L-rhamnose isomerase mutant and method for preparing d-allose

Abstract

This invention discloses an L-rhamnose isomerase mutant and a method for preparing D-allose, belonging to the field of genetic engineering technology. The amino acid sequence of the L-rhamnose isomerase mutant is shown in SEQ ID NO.3. This invention significantly increases the enzyme activity of the L-rhamnose isomerase mutant by mutating amino acid position 268 of the wild-type L-rhamnose isomerase sequence from S to T, thereby improving the catalytic efficiency in the preparation of D-allose from D-allulose and ultimately increasing the yield of D-allose.

Description

A method for preparing an L-rhamnose isomerase mutant and D-allose. Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an L-rhamnosyl isomerase mutant and a method for preparing 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 achieves a one-step synthesis of D-allose from inexpensive sugar sources using engineered microorganisms. However, this method is technically complex, with difficult strain construction requiring modification of the microbial metabolic network. This necessitates not only the introduction of exogenous key enzymes such as D-allulose-3-epimerase and L-rhamnose isomerase, but also the precise knockout of competing metabolic pathways to prevent substrate flow into byproducts. The entire process of constructing and optimizing engineered strains is lengthy and costly. Secondly, this method produces numerous byproducts, leading to high separation and purification costs. Furthermore, parameters such as pH, temperature, dissolved oxygen, and substrate concentration during fermentation require precise control to balance cell growth and product synthesis, making process control challenging during scale-up production.

[0004] The core of the enzymatic conversion preparation of D-allose is to use L-rhamnose isomerase to catalyze the conversion of D-allulose into D-allose in vitro. However, the yield of D-allose is limited by the catalytic efficiency of L-rhamnose isomerase. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an L-rhamnose isomerase mutant and a method for preparing D-allose, so as to overcome the problem that the yield of D-allose is limited by the catalytic efficiency of L-rhamnose isomerase in the prior art.

[0006] In a first aspect, the present invention provides an L-rhamnosyl isomerase mutant, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] Compared with the wild-type L-rhamnose isomerase in the prior art, the present invention mutates the wild-type L-rhamnose isomerase shown in SEQ ID NO.1 by changing the 268th amino acid of its amino acid sequence from S to T. The resulting L-rhamnose isomerase mutant S268T has significantly increased enzyme activity, thereby improving catalytic efficiency and increasing the yield of D-allose when catalyzing the reaction of D-allulose to prepare D-allose.

[0008] Furthermore, the gene sequence encoding the above-mentioned L-rhamnosyl isomerase mutant is shown in SEQ ID NO.4.

[0009] In a second aspect, the present invention provides a biomaterial comprising any one of the following:

[0010] (1) The gene encoding the above-mentioned L-rhamnose isomerase mutant, or a nucleic acid molecule containing the gene encoding the above-mentioned L-rhamnose isomerase mutant;

[0011] (2) An expression vector containing the nucleic acid molecule described in (1);

[0012] (3) A recombinant strain containing the nucleic acid molecule described in (1) or the expression vector described in (2).

[0013] Thirdly, the present invention provides a method for preparing an L-rhamnose isomerase mutant, which is used to prepare the above-mentioned L-rhamnose isomerase mutant by fermentation culture of a recombinant strain containing the L-rhamnose isomerase mutant gene and inducing the recombinant strain to express the L-rhamnose isomerase mutant.

[0014] Furthermore, fermentation is carried out in a high-density manner, including the following steps:

[0015] The recombinant strains were inoculated into a culture medium for seed culture and cultured until OD500. 600 The solution was diluted to 1.5-2.5 to obtain the seed solution.

[0016] The seed culture was inoculated into the fermenter at an inoculation rate of 1% to 10% by volume for fermentation culture until the OD reached the target value. 600 Cool to 30-35°C and continue culturing until OD (October Expiratory Rate). 600 The dissolved oxygen (DO) level is initially set to 40-50%, followed by the addition of an inducing agent for induction culture. When dissolved oxygen rebounds, supplemental culture medium is added to maintain a DO level of 20-40%, until the OD level is reached. 600 Fermentation ended when growth ceased, yielding a fermentation broth containing an L-rhamnosyl isomerase mutant.

[0017] Centrifuge the fermentation broth, collect the bacterial cells, resuspend the bacterial cells, break the bacterial cells, centrifuge again, and the resulting supernatant is the crude enzyme solution of L-rhamnosyl isomerase mutant.

