structure of 50% racemic, 25% levorotatory, and 25% dextrorotatory, significantly different from natural astaxanthin in structure, function, application, and safety. Although chemical synthesis offers high yield and low cost, its stability, antioxidant activity, and coloring properties are significantly lower than natural astaxanthin, resulting in lower absorption and utilization rates in organisms, and potential safety issues. Pharfogel's yeast is considered the most suitable source of astaxanthin for fungal fermentation production. However, unlike the 100% levorotatory structure of astaxanthin derived from Haematococcus pluvialis, astaxanthin derived from Pharfogel's yeast has a 100% dextral structure. Furthermore, the fermentation level of Pharfogel's yeast astaxanthin is low (0.6~0.8 g / L), the fermentation cycle is long (approximately 7 days), the finished product content is relatively low (0.4%~0.6%), and the extraction cost is high, resulting in a high cost for the astaxanthin products produced from it, which greatly limits its large-scale production and market application. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a method for producing astaxanthin using immobilized enzyme technology. This method improves the conversion efficiency of β-carotene by optimizing the catalytic system of the immobilized enzyme, thereby increasing the yield of astaxanthin. It is a highly efficient, low-cost, and industrially promising biological method for synthesizing astaxanthin.

[0005] The present invention also proposes an application.

[0006] According to a first aspect of the present invention, a method for producing astaxanthin using immobilized enzyme technology is provided, the method comprising: S1: The macroporous resin and nickel chloride are mixed and chelated to obtain the pretreated macroporous resin; the final concentration of nickel chloride in the chelation system is 0.15~0.3 mol / L; S2: The enzyme solution containing β-carotene hydroxylase and β-carotene ketolase is mixed with the pretreated macroporous resin obtained in step S1 at a volume-to-mass ratio of (5~8):1 (L:kg) to fix the resin and obtain the immobilized resin. S3: The β-carotene substrate solution with added metal ions is mixed with the immobilized resin obtained in step S2 to carry out a catalytic reaction. After the reaction is completed, astaxanthin solution is produced.

[0007] In some embodiments of the present invention, the macroporous resin in step S1 is a pretreated macroporous resin, the pretreatment including sequential alcohol washing, acid washing and water washing.

[0008] In some embodiments of the present invention, the alcohol washing is performed using 15% to 25% isopropanol, and the volume of the alcohol is 5 to 10 times the volume of the macroporous resin.

[0009] In some embodiments of the present invention, the acid washing is performed using 0.05~0.2 mol / L hydrochloric acid, and the volume of the acid is 1~3 times that of the macroporous resin.

[0010] In some embodiments of the present invention, the water used for washing is deionized water.

[0011] In some embodiments of the present invention, the chelation time in step S1 is 20 to 40 minutes.

[0012] In some embodiments of the present invention, after the chelation in step S1 is completed, the macroporous resin is cleaned with a 0.1 mol / L phosphate buffer solution at pH 6.5.

[0013] In some embodiments of the present invention, the enzyme solution in step S2 is prepared by a method comprising the following steps: mixing engineered bacteria expressing β-carotene hydroxylase and β-carotene ketolase with a buffer solution and then breaking them up to obtain the enzyme solution.

[0014] In some embodiments of the present invention, the volume-to-mass ratio of the buffer solution to the engineered bacteria is (5~15):1 (L:kg).

[0015] In some embodiments of the present invention, the volume-to-mass ratio of the buffer solution to the engineered bacteria is (8~12):1 (L:kg).

[0016] In some embodiments of the present invention, the buffer solution comprises a 0.1 mol / L Tris-HCl buffer solution with pH 6 to pH 7.5.

[0017] In some embodiments of the present invention, the fixed temperature condition in step S2 is 35°C to 39°C.

[0018] In some embodiments of the present invention, the fixed temperature condition in step S2 is 36°C to 38°C.

[0019] In some embodiments of the present invention, the fixed rotational speed condition in step S2 is 80~120 rpm.

[0020] In some embodiments of the present invention, the fixed rotational speed condition in step S2 is 90~110 rpm.

[0021] In some embodiments of the present invention, the fixed time in step S2 is 20 to 40 minutes.

[0022] In some embodiments of the present invention, the fixed time in step S2 is 25 to 35 minutes.

