L-rhamnose isomerase and application thereof in preparation of D-allose

Abstract

The invention discloses L-rhamnose isomerase and application thereof in preparation of D-allose, and belongs to the technical field of genetic engineering. The amino acid sequence of the L-rhamnose isomerase is obtained by performing any one of the following mutations on an amino acid sequence as shown in SEQ ID NO.1: (1) mutating the 132 amino acid from P to K; and (2) the 132 amino acid is mutated from P to K, and the 258 amino acid is mutated from D to Q. Compared with the prior art, two L-rhamnose isomerase mutants are obtained by mutating wild type L-rhamnose isomerase, and compared with the wild type L-rhamnose isomerase, the two L-rhamnose isomerase mutants are higher in enzyme activity and longer in half-life period, so that when the D-psicose is catalyzed to be converted into D-allose, the two L-rhamnose isomerase mutants have the advantages that the enzyme activity is higher, and the half-life period is longer; the catalytic capability is stronger and more durable, and the conversion efficiency of the reaction and the yield of D-allose are improved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an L-rhamnosyl isomerase and its application in the preparation of D-allose. Background Technology

[0002] D-Allose is a type D-dextrose, one of the eight configurations of hexose aldoses. D-Allose has various physiological functions such as antioxidation, anti-inflammation, anti-cancer and immunosuppression. It can also reduce cerebral ischemia-reperfusion injury by downregulating galactolectin-3 and inhibiting the AMPK / mTOR pathway.

[0003] Currently, the industrial production of D-allose mainly relies on L-rhamnose isomerase to catalyze the conversion of D-allulose to D-allose. However, the conversion efficiency is low due to the limited catalytic activity of L-rhamnose isomerase. Existing enzyme modification research mostly focuses on improving the activity of L-rhamnose isomerase, neglecting the stability and duration of action of L-rhamnose isomerase in the working environment. L-rhamnose isomerase activity often declines rapidly during catalysis, limiting the yield of D-allose. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an L-rhamnose isomerase and its application in the preparation of D-allose, aiming to improve the catalytic activity of L-rhamnose isomerase while prolonging its half-life, enhance the sustained catalytic ability of L-rhamnose isomerase in the reaction process, and further improve the conversion efficiency.

[0005] In a first aspect, the present invention provides an L-rhamnosyl isomerase, the amino acid sequence of which is obtained by any one of the following mutations of the amino acid sequence shown in SEQ ID NO.1: (1) The amino acid at position 132 is mutated from P to K; (2) The amino acid at position 132 is mutated from P to K and the amino acid at position 258 is mutated from D to Q.

[0006] In a second aspect, the present invention provides a biomaterial comprising any one of the following: (A) A nucleic acid molecule having a gene sequence encoding the above-mentioned L-rhamnosyl isomerase; (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).

[0007] Optionally, the gene sequence encoding the L-rhamnose isomerase mutant P132K is shown in SEQ ID NO.3, and the gene sequence encoding the L-rhamnose isomerase mutant P132K / D258Q is shown in SEQ ID NO.5.

[0008] Thirdly, the present invention provides a method for preparing L-rhamnose isomerase, which is used to prepare the above-mentioned L-rhamnose isomerase by fermenting and culturing a recombinant strain containing the L-rhamnose isomerase gene and inducing the recombinant strain to express L-rhamnose isomerase.

[0009] Furthermore, it includes the following steps: The recombinant strain was seed cultured to obtain a seed solution; The seed culture was inoculated into the fermentation medium at an inoculation rate of 1% to 10% by volume for fermentation culture until the OD 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 recovered. 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 again. The resulting supernatant is the crude enzyme solution of L-rhamnosyl isomerase.

[0010] Compared with the prior art, the fermentation culture of the present invention is a high-density fermentation, which can obtain a high concentration of crude L-rhamnose isomerase solution.

[0011] Furthermore, the seed culture temperature is 35~40℃ and the rotation speed is 120~250 rpm.

[0012] 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.

[0013] Furthermore, the temperature drop is to 30-35℃.

[0014] 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.

