A long half-life l-rhamnose isomerase and its application for preparing d-allose
By mutating the amino acid sequence of L-rhamnose isomerase and fermenting it, a highly efficient L-rhamnose isomerase mutant was prepared, which solved the problems of low enzyme activity and short half-life, improved the preparation efficiency and stability of D-allose, and reduced costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHUCHENG HAOTIAN PHARMA CO LTD
- Filing Date
- 2026-01-29
- Publication Date
- 2026-07-10
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Figure CN121574975B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and in particular to a long-half-life L-rhamnose isomerase and its application in the preparation of D-allose. Background Technology
[0002] D-Allose is a rare aldose with extremely low calories. It serves as both a sugar substitute and a functional factor, possessing multiple functions such as anti-inflammatory, antioxidant, anti-tumor, and immunomodulatory effects.
[0003] The biosynthesis of D-allose mainly falls into two major technical pathways: microbial fermentation and enzymatic conversion. Microbial fermentation utilizes engineered microorganisms to achieve a one-step synthesis of D-allose from an inexpensive sugar source. However, this method is technically complex, difficult to construct, requires modification of the microbial metabolic network, and involves a long and costly construction and optimization cycle for the entire engineered strain. Furthermore, it produces numerous byproducts, leading to high separation and purification costs. In addition, the strong expression of exogenous metabolic pathways can impose a metabolic burden on host cells, affecting their growth and stability.
[0004] The core of enzymatic conversion lies in the in vitro catalytic conversion of the intermediate D-allulose to D-allose using L-rhamnosyl isomerase. A key limitation of this catalytic process is the low catalytic efficiency of the enzyme, and the gradual decline in enzyme activity as the reaction proceeds, affecting conversion rate and process stability. Therefore, improving the catalytic activity of L-rhamnosyl isomerase and extending its half-life are of great significance for improving substrate conversion efficiency, reducing enzyme usage costs, and achieving sustainable industrial production. Summary of the Invention
[0005] In view of this, the purpose of the present invention is to provide an L-rhamnose isomerase with a long half-life and its application in the preparation of D-allose, in order to overcome the problems of low enzyme activity and short half-life of L-rhamnose isomerase in the prior art.
[0006] In a first aspect, the present invention provides an L-rhamnose isomerase with a long half-life, the amino acid sequence of which is shown in any of the following:
[0007] (1) The amino acid sequence as shown in SEQ ID NO.4;
[0008] (2) The amino acid sequence as shown in SEQ ID NO.6;
[0009] (3) The amino acid sequence shown in SEQ ID NO.8.
[0010] Compared with the prior art, the present invention mutates wild-type L-rhamnose isomerase by changing position 52 of the amino acid sequence shown in SEQ ID NO.2 from G to V, resulting in the L-rhamnose isomerase mutant G52V with the amino acid sequence shown in SEQ ID NO.4; by changing position 294 of the amino acid sequence shown in SEQ ID NO.2 from D to R, resulting in the L-rhamnose isomerase mutant D294R with the amino acid sequence shown in SEQ ID NO.6; and by changing position 52 of the amino acid sequence shown in SEQ ID NO.2 from G to V and position 294 from D to R, resulting in the L-rhamnose isomerase mutant G52V / D294R with the amino acid sequence shown in SEQ ID NO.8. These three L-rhamnose isomerase mutants exhibit higher enzyme activity and longer half-life, thereby effectively improving the conversion rate and yield of D-allose when used to catalyze the D-allulose reaction to prepare D-allose.
[0011] In a second aspect, the present invention provides a biomaterial comprising any one of the following:
[0012] (A) A nucleic acid molecule having a gene sequence encoding the aforementioned long-half-life L-rhamnosyl isomerase;
[0013] (B) An expression vector containing the nucleic acid molecule described in (A);
[0014] (C) A recombinant strain containing the nucleic acid molecule described in (A) or the expression vector described in (B).
[0015] Optionally, the gene sequence encoding the L-rhamnose isomerase mutant G52V is shown in SEQ ID NO.3; the gene sequence encoding the L-rhamnose isomerase mutant D294R is shown in SEQ ID NO.5; and the gene sequence encoding the L-rhamnose isomerase mutant G52V / D294R is shown in SEQ ID NO.7.