[0023] In some embodiments of the present invention, after the fixation described in step S2 is completed, the macroporous resin is cleaned with a 0.1 mol / L phosphate buffer solution with a pH of 6.5.

[0024] In some embodiments of the present invention, the metal ion mentioned in step S3 is a magnesium ion.

[0025] In some embodiments of the present invention, the magnesium ion concentration in the β-carotene substrate solution is 1~4 mmol / L.

[0026] In some embodiments of the present invention, the magnesium ion concentration in the β-carotene substrate solution is 1~3 mmol / L.

[0027] In some embodiments of the present invention, the concentration of β-carotene in the β-carotene substrate solution in step S3 is 5~9 g / L.

[0028] In some embodiments of the present invention, the solvent for the β-carotene substrate solution in step S3 is a 0.1 mol / L Tris-HCl buffer solution with pH 6 to pH 7.5.

[0029] In some embodiments of the present invention, the solvent for the β-carotene substrate solution in step S3 is a 0.1 mol / L Tris-HCl buffer solution with pH 6 to pH 7.

[0030] In some embodiments of the present invention, the catalytic temperature in step S3 is 30°C to 35°C.

[0031] In some embodiments of the present invention, the catalytic time in step S3 is 20-28 h.

[0032] In some embodiments of the present invention, the β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) mentioned in step S2 include recombinant Escherichia coli (E. coli). E.coli / CrtZ and E.coli / CrtW) produces highly active CrtZ and CrtW through liquid fermentation; the CrtZ and CrtW genes are derived from genomic DNA in Haematococcus pluvialis.

[0033] In some embodiments of the present invention, the liquid fermentation includes: Engineered strains E. coli / CrtZ and E. coli / CrtW were inoculated into fermentation medium (20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 5 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 4 g / L potassium dihydrogen phosphate, 2 g / L dipotassium hydrogen phosphate, 0.1 g / L manganese sulfate, pH 7.0, sterilized at 121℃ for 20 min) and cultured at 37℃ and 220 rpm with shaking until mid-log phase. IPTG was then added to a final concentration of 1 mmol / L for induction for 10–16 h.

[0034] In some embodiments of the present invention, the recombinant Escherichia coli comprises: recombinant E. coli / CrtZ and E. coli / CrtW that produce high levels of CrtZ and CrtW, using Escherichia coli BL21 as the host cell and expressing CrtZ and CrtW on plasmid pET-28a(+), respectively.

[0035] The present invention has at least the following beneficial effects: This invention provides a method for producing astaxanthin using immobilized enzyme technology. The method uses CrtZ and CrtW, which are immobilized, to catalyze the production of astaxanthin using β-carotene as a substrate. By optimizing the catalytic system of the immobilized enzyme, the conversion efficiency of β-carotene is improved, thereby increasing the yield of astaxanthin. This is a highly efficient, low-cost biological method for synthesizing astaxanthin with certain industrialization prospects. Attached Figure Description

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram of the construction process of the recombinant Escherichia coli genetically engineered bacteria in Example 1 of the present invention. Detailed Implementation

[0037] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0038] Example 1 This embodiment provides a method for producing astaxanthin using immobilized enzyme technology, as detailed below: 1. Construction of recombinant Escherichia coli genetically engineered bacteria Recombinant Escherichia coli genetically engineered bacteria ( E.coli / CrtZ and E.coli The build process of / CrtW is as follows Figure 1 As shown, the specific construction method is as follows: Genomic DNA was extracted from Haematococcus pluvialis using a DNA extraction kit. Using this genomic DNA as a template, the CrtZ and CrtW genes were amplified using primers. The primer sequences and the amplified gene fragment sequences are shown in Table 1. The PCR product recovery kit was used to purify the CrtZ and CrtW gene fragments. The purified PCR products were ligated into the pET-28a(+) vector, and the ligation product was transformed into Escherichia coli BL21. Blue-white screening plates were used, and plasmids were extracted from the white colonies on the plates. The plasmids were identified by double enzyme digestion, and recombinant engineered bacteria were screened. E.coli / CrtZ and E.coli / CrtW. The plasmid was extracted and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing verification.