[0018] Compared with existing technologies, this invention uses high-density fermentation technology to cultivate recombinant strains to prepare crude enzyme solution of L-rhamnosyl isomerase mutant, which effectively improves enzyme yield and production efficiency, reduces production costs, and is suitable for industrial production.

[0019] Furthermore, the seed culture temperature is 35~40℃.

[0020] Furthermore, the fermentation culture temperature is 35~40℃, the pH value is 6.5~7.5, the tank pressure is 0.03~0.05 MPa, and the initial rotation speed is 200~400 rpm.

[0021] Furthermore, the temperature will be lowered to 25-30℃.

[0022] Furthermore, the induction culture temperature is 25~30℃, and the initial air volume is 200~300m³. 3 / h, when the speed reaches 350~400rpm, adjust the air volume to 350~450m³ / h. 3 / h.

[0023] Fourthly, the present invention provides the application of the above-mentioned L-rhamnose isomerase mutant or the above-mentioned biological material in the preparation of D-allose.

[0024] Fifthly, the present invention provides a method for preparing D-allose, which utilizes the above-mentioned L-rhamnosyl isomerase mutant to catalyze the reaction of substrate D-allulose to generate D-allose.

[0025] Compared with the prior art, the L-rhamnose isomerase mutant obtained by the present invention has higher enzyme activity and improved catalytic efficiency when catalyzing the reaction of substrate D-allulose to generate D-allose, thereby further improving the conversion rate and the yield of D-allose.

[0026] Furthermore, buffer solution, metal ions, crude enzyme solution of L-rhamnosyl isomerase mutant, and D-alulose were added to the reaction system to make the concentration of buffer solution 20-50 mM, metal ions 0.5-2 mM, crude enzyme solution of L-rhamnosyl isomerase mutant 10-30 mg / mL, and D-alulose 400-600 g / L.

[0027] Furthermore, the metal ions include magnesium ions, nickel ions, or copper ions.

[0028] Furthermore, the reaction temperature is 60~75℃, and the pH value is 7.0~8.0. Detailed Implementation

[0029] 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.

[0030] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0031] Example 1

[0032] Construction of recombinant plasmids and mutant plasmids

[0033] Wild-type L-rhamnose isomerase derived from *Thermobacillus composti* was selected, and its amino acid sequence is shown in SEQ ID NO.1. The gene encoding the above-mentioned wild-type L-rhamnose isomerase was optimized according to the codon preference of *Escherichia coli* to obtain the optimized wild-type L-rhamnose isomerase gene, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0034] Mutations were made at the enzyme activity-related sites of the wild-type L-rhamnosyl isomerase. Amino acid position 268 of the amino acid sequence shown in SEQ ID NO.1 was mutated from S to T, yielding the L-rhamnosyl isomerase mutant S268T, whose amino acid sequence is shown in SEQ ID NO.3. The amino acid sequence of the L-rhamnosyl isomerase mutant S268T was reverse-translated into a DNA sequence and optimized according to the codon bias of *E. coli*, resulting in the optimized L-rhamnosyl isomerase mutant S268T gene, whose nucleotide sequence is shown in SEQ ID NO.4.

[0035] Using the optimized wild-type L-rhamnose isomerase gene as a template, PCR amplification was performed with F1 as the upstream primer and R1 as the downstream primer to obtain the wild-type L-rhamnose isomerase gene rhaA with homologous arms at both ends.

[0036] Using the optimized L-rhamnose isomerase mutant S268T gene as a template, and with F1 as the upstream primer and R1 as the downstream primer, PCR amplification was performed to obtain the L-rhamnose isomerase mutant S268T gene rhaA with homologous arms at both ends. S268T .

[0037] F1: 5'-cagcaaatgggtcgcggatccATGGAACGTAACATTGAACAAGCA-3' (SEQ ID NO. 5);

[0038] R1: 5'-gtggtggtggtggtgctcgagTTAGCCGTTGCGCTTGGC-3' (SEQ ID NO. 6).

[0039] The reaction systems for the two PCR amplification reactions are shown in Table 1, and the reaction procedures are shown in Table 2.

[0040] Table 1

[0041]

[0042] Table 2

[0043]

[0044] It should be understood that the first step of the PCR reaction, pre-denaturation at 98°C, and the final step, extension at 72°C, do not participate in the cycling process, and the entire PCR process is performed only once.