[0015] Fourthly, the present invention provides the application of the above-mentioned L-rhamnose isomerase or the above-mentioned biological material in the preparation of D-allose.

[0016] Compared with the prior art, the L-rhamnose isomerase in this invention has higher enzyme activity and a longer half-life. When catalyzing the conversion of D-allulose to D-allose, it has stronger and more sustained catalytic ability, thereby further improving the conversion efficiency and the yield of D-allose.

[0017] Furthermore, the L-rhamnosyl isomerase described above is used to catalyze the reaction of the substrate D-allulose to generate D-allose. The reaction system is prepared as follows: Add crude L-rhamnosyl isomerase solution, metal ions, and D-alulose to a 20-50 mM buffer solution to make the concentration of crude L-rhamnosyl isomerase 10-120 mg / mL, the concentration of metal ions 0.5-2 mM, and the concentration of D-alulose 400-600 g / L in the reaction system.

[0018] Furthermore, the buffer solution includes HEPES buffer, glycine-sodium hydroxide buffer, or sodium carbonate-sodium bicarbonate buffer.

[0019] Furthermore, the metal ions include cobalt ions, manganese ions, or magnesium ions.

[0020] Furthermore, the reaction temperature is 60~80℃, and the pH value of the reaction is 7.0~8.5.

[0021] Compared with the prior art, the present invention mutates wild-type L-rhamnose isomerase by changing amino acid P to K at position 132 of the amino acid sequence shown in SEQ ID NO.1 to obtain L-rhamnose isomerase mutant P132K (amino acid sequence shown in SEQ ID NO.4); and by changing amino acid P to K at position 132 of the amino acid sequence shown in SEQ ID NO.1 and changing amino acid D to Q at position 258 to obtain L-rhamnose isomerase mutant P132K / D258Q (amino acid sequence shown in SEQ ID NO.6). The two L-rhamnose isomerase mutants obtained by the above mutations have higher enzyme activity and longer half-life. When catalyzing the conversion of D-allulose to D-allose, the catalytic ability is stronger and more persistent, effectively improving the conversion efficiency and the yield of D-allose. Attached Figure Description

[0022] Figure 1 The residual enzyme activity of wild-type L-rhamnose isomerase and L-rhamnose isomerase mutant P132K / D258Q in Example 5 after incubation at 65°C for different times.

[0023] Figure 2 The residual enzyme activity of wild-type L-rhamnose isomerase and L-rhamnose isomerase mutant P132K / D258Q in Example 5 after incubation at 70°C for different times. Detailed Implementation

[0024] 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.

[0025] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0026] Example 1 Construction of recombinant plasmid pET-28a(+)-Bs-L-RI Wild-type L-rhamnose isomerase derived from Bacillus subtilis was selected, and its amino acid sequence is shown in SEQ ID NO.1. The gene sequence expressing the wild-type L-rhamnose isomerase was codon-optimized, and the optimized gene sequence of the wild-type L-rhamnose isomerase is shown in SEQ ID NO.2.

[0027] Using the optimized wild-type L-rhamnose isomerase gene as a template, primers F1 and R1 were designed to amplify the optimized wild-type L-rhamnose isomerase gene by PCR, yielding the target gene Bs-L-RI with homologous arms at both ends. The PCR amplification reaction system is shown in Table 1, and the reaction procedure is shown in Table 2.

[0028] F1:5'-atgggtcgcggatccgaattcATGACTATCAAAGCTAATTATGACAGCG-3' (SEQ ID NO. 7).

[0029] R1:5'-gtggtggtggtggtgctcgagTTAAACAATTGGGGAAGACGCT-3' (SEQ ID NO. 8).

[0030] Table 1

[0031] Table 2

[0032] It should be understood that the first step of the PCR reaction, pre-denaturation at 95°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.

[0033] After the PCR amplification was completed, the reaction product was recovered by agarose gel extraction to obtain the target gene fragment Bs-L-RI with high purity.

[0034] The expression vector pET-28a(+) was double-digested with restriction endonucleases EcoRI and XhoI at 37℃ for 20 min. The double-digested products were recovered and purified to obtain the linearized vector pET-28a(+). The double-digestion system is shown in Table 3.