[0039] Table 1 Primer and amplification product sequences

[0040] 2. Engineered bacteria E.coli / CrtZ and E.coli / CrtW fermentation expression engineered bacteria E.coli / CrtZ and E.coli / CrtW were inoculated into fermentation medium (20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 5 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 4 g / L potassium dihydrogen phosphate, 2 g / L dipotassium hydrogen phosphate, 0.1 g / L manganese sulfate, pH 7.0, sterilized at 121℃ for 20 min), and cultured with shaking at 37℃ and 220 rpm until mid-log phase. IPTG was added to a final concentration of 1 mmol / L for induction for 13 h (10-16 h is acceptable), centrifuged at 10000 r / min for 20 min, the supernatant was discarded, and the cells were collected.

[0041] 3. Immobilization of CrtZ and CrtW enzymes The bacterial cells collected in step 2 were mixed thoroughly with Tris-HCl buffer (0.1 mol / L, pH 6.5) at a ratio of 10:1 (L:kg). The mixture was then homogenized using a high-pressure homogenizer at 950 Bar (900-1000 Bar is also acceptable) to obtain the bacterial cell lysate. The centrifuged bacterial cells described above are engineered bacteria. E.coli / CrtZ and E.coli It is obtained by mixing / CrtW in equal proportions.

[0042] The iminodiacetic acid (IDA) chelating resin was washed sequentially with 7 volumes (5 to 10 volumes) of 20% (V:V) isopropanol, followed by 2 volumes of 0.1 mol / L hydrochloric acid. After rinsing with deionized water, nickel chloride was added to a final concentration of 0.2 mol / L, and chelation was carried out at 37°C and 100 rpm for 30 min. The resin was then washed with 0.1 mol / L phosphate buffer at pH 6.5 to obtain the pretreated macroporous resin.

[0043] The above-mentioned bacterial cell lysate was mixed with pretreated macroporous resin at a ratio of 6:1 (L:kg), fixed at 37°C and 100 rpm for 30 min, and then repeatedly washed with 0.1 mol / L, pH 6.5 phosphate buffer to obtain immobilized resin. For long-term storage, the immobilized resin can be placed in 0.1 mol / L, pH 6.5 phosphate buffer and stored at 4°C.

[0044] 4. Catalyzes the production of astaxanthin A substrate solution containing 7 g / L β-carotene and 2 mmol / L MgCl2 was prepared in a 0.1 mol / L Tris-HCl buffer solution at pH 6.5. The immobilized resin obtained in step 3 was mixed with the substrate solution at a ratio of 1:10 (kg:L) for catalytic reaction at 35°C for 24 h. After the reaction, a catalytic system containing astaxanthin was obtained. The astaxanthin concentration in the system reached 5.20 g / L, and the conversion rate was 74.29%.

[0045] Test case This experiment tested the effects of different catalytic conditions and catalytic reaction system compositions on the astaxanthin yield of the method provided in Example 1. The specific experimental methods and results are as follows: 1. Selection of nickel chloride concentration The bacterial cells collected in step 2 of Example 1 were mixed evenly with Tris-HCl buffer (0.1 mol / L, pH 6.5): centrifuged bacterial cells = 10:1 (L:kg), and then homogenized using a high-pressure homogenizer at 950 Bar (900~1000 Bar is also acceptable) to obtain the bacterial cell lysate.

[0046] The iminodiacetic acid (IDA) chelating resin was washed sequentially with 7 volumes (5 to 10 volumes) of 20% (V:V) isopropanol, 2 volumes of 0.1 mol / L hydrochloric acid, and rinsed thoroughly with deionized water. Then, nickel chloride was added to final concentrations of 0.05, 0.1, 0.2, and 0.3 mol / L, respectively, and chelated at 37°C and 100 rpm for 30 min. The resin was then washed thoroughly with 0.1 mol / L phosphate buffer (pH 6.5) to obtain the pretreated iminodiacetic acid (IDA) chelating resin.

[0047] The bacterial cell lysate was mixed with pretreated iminodiacetic acid (IDA) chelating resin at a ratio of 6:1 (L:kg), fixed at 37°C and 100 rpm for 30 min, and then repeatedly washed with 0.1 mol / L, pH 6.5 phosphate buffer to obtain the immobilized resin.