[0016] Thirdly, the present invention provides a method for preparing L-rhamnose isomerase, which is used to prepare the above-mentioned long-half-life L-rhamnose isomerase by fermentation culture of a recombinant strain containing the L-rhamnose isomerase gene and inducing the recombinant strain to express L-rhamnose isomerase.
[0017] Furthermore, it includes the following steps:
[0018] The recombinant strain was seed cultured to obtain a seed solution;
[0019] The seed culture was inoculated into the fermentation medium at an inoculation rate of 1%–10% by volume and cultured until the OD reached the specified level. 600Cool to 30-40°C and continue culturing until OD (October Expiratory Rate). 600 The dissolved oxygen (DO) value was set at 45-55°C. An inducing agent was added for induction culture. During dissolved oxygen rebound, supplemental culture medium was added to maintain the DO value at 20-40% until the OD value recovered. 600 Fermentation ends when the temperature reaches 110-130°C, yielding the fermentation liquid.
[0020] Centrifuge the fermentation broth, resuspend it, break up the cells, and centrifuge again. The resulting supernatant is the crude enzyme solution of L-rhamnosyl isomerase.
[0021] Compared with the prior art, the fermentation culture of the present invention is a high-density fermentation carried out in a fermenter, which can obtain a high concentration of crude L-rhamnosyl isomerase solution.
[0022] Furthermore, the seed culture temperature is 35~40℃ and the rotation speed is 120~250 rpm.
[0023] Furthermore, the fermentation tank pressure was 0.035~0.045 MPa, the initial rotation speed was 300~350 rpm, the temperature was 35~40℃, and the pH value was 6.5~7.5.
[0024] Furthermore, the temperature drop is to 30-35℃.
[0025] Furthermore, the initial airflow for induction culture is 3.8~4 L / min. When the rotation speed reaches 400~450 rpm, the airflow is adjusted to 4.2~4.5 L / min. When the rotation speed reaches 600~700 rpm, the airflow is adjusted to 5.5~6 L / min.
[0026] Fourthly, the present invention provides the application of the above-mentioned long-half-life L-rhamnosyl isomerase or biological material in the preparation of D-allose.
[0027] Fifthly, the present invention provides a method for preparing D-allose, which utilizes the long-half-life L-rhamnosyl isomerase to catalyze the reaction of substrate D-allulose to generate D-allose.
[0028] Compared with the prior art, the L-rhamnose isomerase obtained by mutation in this invention has significantly improved enzyme activity and significantly prolonged half-life. When catalyzing the reaction of substrate D-allulose to produce D-allose, it effectively improves catalytic efficiency, thereby further increasing the yield of D-allose.
[0029] Furthermore, the preparation method for the reaction system that generates D-allose is as follows:
[0030] Add crude L-rhamnosyl isomerase solution, metal ions, and D-alulose to a 20-50 mM buffer solution with a pH of 7.0-8.0, so that the concentration of crude L-rhamnosyl isomerase in the reaction system is 10-120 mg / mL, the concentration of metal ions is 0.5-2 mM, and the concentration of D-alulose is 400-600 g / L.
[0031] Furthermore, the buffer solution includes HEPES buffer, glycine-sodium hydroxide buffer, or Tris-HCl buffer.
[0032] Furthermore, the metal ions include manganese ions, cobalt ions, or magnesium ions.
[0033] Furthermore, the reaction temperature is 60~80℃. Attached Figure Description
[0034] Figure 1 The residual enzyme activity of wild-type L-rhamnose isomerase and L-rhamnose isomerase mutant G52V / D294R incubated at 65°C for different times in Example 5 is shown.
[0035] Figure 2 The residual enzyme activity of wild-type L-rhamnose isomerase and L-rhamnose isomerase mutant G52V / D294R incubated at 75°C for different times in Example 5 is shown. Detailed Implementation
[0036] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.
[0037] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.
[0038] Example 1
[0039] Construction of recombinant plasmid pET-28a(+)-Tc-rhaA
[0040] Wild-type L-rhamnosyl isomerase derived from *Thermobacillus composti* was selected. Based on the codon bias of *Escherichia coli* BL21(DE3), the coding gene of the wild-type L-rhamnosyl isomerase was optimized, and the optimized gene was then synthesized artificially. The coding gene of the optimized wild-type L-rhamnosyl isomerase is shown in SEQ ID NO.1, and the amino acid sequence of the encoded wild-type L-rhamnosyl isomerase is shown in SEQ ID NO.2.
