L-rhamnose isomerase mutant, recombinant plasmid, mutant strain and application

Abstract

The invention relates to the technical field of gene engineering, in particular to an L-rhamnose isomerase mutant, a recombinant plasmid, a mutant strain and application. The amino acid sequence of the mutant is as shown in SEQ ID NO.4, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.3. A specific mutant gene sequence is designed, a recombinant plasmid pET28a (+)-L-Rhi is successfully constructed, the gene sequence is connected between enzyme cutting sites BamH I and Hind III of an escherichia coli expression vector pET-28a (+) to construct a mutant expression vector (the recombinant plasmid pET-28a (+)-L-Rhi), the mutant expression vector is transformed into an escherichia coli BL21 competent cell, a mutant strain is obtained, and the recombinant plasmid pET-28a (+)-L-Rhi is obtained. The obtained mutant strain can overexpress L-rhamnose isomerase, the conversion rate of D-allose is remarkably improved, the thermal stability of the enzyme is remarkably improved through the design of mutation sites, and the half-life period is effectively prolonged.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more particularly to an L-rhamnose isomerase mutant, recombinant plasmid, mutant strain, and its applications. This application is a divisional application of patent application 2025110899820, filed on August 5, 2025, entitled "L-rhamnose isomerase mutant, recombinant plasmid, mutant strain, and its applications". Background Technology

[0002] D-Allose, a functional rare sugar, has significant application value in the fields of food, medicine, and biomaterials. Currently, the biosynthetic pathway using D-glucose as a starting material is one of the emerging directions for the industrial production of D-Allose. Using enzymes as catalysts results in fewer byproducts, making it more conducive to industrial production of D-Allose. Existing enzyme-catalyzed methods for producing D-Allose mainly involve three enzymatic reactions: the first step uses inexpensive D-glucose as a starting material to form D-fructose under the catalysis of xylose isomerase; the second step uses D-fructose as a substrate, and under the influence of the D-tagatose-3-epimerase family, epimerization occurs at the C3 position of D-fructose to obtain D-allulose; the third step uses D-allulose as a substrate, and under the catalysis of aldose isomerase, it is converted into the target product, D-Allose. However, existing synthesis methods require multiple enzymatic catalytic reactions, which are cumbersome and affected by the catalytic efficiency of various enzymes. Furthermore, existing enzymes have low conversion rates for D-glucose, D-fructose, or D-allulose and poor thermal stability, which limits the industrial production of D-allose.

[0003] Chinese invention patent application CN117384985A discloses a method for synthesizing D-ribose and D-allose using whole-cell catalysis with D-xylose and D-glucose, respectively. This method involves constructing a recombinant *E. coli* engineered strain and using D-glucose isomerase and D-allulose-3-epimerase for whole-cell catalysis, achieving inexpensive and environmentally friendly industrial production. However, the conversion rate of D-allose synthesized by this technology is only 8.37%, which still cannot meet the requirements for industrial production.

[0004] Against this backdrop, it is imperative to provide an isomerase with high conversion rate and good thermal stability to achieve efficient conversion and industrial production of D-allose. Summary of the Invention

[0005] To address the technical problems of existing D-allose enzymes requiring multiple catalytic steps, resulting in cumbersome processes and low conversion rates, as well as the poor heat resistance of existing isomerases, this invention provides an L-rhamnose isomerase mutant, recombinant plasmid, mutant strain, and its applications. A mutant with high catalytic activity and high heat resistance is constructed. The resulting mutant strain overexpresses L-rhamnose isomerase, significantly improving the conversion rate of D-allose, and exhibits a long half-life and good thermal stability at high temperatures.

[0006] The first aspect of the present invention provides an L-rhamnose isomerase mutant, the amino acid sequence of which is shown in SEQ ID NO.4 and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3; the mutant is obtained by mutating the 281st amino acid of L-rhamnose isomerase from H to R, specifically the mutant H281R.