[0035] Table 3

[0036] The target gene fragment Bs-L-RI obtained by PCR amplification was ligated into the linearized vector pET-28a(+) at 37℃ for 20 min to obtain the ligation product. The ligation system is shown in Table 4.

[0037] Table 4

[0038] After the above ligation was completed, the ligation product was transformed into E. coli DH5α competent cells using chemical transformation. Single colonies were picked for plasmid extraction, and the extracted plasmids were sequenced for DNA sequencing. The plasmid with correct sequencing was the recombinant plasmid pET-28a(+)-Bs-L-RI.

[0039] Example 2 Constructing mutant plasmids In some embodiments, using the recombinant plasmid pET-28a(+)-Bs-L-RI as a template, and P132K-F as the upstream primer and P132K-R as the downstream primer, a reverse PCR amplification reaction is performed. After removing the template from the PCR reaction product, self-circularization ligation is performed to obtain a mutant plasmid containing the L-rhamnose isomerase mutant P132K gene, named plasmid 1.

[0040] P132K-F: CaaaACTCTGTTCAGCCACGAGAAAGCTGCTG (SEQ ID NO. 9).

[0041] P132K-R: GGCTGAACAGAGTtttGTTAAAGTCCAGGCCCAGGC (SEQ ID NO. 10).

[0042] The nucleotide sequence of the L-rhamnose isomerase mutant P132K gene in plasmid 1 is shown in SEQ ID NO.3, and the amino acid sequence of the L-rhamnose isomerase mutant P132K encoded by it is shown in SEQ ID NO.4.

[0043] In some embodiments, using plasmid 1 as a template, D258Q-F as the upstream primer and D258Q-R as the downstream primer, a reverse PCR amplification reaction is performed. After removing the template from the PCR reaction product, self-circularization ligation is performed to obtain a mutant plasmid containing the L-rhamnose isomerase mutant P132K / D258Q gene, which is named plasmid 2.

[0044] The nucleotide sequence of the L-rhamnose isomerase mutant P132K / D258Q gene in plasmid 2 is shown in SEQ ID NO.5, and the amino acid sequence of the L-rhamnose isomerase mutant P132K / D258Q encoded by it is shown in SEQ ID NO.6.

[0045] D258Q-F: TGcagACCGGTCACTTTCACCCAACCGAAACC (SEQ ID NO. 11).

[0046] D258Q-R: AAAGTGACCGGTctgCAGCAGACACAGTTTATGGTTGG (SEQ ID NO. 12).

[0047] The reaction systems for each of the above PCR amplification reactions are shown in Table 5, and the reaction procedures are shown in Table 6.

[0048] Table 5

[0049] Table 6

[0050] It should be understood that the pre-denaturation, final extension, and storage steps in the above PCR reaction do not participate in the cycling process, and the entire PCR process is performed only once.

[0051] Template elimination: After each of the above reverse PCR reactions was completed, two reaction solutions were obtained. 1 μL of restriction endonuclease DpnⅠ was added to each of the two reaction solutions (25 μL), and the mixture was gently blown and aspirated. The solutions were then reacted at 37 °C for 1 h to obtain two enzyme digestion solutions. The enzyme digestion solutions were verified by agarose gel electrophoresis.

[0052] Self-circularization of reverse PCR products: Using the two verified enzyme digestion solutions obtained above, reaction solutions were prepared according to Table 7, gently mixed, and reacted at 16℃ for 1 h to obtain plasmid 1 and plasmid 2, respectively.

[0053] Table 7

[0054] Example 3 Preparation of crude enzyme solution The recombinant plasmid pET-28a(+)-Bs-L-RI successfully constructed in Example 1, and plasmids 1 and 2 successfully constructed in Example 2, were chemically transformed into E. coli competent cells BL21(DE3). The cells were then plated on LB plates containing kanamycin (50 μg / mL) and cultured overnight at 37°C to obtain the recombinant strain E. coli BL21-pET-28a(+)-Bs-L-RI (containing recombinant plasmid pET-28a(+)-L-RI), the mutant strain E. coli BL21-1 (containing plasmid 1), and the mutant strain E. coli BL21-2 (containing plasmid 2).