[0045] After each of the above PCR amplifications was completed, the reaction products were recovered by agarose gel extraction to obtain gene fragments with high purity.

[0046] The expression vector pET-28a(+) was double-digested using restriction endonucleases BamHI and XhoI at 37°C for 20 min. The double-digested products were then recovered and purified by agarose gel electrophoresis to obtain the linearized vector pET-28a(+). The double-digestion system is shown in Table 3.

[0047] Table 3

[0048]

[0049] The genes rhaA and rhaA obtained after the above PCR were used. S268T The plasmids pET28a-rhaA and pET28a-rhaA were respectively ligated into the linearized vector pET-28a(+) to construct the recombinant plasmid pET28a-rhaA and the mutant plasmid pET28a-rhaA. S268T The bonding system is shown in Table 4. The bonding reaction temperature was 37℃, and the reaction time was 20 min.

[0050] Table 4

[0051]

[0052] The two ligation products were transformed into E. coli DH5α competent cells, and positive clones were screened on LB plates containing kanamycin (50 μg / mL) and verified. Plasmids were extracted and DNA was sequenced to ensure that the gene sequence was completely correct and free of error mutations.

[0053] Example 2

[0054] Preparation of crude enzyme solution

[0055] The recombinant plasmid pET28a-rhaA and the mutant plasmid pET28a-rhaA that were correctly sequenced were used. S268T Recombinant strains BL21-pET28a-rhaA and BL21-pET28a-rhaA were obtained by chemical transformation into E. coli BL21(DE3) competent cells.S268T .

[0056] The recombinant strains BL21-pET28a-rhaA and BL21-pET28a-rhaA S268T Spread the cultures onto LB agar plates containing kanamycin (50 μg / mL) and incubate overnight at 37°C. Single colonies were picked and inoculated into liquid medium for seed culture. The seed culture medium was LB liquid medium containing kanamycin (50 μg / mL). The cultures were shaken at 37°C and 180 rpm for 12 h to obtain two seed solutions.

[0057] The two seed solutions were inoculated into two separate liquid 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 incubated at 37°C and 180 rpm until OD200. 600 The concentration reached 0.7, and then induction culture was performed separately. The induction culture conditions were as follows: the temperature was lowered to 20℃, IPTG was added to a final concentration of 0.5mM, and the culture was induced to OD. 600 Fermentation broth containing wild-type L-rhamnose isomerase and fermentation broth containing L-rhamnose isomerase mutant S268T were obtained at 3.5.

[0058] The two fermentation broths were centrifuged at 4℃ and 8000 rpm for 10 minutes each, the supernatant was discarded, and the bacterial cells were collected separately. The bacterial cells were resuspended in 100mM phosphate buffer (pH 7.5), and then the cells were lysed using sonication at 400W for 15 minutes (2 seconds per sonication, 3 seconds per pause). After cell lysis, the cell lysate was centrifuged, and the supernatant was collected separately. These were the crude enzyme solutions of wild-type L-rhamnose isomerase and L-rhamnose isomerase mutant S268T, respectively.

[0059] Example 3

[0060] Enzyme activity measurement

[0061] Enzyme activity unit 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 enzyme activity unit (U).

[0062] Standard reaction system 1:

[0063] 50 mM Tris-HCl buffer (pH 7.5), 1 mM 100 mM D-allulose was added to a solution of crude wild-type L-rhamnose isomerase prepared in Example 2 above, so that the concentration of crude wild-type L-rhamnose isomerase in the reaction system was 0.5 mg / mL, and the total reaction volume was 1 mL. Reaction conditions: 65℃ for 10 minutes.

[0064] Standard reaction system 2:

[0065] 50 mM Tris-HCl buffer (pH 7.5), 1 mM 100 mM D-allulose was added to a solution of crude enzyme solution of L-rhamnose isomerase mutant S268T prepared in Example 2 above, so that the concentration of crude enzyme of L-rhamnose isomerase mutant S268T in the reaction system was 0.5 mg / mL, and the total reaction volume was 1 mL. Reaction conditions: 65℃ for 10 minutes.

[0066] Termination and Detection: The reactions of the two reaction systems were terminated by heating in a boiling water bath for 10 minutes. After the reaction was completed, each reaction solution was centrifuged at 12000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm microporous membrane. The filtrate was diluted 10 times and the D-allose content was detected by HPLC.