[0041] Primer design: Upstream primer F1 contains a homologous arm of the EcoRI restriction site; downstream primer R1 contains a homologous arm of the Not I restriction site.
[0042] F1: 5'-atgggtcgcggatccgaattcATGGAACGTAACATCGAACAAGC-3' (SEQ ID NO. 9).
[0043] R1: 5'-tggtggtgctcgagtgcggccgcTTAACCATTACGCTTTGCCAGTA-3' (SEQ ID NO. 10).
[0044] Using the optimized wild-type L-rhamnosyl isomerase encoding gene as a template, PCR amplification was performed using F1 and R1 primers to obtain the target gene Tc-rhaA with homologous arms. The PCR reaction system is shown in Table 1, and the reaction procedure is shown in Table 2.
[0045] Table 1
[0046]
[0047] Table 2
[0048]
[0049] It should be understood that the first step of the PCR amplification reaction, pre-denaturation at 95°C, and the last step, final extension at 72°C, are not included in the cycle and are performed only once in the entire PCR amplification reaction.
[0050] Vector linearization: The pET-28a(+) expression vector was double-digested with restriction endonucleases EcoRI and NotI to obtain the linearized vector pET-28a(+). The double digestion temperature was 37℃ and the time was 20 min. After double digestion, the linearized vector pET-28a(+) was recovered by agarose gel electrophoresis. The double digestion system is shown in Table 3.
[0051] Table 3
[0052]
[0053] Seamless cloning assembly: The target gene Tc-rhaA obtained after the above PCR amplification was ligated with the linearized vector pET-28a(+) and ligated at 37℃ for 30 min. The ligation system is shown in Table 4.
[0054] Table 4
[0055]
[0056] After the above ligation is completed, the ligation product is transformed into Escherichia coli DH5α clonal competent cells. Positive clones are screened on LB plates containing kanamycin (50 μg / mL). Plasmids are extracted from the positive clones and sent for verification. The plasmid pET-28a(+)-Tc-rhaA is the recombinant plasmid that is verified correctly.
[0057] Example 2
[0058] Constructing mutant plasmids
[0059] Using the recombinant plasmid pET-28a(+)-Tc-rhaA as a template, and with G52V-F as the upstream primer and G52V-R as the downstream primer, a reverse PCR amplification reaction was performed. After removing the template from the reaction product, circularization and ligation were performed to obtain a mutant plasmid containing the L-rhamnose isomerase mutant G52V gene, named pET-28a(+)-Tc-rhaA. G52V .
[0060] G52V-F: 5'-TGTTTCGCgtgGGCGAACTGGGTGGCGGCCTG-3' (SEQ ID NO. 11).
[0061] G52V-R: 5'-TTCGCCcacGCGAAACAGGAAGCCCTGCACGT-3' (SEQ ID NO. 12).
[0062] The sequence of the L-rhamnosyl isomerase mutant G52V gene is shown in SEQ ID NO.3, and the amino acid sequence of the L-rhamnosyl isomerase mutant G52V encoded by it is shown in SEQ ID NO.4.
[0063] Using the recombinant plasmid pET-28a(+)-Tc-rhaA as a template, and with D294R-F as the upstream primer and D294R-R as the downstream primer, a reverse PCR amplification reaction was performed. After removing the template from the reaction product, circularization and ligation were performed to obtain a mutant plasmid containing the L-rhamnose isomerase mutant D294R gene, named pET-28a(+)-Tc-rhaA. D294R .
[0064] D294R-F: 5'-TGCGTTGGcgcAGCGATTCACGTCGTTATTCTGG-3' (SEQ ID NO. 13).
[0065] D294R-R: 5'-ATCGCTgcgCCAACGCATCGGGCGAGATACAT-3' (SEQ ID NO. 14).
[0066] The sequence of the L-rhamnose isomerase mutant D294R gene is shown in SEQ ID NO.5, and the amino acid sequence of the L-rhamnose isomerase mutant D294R encoded by it is shown in SEQ ID NO.6.