[0007] A second aspect of the present invention provides a recombinant plasmid, which is obtained by inserting the coding gene of the mutant described above into a plasmid vector.

[0008] Optionally, the plasmid vector is pET-28a(+).

[0009] Optionally, the preparation steps of the recombinant plasmid include: ligating the gene sequence between the restriction sites BamHI and HindIII of the plasmid vector pET-28a(+) to complete the construction of the recombinant plasmid pET-28a(+)-L-Rhi.

[0010] This invention utilizes the L-Rhi amino acid sequence (PDB: 2i57) from the PDB (Protein Database) to optimize the codons of the nucleotide sequence derived from *Pseudomonas stutzeri* and synthesize the target gene (corresponding to the wild-type plasmid template). Based on the wild-type plasmid template, mutant primers were designed, and the mutant was obtained through site-directed mutagenesis PCR amplification. After extensive experimentation, it was found that when the 281st amino acid of the L-rhamnose isomerase was mutated from H to R, the resulting mutant (H281R) exhibited high catalytic activity in the preparation of D-allose, significantly improving the conversion rate of D-allose, and possessing reliable thermal stability, while also extending the half-life of the isomerase.

[0011] A third aspect of the present invention provides a mutant strain, comprising constructing a mutant strain by transforming the recombinant plasmid into the original bacteria.

[0012] Optionally, the recombinant plasmid can be transformed into Escherichia coli BL21 using a chemical transformation method to obtain a mutant strain.

[0013] This invention does not impose particular limitations on the operational details of the chemical transformation method; any basic method that is operable in the field and can achieve the cultivation objective is acceptable.

[0014] Optionally, the original bacterium is *Escherichia coli* BL21, and the mutant strain is... E. coli BL21-pET28a(+)-L-Rhi.

[0015] The fourth aspect of the present invention provides an enzyme solution of L-rhamnose isomerase mutant, which is obtained by inducing culture of mutant strains to obtain heterologously expressed L-rhamnose isomerase mutant enzyme solution.

[0016] Optionally, the induction culture step specifically includes: placing the mutant strain in a shaking culture at 30-40℃ and 100-300rpm, shaking culture until the OD value (Optical Density) is 0.6-0.8, adding an inducer, inducing culture at 15-25℃ for 10-20h, collecting the bacterial cells by centrifugation, resuspending them in buffer, then sonicating and centrifuging, collecting the supernatant, and purifying to obtain the L-rhamnosyl isomerase mutant enzyme solution.

[0017] Alternatively, the mutant strain can be cultured with shaking at 37°C and 120 rpm.

[0018] Alternatively, the cells can be induced and cultured at 18-20°C for 12-16 hours.

[0019] Alternatively, the buffer solution may be PBS (phosphate) buffer.

[0020] Optionally, the purification method is as follows: the supernatant is purified and eluted using a nickel column, and the purified product is an L-rhamnosyl isomerase mutant enzyme solution.

[0021] Optionally, the inducing agent is IPTG (isopropyl-β-D-thiogalactoside), and the final concentration of the inducing agent after addition is 0.1-0.5 mM; more preferably, it is 0.2 mM.

[0022] The sixth aspect of this invention provides the application of the L-rhamnose isomerase mutant enzyme solution as described above in the preparation of D-allose; the method for preparing D-allose includes: The L-rhamnose isomerase mutant enzyme solution, D-allulose, and metal salt solution were sequentially added to an alkaline buffer solution to carry out a catalytic reaction. The reaction was then terminated by boiling in water to obtain a reaction solution containing D-allulose.

[0023] In some embodiments, the metal salt may include zinc chloride, ferrous sulfate, nickel chloride, cobalt chloride, copper sulfate, etc.

[0024] To improve the conversion rate of D-allose, the metal salt may optionally be zinc chloride.

[0025] Optionally, the final concentration of the metal salt solution after addition is 0.5-5 mM, and more preferably 1 mM.