[0048] A substrate solution containing 7 g / L β-carotene was prepared using water as the solvent. The immobilized resin and the substrate solution were mixed at a ratio of 1:10 (kg:L) to carry out the catalytic reaction. The reaction was carried out at room temperature for 24 h. After the reaction was completed, a catalytic system containing astaxanthin was obtained.

[0049] The astaxanthin content in the catalytic system after the reaction was determined according to the high performance liquid chromatography method described in national standard GB / T 31520-2015. The results showed that the astaxanthin yields catalyzed by immobilized resin with final concentrations of 0.05, 0.1, 0.2, and 0.3 mol / L nickel chloride were 1.46, 2.92, 3.58, and 3.55 g / L, respectively. Therefore, 0.2 mol / L nickel chloride is preferred for addition to the macroporous resin.

[0050] 2. Selection of the mixing ratio of bacterial cell lysis broth and immobilization resin The bacterial cells collected in step 2 of Example 1 were mixed evenly with Tris-HCl buffer (0.1 mol / L, pH 6.5): centrifuged bacterial cells = 10:1 (L:kg), and then homogenized using a high-pressure homogenizer at 950 Bar (900~1000 Bar is also acceptable) to obtain the bacterial cell lysate.

[0051] The iminodiacetic acid (IDA) chelating resin was washed sequentially with 7 volumes (5 to 10 volumes) of 20% (V:V) isopropanol, 2 volumes of 0.1 mol / L hydrochloric acid, and rinsed thoroughly with deionized water. Then, nickel chloride with a final concentration of 0.2 mol / L was added, and chelation was carried out at 37°C and 100 rpm for 30 min. Finally, the resin was washed with 0.1 mol / L phosphate buffer at pH 6.5 to obtain the pretreated iminodiacetic acid (IDA) chelating resin.

[0052] The bacterial cell lysate was mixed with pretreated iminodiacetic acid (IDA) chelating resin at ratios of 4:1, 5:1, 6:1, 7:1, and 8:1 (L:kg), respectively. After immobilization at 37°C and 100 rpm for 30 min, the mixture was repeatedly washed with 0.1 mol / L phosphate buffer at pH 6.5 to obtain the immobilized resin.

[0053] A substrate solution containing 7 g / L β-carotene was prepared; the immobilized resin and the substrate solution were mixed at a ratio of 1:10 (kg:L) to carry out the catalytic reaction, and the reaction was carried out for 24 h. After the reaction was completed, a catalytic system containing astaxanthin was obtained.

[0054] The astaxanthin content in the catalytic system after the reaction was determined according to the high performance liquid chromatography method described in national standard GB / T 31520-2015. The results showed that the astaxanthin yields catalyzed by the immobilized resin when the bacterial cell lysate and the immobilized resin were mixed at ratios of 4:1, 5:1, 6:1, 7:1, and 8:1 (L:kg) were 1.91, 3.22, 3.53, 3.55, and 3.50 g / L, respectively. Therefore, a mixing ratio of 6:1 is preferred.

[0055] 3. Selection of buffer solution for catalytic reaction system The bacterial cells collected in step 2 of Example 1 were mixed evenly with Tris-HCl buffer (0.1 mol / L, pH 6.5): centrifuged bacterial cells = 10:1 (L:kg), and then homogenized using a high-pressure homogenizer at 950 Bar (900~1000 Bar is also acceptable) to obtain the bacterial cell lysate.

[0056] The iminodiacetic acid (IDA) chelating resin was washed sequentially with 7 volumes (5 to 10 volumes) of 20% (V:V) isopropanol, 2 volumes of 0.1 mol / L hydrochloric acid, and rinsed thoroughly with deionized water. Then, nickel chloride with a final concentration of 0.2 mol / L was added, and chelation was carried out at 37°C and 100 rpm for 30 min. Finally, the resin was washed with 0.1 mol / L phosphate buffer at pH 6.5 to obtain the pretreated iminodiacetic acid (IDA) chelating resin.

[0057] The bacterial cell lysate was mixed with pretreated iminodiacetic acid (IDA) chelating resin at a ratio of 6:1 (L:kg), fixed at 37°C and 100 rpm for 30 min, and then repeatedly washed with 0.1 mol / L, pH 6.5 phosphate buffer to obtain the immobilized resin.