[0067] The relative enzyme activity of the L-rhamnose isomerase mutant S268T was calculated with the wild-type L-rhamnose isomerase activity as 100%, and the results are shown in Table 5.

[0068] 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.

[0069] Table 5

[0070]

[0071] The results above show that, compared with the wild-type L-rhamnose isomerase, the L-rhamnose isomerase mutant S268T obtained by mutation in this invention has significantly improved enzyme activity.

[0072] Example 4

[0073] High-density fermentation for the preparation of crude enzyme solution

[0074] The recombinant strains BL21-pET28a-rhaA and BL21-pET28a-rhaA prepared in Example 2 above were selected. S268TThe samples were inoculated into liquid culture medium for seed culture, with the medium being LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C until OD200. 600 Two seed solutions were obtained at a concentration of 2.0.

[0075] The two seed solutions were inoculated into two fermenters containing culture medium at a volume ratio of 5%. The fermenters were 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 the OD reached [value missing]. 600 At 35°C, the temperature was lowered to 30°C, and the culture was continued until OD (October Expiratory Rate). 600 Induction culture was performed separately at a concentration of 50 μL. The induction culture conditions were as follows: IPTG was added to a final concentration of 0.5 mM, the induction culture temperature was 30 °C, and the initial air volume was 250 m³ / h. 3 / h, when the speed reaches 400rpm, adjust the air volume to 400m³ / h. 3 / h, when dissolved oxygen rebounds, start adding feed medium to maintain the DO value at 20-40% until OD 600 It stops growing and fermentation ends.

[0076] The culture medium in the above fermenter can be composed of: 2 g / L citric acid monohydrate, 14 g / L potassium dihydrogen phosphate, 4.5 g / L dipotassium hydrogen phosphate trihydrate, 4 g / L ammonium sulfate, 20 g / L glucose, 1 g / L magnesium sulfate heptahydrate, 1 g / L yeast extract, 2 mL / L trace element solution, and 50 μg / mL kanamycin.

[0077] The trace element solution consists of: MnSO4·7H2O 0.5 g / L, FeSO4·7H2O 10.0 g / L, CaCl2 2.0 g / L, and (NH4)Mo7O. 24 0.1 g / L, CuSO4·5H2O 3.0 g / L, ZnSO4·7H2O 5.25 g / L, dissolved in sterile water.

[0078] The composition of the supplemental culture medium can be: 600 g / L glucose, 2 g / L magnesium sulfate, and 10 g / L yeast extract.

[0079] After the high-density fermentation was completed, fermentation broth containing wild-type L-rhamnose isomerase and fermentation broth containing L-rhamnose isomerase mutant S268T were obtained, respectively.

[0080] The two fermentation broths were centrifuged separately using a high-speed refrigerated centrifuge at 4°C and 8000 rpm for 15 min. The supernatant was discarded, and the bacterial cells were collected. The collected bacterial cells were resuspended in phosphate buffer, and the OD of the resuspended broth was measured. 600The value was 80. Then, the cells were homogenized at 750 bar for 30 min using a high-pressure homogenizer to break them up. After cell disruption, the cells were centrifuged again at 4°C and 12000 rpm for 60 min using a high-speed refrigerated centrifuge to remove cell debris. The supernatant was collected to obtain crude enzyme solutions of wild-type L-rhamnose isomerase and L-rhamnose isomerase mutant S268T, respectively.

[0081] Example 5

[0082] Preparation of D-allose

[0083] Tris-HCl buffer, magnesium chloride, the crude wild-type L-rhamnose isomerase solution prepared in Example 4 above, and D-alulose were added to the reaction system to make the concentration of Tris-HCl buffer 50 mM, pH 7.5, magnesium chloride 1 mM, crude wild-type L-rhamnose isomerase 20 mg / mL, and D-alulose 500 g / L.

[0084] Tris-HCl buffer, magnesium chloride, crude enzyme solution of L-rhamnosyl isomerase mutant S268T prepared in Example 4 above, and D-alulose were added to the reaction system to make the concentration of Tris-HCl buffer 50 mM, pH 7.5, magnesium chloride 1 mM, crude enzyme of L-rhamnosyl isomerase mutant S268T 20 mg / mL, and D-alulose 500 g / L.