[0055] Single colonies of the recombinant strain E. coli BL21-pET-28a(+)-Bs-L-RI, the mutant strain E. coli BL21-1, and the mutant strain E. coli BL21-2 were picked and inoculated into culture medium for seed culture. The seed culture medium could be LB liquid medium containing kanamycin (50 μg / mL). The culture was carried out at 37℃ and 150 rpm for 8 h with shaking to obtain seed cultures of the recombinant strain E. coli BL21-pET-28a(+)-Bs-L-RI, the mutant strain E. coli BL21-1, and the mutant strain E. coli BL21-2, respectively.

[0056] The three seed solutions were inoculated at a volume ratio of 2% into separate culture media for fermentation. The fermentation medium could be LB liquid medium containing kanamycin (50 μg / mL). The cultures were incubated at 37°C with shaking at 220 rpm until OD reached. 600 The concentration reached 0.6. Then, induction cultures were performed separately under the following conditions: IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.5 mM, and the cultures were induced at 20°C for 16 h, yielding fermentation broths containing wild-type L-rhamnose isomerase, L-rhamnose isomerase mutant P132K, and L-rhamnose isomerase mutant P132K / D258Q, respectively.

[0057] The three fermentation broths were centrifuged at 8000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were collected separately. The bacterial cells were resuspended in 100 mM PBS buffer (pH 7.5), and then the cells were sonicated at 200 W for 3 seconds at 2-second intervals, for a total sonication time of 20 minutes. After sonication, the broths were centrifuged at 12000 rpm for 60 min to obtain three supernatants: crude enzyme solution of wild-type L-rhamnose isomerase, crude enzyme solution of L-rhamnose isomerase mutant P132K, and crude enzyme solution of L-rhamnose isomerase mutant P132K / D258Q.

[0058] Example 4 Measurement of relative enzyme activity Enzyme activity (U): The amount of enzyme required to catalyze the synthesis of 1 μmol of D-allose per unit time (min) under standard reaction conditions.

[0059] Enzyme activity assay method: In the following standard reaction system, react at 70℃ for 30 min, and then boil for 10 min to terminate the reaction.

[0060] Standard reaction system 1: 50 mM HEPES buffer (pH 8.0), 1 mM manganese chloride, 20 mM D-allulose, and the crude wild-type L-rhamnose isomerase prepared in Example 3 were added to make the final concentration of the crude wild-type L-rhamnose isomerase 0.5 mg / mL.

[0061] Standard reaction system 2: 50 mM HEPES buffer (pH 8.0), 1 mM manganese chloride, 20 mM D-allulose, and the crude enzyme solution of L-rhamnose isomerase mutant P132K prepared in Example 3 were added to make the final concentration of crude enzyme of L-rhamnose isomerase mutant P132K 0.5 mg / mL.

[0062] Standard reaction system 3: 50 mM HEPES buffer (pH 8.0), 1 mM manganese chloride, 20 mM D-allulose, and the crude enzyme solution of L-rhamnose isomerase mutant P132K / D258Q prepared in Example 3 were added to make the final concentration of crude enzyme of L-rhamnose isomerase mutant P132K / D258Q 0.5 mg / mL.

[0063] After each of the above reactions was completed, the reaction solutions were centrifuged at 12,000 rpm for 10 min, and the supernatants were collected, diluted 10 times, and filtered through a 0.22 μm microporous membrane. The D-allose content was then detected by HPLC.

[0064] 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.

[0065] With the wild-type enzyme activity as 100%, the relative enzyme activity of each mutant was calculated, and the results are shown in Table 8.

[0066] Table 8

[0067] The results above show that, compared with wild-type L-rhamnose isomerase, the enzyme activities of the L-rhamnose isomerase mutants P132K and P132K / D258Q obtained by mutation in this invention are significantly improved.