[0058] A substrate solution containing 7 g / L β-carotene was prepared, and the solvent was set as 0.1 mol / L Tris-HCl buffer solution with pH 5.5, 6, 6.5, 7, 7.5 and 8, respectively. The immobilized resin and the substrate solution were mixed at a ratio of 1:10 (kg:L) to carry out the catalytic reaction. The reaction was carried out for 24 h. After the reaction was completed, a catalytic system containing astaxanthin was obtained.

[0059] The astaxanthin content in the catalytic system after the reaction was determined according to the high performance liquid chromatography method described in national standard GB / T 31520-2015. The results showed that the astaxanthin yield was 3.95, 4.39, 4.51, 4.36, 4.43, and 1.06 g / L after the pH of the Tris-HCl buffer was set to 5.5, 6, 6.5, 7, 7.5, and 8, respectively. Therefore, the preferred pH was 6.5.

[0060] 4. Selection of catalytic reaction temperature The bacterial cells collected in step 2 of Example 1 were mixed evenly with Tris-HCl buffer (0.1 mol / L, pH 6.5): centrifuged bacterial cells = 10:1 (L:kg), and then homogenized using a high-pressure homogenizer at 950 Bar (900~1000 Bar is also acceptable) to obtain the bacterial cell lysate.

[0061] The iminodiacetic acid (IDA) chelating resin was washed sequentially with 7 volumes (5 to 10 volumes) of 20% (V:V) isopropanol, 2 volumes of 0.1 mol / L hydrochloric acid, and rinsed thoroughly with deionized water. Then, nickel chloride with a final concentration of 0.2 mol / L was added, and chelation was carried out at 37°C and 100 rpm for 30 min. Finally, the resin was washed with 0.1 mol / L phosphate buffer at pH 6.5 to obtain the pretreated iminodiacetic acid (IDA) chelating resin.

[0062] The bacterial cell lysate was mixed with pretreated macroporous resin at a ratio of 6:1 (L:kg), fixed at 37°C and 100 rpm for 30 min, and then repeatedly washed with 0.1 mol / L, pH 6.5 phosphate buffer to obtain immobilized resin.

[0063] A substrate solution containing 7 g / L β-carotene was prepared using a pH 6.5, 0.1 mol / L Tris-HCl buffer solution. The immobilized resin and substrate solution were mixed at a ratio of 1:10 (kg:L) for catalytic reaction. The catalytic reaction temperatures were set at 25℃, 30℃, 35℃, and 40℃, and the reaction was carried out for 24 h. After the reaction was completed, a catalytic system containing astaxanthin was obtained.

[0064] The astaxanthin content in the catalytic system after the reaction was determined according to the high performance liquid chromatography method described in national standard GB / T 31520-2015. The results showed that the astaxanthin yields were 4.47, 4.72, 4.88, and 4.20 g / L when the catalytic reaction temperature was set to 25℃, 30℃, 35℃, and 40℃, respectively. Therefore, the preferred catalytic reaction temperature is 35℃.

[0065] 5. The effect of metal ions on catalytic reactions Based on the method provided in Example 1, different concentrations of different metal ions were set as the test subjects and their effects on the final astaxanthin yield were tested. The metal ions in the substrate solution in step 4 of Example 1 were replaced with 0.5, 1, 2, 3, 4, 5, and 6 mmol / L of MgCl2, CaCl2, MnCl2, CuCl2, and FeSO4, respectively. All other steps and conditions were the same as in Example 1.