[0067] Using the mutant plasmid pET-28a(+)-Tc-rhaA G52V Using D294R-F as the upstream primer and D294R-R as the downstream primer, a reverse PCR amplification reaction was performed. After removing the template from the reaction product, circularization and ligation were performed to obtain a mutant plasmid containing the L-rhamnose isomerase mutant G52V / D294R gene, named pET-28a(+)-Tc-rhaA. G52V / D294R .
[0068] D294R-F: 5'-TGCGTTGGcgcAGCGATTCACGTCGTTATTCTGG-3' (SEQ ID NO. 13).
[0069] D294R-R: 5'-ATCGCTgcgCCAACGCATCGGGCGAGATACAT-3' (SEQ ID NO. 14).
[0070] The sequence of the L-rhamnose isomerase mutant G52V / D294R gene is shown in SEQ ID NO.7, and the amino acid sequence of the L-rhamnose isomerase mutant G52V / D294R encoded by it is shown in SEQ ID NO.8.
[0071] The reaction systems for each of the above reverse PCR amplification reactions are shown in Table 5, and the reaction procedures are shown in Table 6.
[0072] Table 5
[0073]
[0074] Table 6
[0075]
[0076] It should be understood that the pre-denaturation, final extension, and storage steps in each of the above PCR reactions do not participate in the cycling process, and the entire PCR process is performed only once.
[0077] Template elimination: After each of the above reverse PCR reactions was completed, three reaction solutions were obtained. 1 μL of restriction endonuclease DpnⅠ was added to each of the three reaction solutions (25 μL), and the mixture was gently blown and aspirated. The solutions were then reacted at 37℃ for 1 h to obtain three enzyme digestion solutions. The enzyme digestion solutions were verified by agarose gel electrophoresis.
[0078] Reverse PCR product self-circularization: Using the three verified enzyme digestion solutions obtained above, reaction solutions were prepared according to Table 7, gently mixed, and incubated at 16℃ for 1 h to obtain the mutant plasmid pET-28a(+)-Tc-rhaA. G52V Mutant plasmid pET-28a(+)-Tc-rhaA D294R and the mutant plasmid pET-28a(+)-Tc-rhaA G52V / D294R .
[0079] Table 7
[0080]
[0081] Example 3
[0082] Preparation of crude enzyme solution
[0083] The above recombinant plasmid pET-28a(+)-Tc-rhaA and mutant plasmid ET-28a(+)-Tc-rhaA were used. G52V Mutant plasmid pET-28a(+)-Tc-rhaA D294R Mutant plasmid pET-28a(+)-Tc-rhaA G52V / D294R The cells were chemically transformed into E. coli BL21(DE3) competent cells and plated on LB agar plates containing kanamycin (50 μg / mL). After overnight incubation at 37°C, recombinant strains BL21-Tc-rhaA containing the recombinant plasmid pET-28a(+)-Tc-rhaA and mutant strain ET-28a(+)-Tc-rhaA were obtained. G52V The mutant strain BL21-Tc-rhaA G52V Contains the mutant plasmid pET-28a(+)-Tc-rhaA D294R The mutant strain BL21-Tc-rhaA D294R and containing the mutant plasmid pET-28a(+)-Tc-rhaA G52V / D294R The mutant strain BL21-Tc-rhaA G52V / D294R .
[0084] The above recombinant strain BL21-Tc-rhaA and mutant strain BL21-Tc-rhaA were used. G52V Mutant strain BL21-Tc-rhaA D294R Mutant strain BL21-Tc-rhaA G52V / D294RThe seeds were inoculated into culture medium for seed culture. The culture medium for seed culture was LB liquid medium containing kanamycin (50 μg / mL). The seeds were cultured at 37℃ and 220 rpm for 8 h with shaking to obtain four seed solutions.
[0085] The four seed solutions were inoculated into separate culture media at a 2% (v / v) inoculum for fermentation. The fermentation medium was LB liquid medium containing kanamycin (50 μg / mL), and the cultures were shaken at 37°C and 180 rpm until OD reached. 600 The concentration was reached 0.7, and then the temperature was lowered for induction culture. The induction culture conditions were: cooling to 20℃, adding IPTG to a final concentration of 0.5mM for induction culture, until OD... 600 When the value reached 3.0, fermentation broths containing wild-type L-rhamnose isomerase, fermentation broths containing L-rhamnose isomerase mutant G52V, fermentation broths containing L-rhamnose isomerase mutant D294R, and fermentation broths containing L-rhamnose isomerase mutants G52V / D294R were obtained.