[0068] Example 5 Determining half-life The crude enzyme solution of wild-type L-rhamnosyl isomerase prepared in Example 3 above was incubated at 65℃ and 70℃ for 2h, 4h, 6h, 8h, 10h and 12h respectively. Then, the enzyme activity of the crude enzyme solution of wild-type L-rhamnosyl isomerase after incubation at different temperatures and times was measured according to the method in Example 4 above. The enzyme activity after 0h incubation was taken as 100%. The residual enzyme activity of wild-type L-rhamnosyl isomerase after incubation at different times was calculated, and the half-life of wild-type L-rhamnosyl isomerase at 65℃ and 70℃ was obtained respectively.

[0069] The crude enzyme solution of L-rhamnosyl isomerase mutant P132K / D258Q prepared in Example 3 was incubated at 65℃ and 70℃ for 2h, 4h, 6h, 8h, 10h, and 12h, respectively. Then, the enzyme activity of the crude enzyme solution of L-rhamnosyl isomerase mutant P132K / D258Q after incubation at different temperatures and times was measured according to the method in Example 4. The enzyme activity after 0h incubation was taken as 100%, and the residual enzyme activity of L-rhamnosyl isomerase mutant P132K / D258Q after incubation at different times was calculated. The half-life of L-rhamnosyl isomerase mutant P132K / D258Q at 65℃ and 70℃ was obtained.

[0070] The results are as follows Figure 1 , Figure 2 As shown: by Figure 1 It can be seen that, compared with the wild-type L-rhamnose isomerase, the half-life of the L-rhamnose isomerase mutant P132K / D258Q at 65℃ is extended from 6.0 h to 9.1 h. Figure 2It can be seen that, compared with the wild-type L-rhamnose isomerase, the half-life of the L-rhamnose isomerase mutant P132K / D258Q at 70℃ is extended from the original 4.0h to 5.1h.

[0071] The crude enzyme solution of L-rhamnosyl isomerase mutant P132K prepared in Example 3 above was incubated at 65℃ and 70℃ for 2h, 4h, 6h, 8h, 10h and 12h respectively. Then, the enzyme activity of the crude enzyme solution of L-rhamnosyl isomerase mutant P132K after incubation at different temperatures and times was measured according to the method in Example 4 above. The enzyme activity after 0h incubation was taken as 100%. The residual enzyme activity of L-rhamnosyl isomerase mutant P132K after incubation at different times was calculated, and the half-life of L-rhamnosyl isomerase mutant P132K at 65℃ and 70℃ was obtained respectively.

[0072] The results showed that, compared with the wild-type L-rhamnosyl isomerase, the half-life of the L-rhamnosyl isomerase mutant P132K at 65℃ was extended from 6.0h to 7.0h, and the half-life at 70℃ was extended from 4.0h to 4.5h.

[0073] Example 6 High-density fermentation for the preparation of crude enzyme solution Single colonies of the recombinant strain E. coli BL21-pET-28a(+)-Bs-L-RI, the mutant strain E. coli BL21-1, and the mutant strain E. coli BL21-2 constructed in Example 3 were picked and inoculated into a culture medium for seed culture. The seed culture medium can be LB liquid medium containing kanamycin (50 μg / mL), and cultured with shaking at 37°C and 150 rpm until OD. 600 The value was increased to 1.5, and seed cultures of recombinant strain E. coli BL21-pET-28a(+)-Bs-L-RI, mutant strain E. coli BL21-1, and mutant strain E. coli BL21-2 were obtained respectively.

[0074] The three seed solutions were inoculated into fermenters at a volume ratio of 5% and cultured under the conditions of 0.04 MPa, initial rotation speed of 300 rpm, temperature of 37°C, and pH of 7.0 until OD500. 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 between 20% and 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 P132K, and L-rhamnose isomerase mutant P132K / D258Q, respectively.

[0075] The three fermentation broths were centrifuged at 8000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were collected. The cells were resuspended in 100 mM PBS buffer (pH 7.5), and then the cells were sonicated at 200 W for 3 seconds at 2-second intervals for a total sonication time of 20 minutes. After sonication, the cells 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 P132K, and crude enzyme solution of L-rhamnose isomerase mutant P132K / D258Q.