[0066] The astaxanthin content in the catalytic system after the reaction was determined according to the high performance liquid chromatography method described in national standard GB / T 31520-2015. The results were as follows: Mg was added at concentrations of 0.5, 1, 2, 3, 4, 5, and 6 mmol / L. 2+After 24 hours of catalysis, the astaxanthin concentrations were 4.82, 5.01, 5.20, 5.18, 5.05, 4.70, and 3.92 g / L, respectively. Adding 0.5, 1, 2, 3, 4, 5, and 6 mmol / L of Ca... 2+ After 24 h of catalysis, the astaxanthin concentrations were 4.79, 4.86, 4.72, 4.78, 4.61, 4.17, and 4.06 g / L, respectively. Mn was added at concentrations of 0.5, 1, 2, 3, 4, 5, and 6 mmol / L. 2+ After 24 h of catalysis, the astaxanthin concentrations were 2.19, 2.06, 1.80, 1.52, 0.81, 0.77, and 0.52 g / L, respectively. Cu was added at concentrations of 0.5, 1, 2, 3, 4, 5, and 6 mmol / L. 2+ After 24 hours of catalysis, the astaxanthin concentrations were 4.71, 4.88, 4.85, 4.66, 4.42, 4.26, and 4.07 g / L, respectively. Fe was added at concentrations of 0.5, 1, 2, 3, 4, 5, and 6 mmol / L. 2+ The astaxanthin contents generated after 24 h of catalysis were 4.61, 4.60, 4.82, 4.84, 4.80, 4.77, and 4.78 g / L, respectively.

[0067] The results showed that adding 0.5–6 mmol / L of Ca... 2+ Mn 2+ Cu 2+ Fe 2+ Ions have no enhancing effect on catalytic reactions, but the addition of 1-4 mmol / L Mg... 2+ This helps to enhance the conversion of β-carotene to astaxanthin, and the addition of 2 mmol / L Mg... 2+ The astaxanthin content reached 5.20 g / L after 24 hours of reaction.

[0068] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for producing astaxanthin using an immobilized enzyme technology, characterized by, The method comprises: S1: uniformly chelate macroporous resin and nickel chloride to obtain pretreated macroporous resin; the final concentration of nickel chloride in the chelation system is 0.15-0.3 mol / L; S2: mix an enzyme solution containing β-carotene hydroxylase and β-carotene ketolase with the pretreated macroporous resin obtained in step S1 according to a volume-mass ratio of (5-8):1 (L:kg) to perform immobilization, and obtain immobilized resin; S3: mix a β-carotene substrate solution added with metal ions with the immobilized resin obtained in step S2 to perform catalytic reaction, and obtain astaxanthin solution after the reaction.

2. The method for producing astaxanthin using an immobilized enzyme technology according to claim 1, characterized by, The macroporous resin in step S1 is pretreated macroporous resin, and the pretreatment comprises sequentially performing alcohol washing, acid washing and water washing.

3. The method for producing astaxanthin using an immobilized enzyme technology according to claim 1, wherein, The chelation time in step S1 is 20-40 min.

4. The method for producing astaxanthin using an immobilized enzyme technology according to claim 1, wherein, The enzyme solution in step S2 is prepared by a preparation method comprising the following steps: uniformly mixing engineering bacteria expressing β-carotene hydroxylase and β-carotene ketolase with a buffer and crushing, to obtain the enzyme solution; The volume-mass ratio of the buffer to the engineering bacteria is (5-15):1 (L:kg); The buffer comprises 0.1 mol / L Tris-HCl buffer with pH 6-pH 7.

5.

5. The method for producing astaxanthin using an immobilized enzyme technology according to claim 1, wherein, The immobilization temperature condition in step S2 is 35℃-39℃; The immobilization rotation speed condition in step S2 is 80-120 rpm; The immobilization time in step S2 is 20-40 min.

6. The method for producing astaxanthin using an immobilized enzyme technology according to claim 1, wherein, The metal ion in step S3 is magnesium ion; The magnesium ion concentration in the β-carotene substrate solution in step S3 is 1-4 mmol / L.

7. The method for producing astaxanthin using an immobilized enzyme technology according to claim 1, wherein, The β-carotene concentration in the β-carotene substrate solution in step S3 is 5-9 g / L.

8. The method for producing astaxanthin using an immobilized enzyme technology according to claim 1, wherein, The solvent of the β-carotene substrate solution in step S3 is 0.1 mol / L Tris-HCl buffer with pH 6-pH 7.

5.

9. The method for producing astaxanthin using an immobilized enzyme technology according to claim 1, wherein, The catalysis temperature in step S3 is 30℃-35℃; The catalysis time in step S3 is 20-28 h.

10. Use of the method for producing astaxanthin by the use of immobilized enzyme technology as defined in any one of claims 1 to 9 for the preparation of a product containing astaxanthin, characterized in that, The product comprises at least one of food, medicine, cosmetics and health products.

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