[0086] The four fermentation broths were centrifuged at 4℃ and 8000 rpm for 10 minutes to collect the cells. The collected cells were resuspended in 100 mM phosphate buffer (pH 7.5). After resuspending, the cells were lysed using sonication at 400 W for 15 minutes, with a 2-second sonication interval followed by a 3-second pause. After cell lysis, the resulting bacterial solutions were centrifuged at 4℃ and 12000 rpm for 30 minutes. The supernatants were collected to obtain the crude enzyme solutions of wild-type L-rhamnose isomerase, L-rhamnose isomerase mutant G52V, L-rhamnose isomerase mutant D294R, and L-rhamnose isomerase mutants G52V / D294R, respectively.
[0087] Example 4
[0088] Determination of enzyme activity
[0089] Enzyme activity definition: Under standard reaction conditions, the amount of enzyme required to catalyze the production of 1 μmol of D-allose per minute is defined as 1 unit of enzyme activity (U).
[0090] Standard reaction system 1: Add the following to 50 mM HEPES buffer (pH 7.5) The reaction system consisted of D-allulose and crude enzyme solution of wild-type L-rhamnosyl isomerase prepared in Example 3, with a total volume of 1 mL. The concentration of the enzyme was 1 mM, the concentration of D-allulose was 100 mM, and the concentration of crude wild-type L-rhamnosyl isomerase was 0.5 mg / mL.
[0091] Standard reaction system 2: Add to 50 mM HEPES buffer (pH 7.5) The reaction system contained D-allulose, crude enzyme solution of L-rhamnose isomerase mutant G52V prepared in Example 3, and D-allulose, making the total volume of the reaction system 1 mL. The concentration of the enzyme was 1 mM, the concentration of D-allulose was 100 mM, and the concentration of crude enzyme of L-rhamnosyl isomerase mutant G52V was 0.5 mg / mL.
[0092] Standard reaction system 3: Add to 50 mM HEPES buffer (pH 7.5) The reaction system contained D-allulose, crude enzyme solution of the L-rhamnose isomerase mutant D294R prepared in Example 3, and D-allulose, making the total volume of the reaction system 1 mL. The concentration of the enzyme was 1 mM, the concentration of D-allulose was 100 mM, and the concentration of crude enzyme of L-rhamnosyl isomerase mutant D294R was 0.5 mg / mL.
[0093] Standard reaction system 4: Add to 50 mM HEPES buffer (pH 7.5) The reaction mixture contained D-allulose and crude enzyme solution of the L-rhamnose isomerase mutant G52V / D294R prepared in Example 3, making the total volume of the reaction system 1 mL. The concentration of the enzyme was 1 mM, the concentration of D-allulose was 100 mM, and the concentration of crude enzyme of L-rhamnose isomerase mutant G52V / D294R was 0.5 mg / mL.
[0094] Each of the above standard reaction systems was reacted at 65°C for 10 minutes, and then the reaction was terminated by heating in a boiling water bath for 10 minutes.
[0095] After each reaction was completed, the reaction solutions were centrifuged at 12000 rpm for 10 min, and the supernatants were collected, diluted 10-fold, and filtered through a 0.22 μm microporous membrane. The D-allose content was then determined by HPLC. The relative enzyme activities of each L-rhamnose isomerase mutant were calculated with the wild-type L-rhamnose isomerase activity as 100%, and the results are shown in Table 8.
[0096] HPLC detection method: Detector: RID; Analytical column: ZORBAX NH2 (4.6×250 mm, 5 µm), 40℃, eluted with 75% (volume fraction) acetonitrile (acetonitrile:water = 75:25), flow rate: 1 mL / min.
[0097] Table 8
[0098]
[0099] The results above show that, compared with wild-type L-rhamnose isomerase, the enzyme activities of L-rhamnose isomerase mutants G52V, D294R, and G52V / D294R are all significantly increased, with the L-rhamnose isomerase mutant G52V / D294R exhibiting the highest enzyme activity.