[0085] The two reaction systems were reacted at 65℃ for 12 h. After the reaction, each reaction solution was centrifuged at 12000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm microporous membrane. The filtrate was diluted 10 times and the D-allose content was detected by HPLC. The conversion rate was calculated, and the results are shown in Table 6.

[0086] Conversion rate = D-allose content after reaction termination ÷ D-allose content at the beginning of reaction × 100%.

[0087] 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.

[0088] Table 6

[0089]

[0090] The results above show that, compared with the wild-type L-rhamnose isomerase, the L-rhamnose isomerase mutant S268T obtained by mutation in this invention has higher enzyme activity, thereby further improving the conversion rate and D-allose yield when catalyzing the reaction of substrate D-allulose to D-allose.

[0091] 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. An L-rhamnosyl isomerase mutant, characterized in that, The amino acid sequence of the L-rhamnosyl isomerase mutant is shown in SEQ ID NO.

3.

2. The L-rhamnosyl isomerase mutant according to claim 1, characterized in that, The gene sequence of the L-rhamnosyl isomerase mutant is shown in SEQ ID NO.

4.

3. A biomaterial, characterized in that, Includes any one of the following: (1) a gene encoding the L-rhamnose isomerase mutant of claim 1, or a nucleic acid molecule containing a gene encoding the L-rhamnose isomerase mutant of claim 1; (2) an expression vector containing the nucleic acid molecule of (1); (3) a recombinant strain containing the nucleic acid molecule of (1) or containing the expression vector of (2).

4. A method for preparing an L-rhamnosyl isomerase mutant, used to prepare the L-rhamnosyl isomerase mutant according to claim 1, characterized in that, A recombinant strain containing the L-rhamnose isomerase mutant gene was fermented and cultured, and the recombinant strain was induced to express the L-rhamnose isomerase mutant.

5. The preparation method according to claim 4, characterized in that, The fermentation is a high-density fermentation, comprising the following steps: inoculating the recombinant strain into a culture medium for seed culture, and culturing until OD... 600 The seed culture was obtained by adjusting the temperature to 1.5-2.5; the seed culture was then inoculated into a fermenter for fermentation culture until the OD value reached 1.5-2.

5. 600 Cool to 30-35°C and continue culturing until OD (October Expiratory Rate). 600 The dissolved oxygen (DO) level is initially set to 40-50%, followed by the addition of an inducing agent for induction culture. When dissolved oxygen rebounds, supplemental culture medium is added to maintain a DO level of 20-40%, until the OD level is reached. 600 Fermentation ends when growth ceases, yielding a fermentation broth containing the L-rhamnose isomerase mutant. The fermentation broth is centrifuged, the cells are collected, resuspended, and then the cells are broken. The mixture is centrifuged again, and the resulting supernatant is the crude enzyme solution of the L-rhamnose isomerase mutant.

6. The preparation method according to claim 5, characterized in that, The seed culture temperature is 35-40℃; and / or, the fermentation culture temperature is 35-40℃, the pH value is 6.5-7.5, the tank pressure is 0.03-0.05 MPa, and the initial rotation speed is 200-400 rpm; and / or, the cooling is to lower the temperature to 25-30℃; and / or, the induction culture temperature is 25-30℃, and the initial air volume is 200-300 m³ / h. 3 / h, when the speed reaches 350~400rpm, adjust the air volume to 350~450m³ / h. 3 / h.

7. The use of the L-rhamnose isomerase mutant of claim 1 or the biomaterial of claim 3 in the preparation of D-allose.

8. A method for preparing D-allose, characterized in that, The L-rhamnose isomerase mutant of claim 1 is used to catalyze the reaction of substrate D-allulose to generate D-allose.

9. The preparation method according to claim 8, characterized in that, Add buffer solution, metal ions, crude enzyme solution of L-rhamnosyl isomerase mutant, and D-alulose to the reaction system to make the concentration of buffer solution 20-50 mM, metal ions 0.5-2 mM, crude enzyme solution of L-rhamnosyl isomerase mutant 10-30 mg / mL, and D-alulose 400-600 g / L.

10. The preparation method according to claim 9, characterized in that, The metal ions include magnesium ions, nickel ions, or copper ions; and / or, the reaction temperature is 60~75℃, and the pH value is 7.0~8.0.

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