[0076] 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.

[0077] The above-mentioned feed culture medium consists of 500 g / L glucose, 2 g / L magnesium sulfate heptahydrate, and 2 g / L yeast extract.

[0078] Example 7 Preparation of D-allose Configure the reaction system: D-alulose substrate, MnCl2, and crude wild-type L-rhamnose isomerase prepared in Example 6 were added to a 50 mM HEPES buffer with a pH of 8.0 to make the concentration of D-alulose 500 g / L, the concentration of MnCl2 1 mM, and the concentration of crude wild-type L-rhamnose isomerase 80 mg / mL in the reaction system.

[0079] D-allulose substrate, MnCl2, and crude enzyme solution of L-rhamnose isomerase mutant P132K prepared in Example 6 were added to 50 mM HEPES buffer with pH 8.0, so that the concentration of D-allulose in the reaction system was 500 g / L, the concentration of MnCl2 was 1 mM, and the concentration of crude enzyme of L-rhamnose isomerase mutant P132K was 80 mg / mL.

[0080] D-allulose substrate, MnCl2, and crude enzyme solution of L-rhamnose isomerase mutant P132K / D258Q prepared in Example 6 were added to a 50 mM HEPES buffer with a pH of 8.0, so that the concentration of D-allulose in the reaction system was 500 g / L, the concentration of MnCl2 was 1 mM, and the concentration of crude enzyme of L-rhamnose isomerase mutant P132K / D258Q was 80 mg / mL.

[0081] Each of the above reaction systems was reacted at 70℃ for 12 h. After the reaction was completed, each reaction solution was centrifuged at 12000 rpm for 10 min. The supernatant was collected, diluted 10 times, and filtered through a 0.22 μm microporous membrane. The content of D-allose was then detected by HPLC, and the conversion rate of the substrate D-allose was calculated. The results are shown in Table 9.

[0082] 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.

[0083] Conversion rate: D-allose content after the reaction ÷ D-allose content at the beginning of the reaction × 100%.

[0084] Table 9

[0085] The results above show that, compared with wild-type L-rhamnose isomerase, the L-rhamnose isomerase mutants P132K and P132K / D258Q in this invention further improved the conversion rate and the yield of D-allose by increasing enzyme activity and prolonging half-life.

[0086] 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, characterized in that, The amino acids of the L-rhamnosyl isomerase are obtained by any one of the following mutations of the amino acid sequence shown in SEQ ID NO.1: (1) The amino acid at position 132 is mutated from P to K; (2) The amino acid at position 132 is mutated from P to K and the amino acid at position 258 is mutated from D to Q.

2. A biomaterial, characterized in that, Includes any one of the following: (A) A nucleic acid molecule having a gene sequence encoding the 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, characterized in that, To prepare the L-rhamnose isomerase according to claim 1, a recombinant strain containing the L-rhamnose isomerase gene is fermented and cultured, and the recombinant strain is 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 recovered. 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 L-rhamnose isomerase of claim 1 or the biomaterial of claim 2 in the preparation of D-allose.

7. The application according to claim 6, characterized in that, The L-rhamnosyl isomerase catalyzes the reaction of substrate D-allulose to D-allose, and the reaction system is prepared as follows: Add crude L-rhamnosyl isomerase solution, metal ions, and D-alulose to a 20-50 mM buffer solution to make the concentration of crude L-rhamnosyl isomerase 10-120 mg / mL, the concentration of metal ions 0.5-2 mM, and the concentration of D-alulose 400-600 g / L in the reaction system.

8. The application according to claim 7, characterized in that, The buffer solution includes HEPES buffer, glycine-sodium hydroxide buffer, or sodium carbonate-sodium bicarbonate buffer.

9. The application according to claim 7, characterized in that, The metal ions include cobalt ions, manganese ions, or magnesium ions.

10. The application according to claim 7, characterized in that, The reaction temperature is 60~80℃, and the pH value is 7.0~8.5.

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