[0100] Example 5
[0101] Half-life determination
[0102] The crude enzyme solution of wild-type L-rhamnosyl isomerase prepared in Example 3 was incubated at 65℃ and 75℃ for 2h, 4h, 6h, 8h, 10h and 12h respectively. The enzyme activity after 0h incubation was taken as 100%. The relative enzyme activity of the crude enzyme solution of wild-type L-rhamnosyl isomerase after incubation at different temperatures and times was determined according to the method in Example 4 above. The half-life of wild-type L-rhamnosyl isomerase at 65℃ and 75℃ was obtained respectively.
[0103] The crude enzyme solution of L-rhamnose isomerase mutant G52V prepared in Example 3 was incubated at 65℃ and 75℃ for 2h, 4h, 6h, 8h, 10h and 12h respectively. The enzyme activity after 0h incubation was taken as 100%. The relative enzyme activity of the crude enzyme solution of L-rhamnose isomerase mutant G52V after incubation at different temperatures and times was determined according to the method in Example 4 above. The half-life of wild-type L-rhamnose isomerase at 65℃ and 75℃ was obtained respectively.
[0104] The test results showed that, compared with wild-type L-rhamnose isomerase, the half-life of L-rhamnose isomerase mutant G52V at 65℃ was extended from 6h to 7.5h, and the half-life of L-rhamnose isomerase mutant G52V at 75℃ was extended from 4.5h to 5.5h.
[0105] The crude enzyme solution of L-rhamnose isomerase mutant D294R prepared in Example 3 was incubated at 65℃ and 75℃ for 2h, 4h, 6h, 8h, 10h and 12h respectively. The enzyme activity after 0h incubation was taken as 100%. The relative enzyme activity of the crude enzyme solution of L-rhamnose isomerase mutant D294R after incubation at different temperatures and times was determined according to the method in Example 4 above. The half-life of wild-type L-rhamnose isomerase at 65℃ and 75℃ were obtained respectively.
[0106] The test results showed that, compared with wild-type L-rhamnose isomerase, the half-life of L-rhamnose isomerase mutant D294R at 65℃ was extended from 6h to 6.5h, and the half-life of L-rhamnose isomerase mutant D294R at 75℃ was extended from 4.5h to 4.8h.
[0107] The crude enzyme solution of the L-rhamnose isomerase mutant G52V / D294R prepared in Example 3 was incubated at 65℃ and 75℃ for 2h, 4h, 6h, 8h, 10h and 12h respectively. The enzyme activity after 0h incubation was taken as 100%. The relative enzyme activity of the crude enzyme solution of L-rhamnose isomerase mutant G52V / D294R after incubation at different temperatures and times was determined according to the method in Example 4 above. The half-life of wild-type L-rhamnose isomerase at 65℃ and 75℃ was obtained respectively.
[0108] Test results are as follows Figure 1 , Figure 2 As shown, by Figure 1 It can be seen that, compared to the wild-type L-rhamnosyl isomerase, the half-life of the L-rhamnosyl isomerase mutant G52V / D294R at 65℃ is extended from 6 h to 9.5 h. Figure 2 It can be seen that, compared with the wild-type L-rhamnose isomerase, the half-life of the L-rhamnose isomerase mutant G52V / D294R at 75℃ is extended from 4.5h to 7.1h.
[0109] Example 6
[0110] High-density fermentation for the preparation of crude enzyme solution
[0111] The recombinant strain BL21-Tc-rhaA, the mutant strain BL21-Tc-rhaAG52V, the mutant strain BL21-Tc-rhaAD294R, and the mutant strain BL21-Tc-rhaAG52V / D294R obtained in Example 3 were inoculated into a culture medium for seed culture. The seed culture medium was LB liquid medium containing kanamycin (50 μg / mL), and the cultures were shaken at 37°C and 220 rpm until OD200. 600 The value was increased to 1.5, and four seed solutions were obtained respectively.
[0112] The above four seed solutions were inoculated into fermenters at a volume ratio of 5%, and cultured at a pressure of 0.04 MPa, an initial rotation speed of 300 rpm, a temperature of 37°C, and a pH of 7.0 until OD. 600 At 35°C, the temperature was lowered to 30°C, and the culture was continued until OD (October Expiratory Rate). 600Induction culture was performed at 45°C. The induction culture conditions were as follows: IPTG was added to a final concentration of 0.05 mM; the initial airflow rate was 4 L / min; when the rotation speed reached 400 rpm, the airflow rate was adjusted to 4.2 L / min; when the rotation speed reached 600 rpm, the airflow rate was adjusted to 5.5 L / min; when dissolved oxygen rebounded, supplemental culture medium was added to maintain the DO value at 20-40% until OD... 600 Fermentation was stopped when the temperature reached 120°C, yielding fermentation broths containing wild-type L-rhamnose isomerase, L-rhamnose isomerase mutant G52V, L-rhamnose isomerase mutant D294R, and L-rhamnose isomerase mutants G52V / D294R, respectively.
[0113] The four fermentation broths were centrifuged at 8000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were collected. The bacterial cells were resuspended in 100 mM PBS buffer (pH 7.5), and then the bacterial cells were sonicated at 200 W for 3 seconds at 2-second intervals for a total of 20 minutes. After sonication, the broths were centrifuged at 12000 rpm for 60 min to obtain four supernatants: crude enzyme solution of wild-type L-rhamnose isomerase, crude enzyme solution of L-rhamnose isomerase mutant G52V, crude enzyme solution of L-rhamnose isomerase mutant D294R, and crude enzyme solution of L-rhamnose isomerase mutant G52V / D294R.
[0114] The fermentation medium in the above fermenter consisted of: 8 g / L yeast extract, 2.5 g / L ammonium sulfate, 12 g / L peptone, 0.5 g / L magnesium sulfate heptahydrate, 12 g / L glucose, 3 g / L sodium chloride, 2.1 g / L citric acid monohydrate, and 4 g / L potassium phosphate.
[0115] The above-mentioned feed culture medium consists of 500 g / L glucose, 2 g / L magnesium sulfate heptahydrate, and 2 g / L yeast extract.
[0116] Example 7
[0117] Preparation of D-allose
[0118] Add to 50 mM HEPES buffer (pH 7.5) D-allulose and the crude enzyme solution of wild-type L-rhamnosyl isomerase prepared in Example 6 above, so that the reaction system contains D-allulose and the crude enzyme solution of wild-type L-rhamnosyl isomerase prepared in Example 6 above. The concentration of the enzyme was 1 mM, the concentration of D-allulose was 500 g / L, and the concentration of crude wild-type L-rhamnosyl isomerase was 80 mg / mL.
[0119] Add to 50 mM HEPES buffer (pH 7.5) D-allulose and the crude enzyme solution of the L-rhamnosyl isomerase mutant G52V prepared in Example 6 above were used to make the reaction system contain D-allulose and D-allulose. The concentration of the enzyme was 1 mM, the concentration of D-allulose was 500 g / L, and the concentration of crude enzyme of L-rhamnosyl isomerase mutant G52V was 80 mg / mL.
[0120] Add to 50 mM HEPES buffer (pH 7.5) D-allulose and the crude enzyme solution of the L-rhamnose isomerase mutant D294R prepared in Example 6 above were used to make the reaction system contain D-allulose and crude enzyme solution of D-allulose and L-rhamnose isomerase mutant D294R. The concentration of the enzyme was 1 mM, the concentration of D-allulose was 500 g / L, and the concentration of crude enzyme of L-rhamnose isomerase mutant D294R was 80 mg / mL.
[0121] Add to 50 mM HEPES buffer (pH 7.5) D-allulose and the crude enzyme solution of the L-rhamnose isomerase mutant G52V / D294R prepared in Example 6 above were used to make the reaction system contain D-allulose and the crude enzyme solution of the L-rhamnose isomerase mutant G52V / D294R. The concentration of the enzyme was 1 mM, the concentration of D-allulose was 500 g / L, and the concentration of crude enzyme of L-rhamnose isomerase mutant G52V / D294R was 80 mg / mL.
[0122] Each of the above standard reaction systems was reacted at 65°C for 12 hours.
[0123] After each of the above reactions was completed, the reaction solutions were centrifuged at 12000 rpm for 10 min, and the supernatant was collected, diluted 10 times, and filtered through a 0.22 μm microporous membrane. The D-allose content was then detected by HPLC, and the conversion rate was calculated. The results are shown in Table 9.
[0124] HPLC detection method: Detector: RID; Analytical column: ZORBAX NH2 (4.6×250 mm, 5 µm), 40℃, eluted with 75% (volume fraction) acetonitrile (acetonitrile:water = 75:25), flow rate: 1 mL / min.
[0125] Conversion rate: D-allose content after the reaction ÷ D-allose content at the beginning of the reaction × 100%.
[0126] Table 9
[0127]
[0128] The results above show that, compared with wild-type L-rhamnose isomerase, the L-rhamnose isomerase mutants G52V, D294R, and G52V / D294R in this invention can further improve the conversion rate and the yield of D-allose.
[0129] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A long-half-life L-rhamnosyl isomerase, characterized in that, The amino acid sequence of the L-rhamnosyl isomerase is shown in any of the following examples: (1) The amino acid sequence as shown in SEQ ID NO.4; (2) The amino acid sequence as shown in SEQ ID NO.6; (3) The amino acid sequence shown in SEQ ID NO.
8.
2. A biomaterial, characterized in that, Includes any one of the following: (A) A nucleic acid molecule having a gene sequence encoding the long-half-life L-rhamnosyl isomerase of claim 1; (B) An expression vector containing the nucleic acid molecule described in (A); (C) A recombinant strain containing the nucleic acid molecule described in (A) or the expression vector described in (B).
3. A method for preparing L-rhamnosyl isomerase, used to prepare the long-half-life L-rhamnosyl isomerase as described in claim 1, characterized in that, A recombinant strain containing the L-rhamnose isomerase gene was fermented and cultured, and the recombinant strain was induced to express the L-rhamnose isomerase.
4. The preparation method according to claim 3, characterized in that, Includes the following steps: The recombinant strain was subjected to seed culture to obtain seed solution; The seed culture was inoculated into a fermentation medium for fermentation culture until the OD value reached... 600 Cool to 30-40°C and continue culturing until OD (October Expiratory Rate). 600 The dissolved oxygen (DO) value was set at 45-55°C. An inducing agent was added for induction culture. During dissolved oxygen rebound, supplemental culture medium was added to maintain the DO value at 20-40% until the OD value reached its maximum. 600 Fermentation ends when the temperature reaches 110-130°C, yielding the fermentation liquid. Centrifuge the fermentation broth, resuspend it, break up the cells, and centrifuge it again. The resulting supernatant is the crude enzyme solution of L-rhamnosyl isomerase.
5. The preparation method according to claim 4, characterized in that, The seed culture temperature is 35~40℃, and the rotation speed is 120~250 rpm; and / or, The fermentation culture was carried out at a tank pressure of 0.035~0.045 MPa, an initial rotation speed of 300~350 rpm, a temperature of 35~40℃, and a pH value of 6.5~7.5; and / or, The cooling is described as reducing the temperature to 30~35℃; and / or, The initial airflow for the induction culture is 3.8~4 L / min. When the rotation speed reaches 400~450 rpm, the airflow is adjusted to 4.2~4.5 L / min. When the rotation speed reaches 600~700 rpm, the airflow is adjusted to 5.5~6 L / min.
6. The use of the long-half-life L-rhamnose isomerase of claim 1 or the biomaterial of claim 2 in the preparation of D-allose.
7. A method for preparing D-allose, characterized in that, The long-half-life L-rhamnosyl isomerase described in claim 1 is used to catalyze the reaction of substrate D-allulose to generate D-allose.
8. The preparation method according to claim 7, characterized in that, The preparation method for the reaction system that generates D-allose is as follows: Add crude L-rhamnosyl isomerase solution, metal ions, and D-alulose to a 20-50 mM buffer solution with a pH of 7.0-8.0, so that the concentration of crude L-rhamnosyl isomerase in the reaction system is 10-120 mg / mL, the concentration of metal ions is 0.5-2 mM, and the concentration of D-alulose is 400-600 g / L.
9. The preparation method according to claim 8, characterized in that, The buffer solution includes HEPES buffer, glycine-sodium hydroxide buffer, or Tris-HCl buffer; and / or, The metal ions include manganese ions, cobalt ions, or magnesium ions.
10. The preparation method according to claim 8 or 9, characterized in that, The reaction temperature is 60~80℃.