[0026] Optionally, the final concentration of the added D-allulose is 1-50 g / L, and more preferably 10 g / L.

[0027] Optionally, the final concentration of the L-rhamnosyl isomerase mutant enzyme solution after addition is 1-10 mg / mL; more preferably, it is 2 mg / mL.

[0028] In some embodiments, the alkaline buffer solution may include, for example, glycine-sodium hydroxide buffer solution, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, etc.

[0029] Optionally, the pH of the alkaline buffer solution is 7.5-10, and may include 7.5, 8.0, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 10, and may further be 9.

[0030] To improve the conversion rate of D-allose, the alkaline buffer solution may optionally be a glycine-sodium hydroxide buffer solution.

[0031] In some embodiments, the catalytic reaction is carried out at 50-70°C for 15-60 min; to improve the conversion rate of D-allose, it can be further selected to carry out the catalytic reaction at 60°C for 30 min.

[0032] Optionally, the boiling time of the water is 5-15 minutes; more preferably 8-10 minutes.

[0033] This invention further optimizes the preparation process of D-allose, discovering that in addition to the expression effect of the mutant, factors such as the choice of alkaline buffer solution, the choice of metal salt, and the catalytic reaction time all affect the final D-allose conversion rate. Experimental investigation revealed that when the alkaline buffer solution is a glycine-sodium hydroxide buffer solution with pH=9, the metal salt is zinc chloride, and the catalytic reaction temperature is 60℃, the compatibility with L-rhamnosyl isomerase is good, resulting in the highest final D-allose conversion rate.

[0034] Beneficial effects This invention provides an L-rhamnose isomerase mutant, a recombinant plasmid, a mutant strain, and its applications, which have the following advantages: (1) Based on the L-Rhi amino acid sequence (PDB: 2i57) in the PDB (Protein Database), the present invention optimizes the codons of the nucleotide sequence derived from Pseudomonas stutzeri to obtain a wild-type plasmid template. Based on the wild-type template, mutant primers are designed to carry out PCR reaction to obtain mutants. After experimentation, it was found that when the 281st amino acid of L-rhamnose isomerase is mutated from H to R to obtain the mutant (H281R), the obtained mutant exhibits excellent heterologous expression and catalytic activity, and significantly improves the product conversion rate when applied to the preparation of D-allose.

[0035] (2) The present invention further discovers that mutants obtained by site-directed mutagenesis using specific mutagenesis primers not only exhibit high conversion rates but also have reliable thermal stability.

[0036] (3) In this invention, the reaction product after PCR amplification is ligated between the restriction sites BamHI and HindIII of the Escherichia coli expression vector pET-28a(+), and the mutant expression vector (recombinant plasmid pET-28a(+)-L-Rhi) is successfully constructed.

[0037] (4) In this invention, the mutant expression vector was further transformed into Escherichia coli competent cells BL21 by chemical transformation to obtain the mutant strain ( E. coli The mutant strain BL21-pET28a(+)-L-Rhi was able to overexpress L-rhamnose isomerase, which significantly improved the conversion rate of D-allose.

[0038] (5) The process of the present invention is simple and does not require multi-step enzymatic hydrolysis. It only requires the application of a specific L-rhamnose isomerase mutant to the preparation of D-allose, and the substrate D-allulose is reacted under mild catalytic reaction conditions to obtain a high content of D-allose. The method of the present invention can significantly increase the yield of D-allose and realize the industrial production of D-allose. It has extremely high practical value and broad application prospects. Attached Figure Description

[0039] Figure 1 The residual enzyme activity assay results of the L-rhamnose isomerase mutant enzyme solutions of Examples 1-5 and the wild-type L-rhamnose isomerase of Control Example 1 at 65°C; Figure 2 The residual enzyme activity assay results at 70°C for the L-rhamnose isomerase mutant enzyme solutions of Examples 1-5 and the wild-type L-rhamnose isomerase of Control Example 1. Detailed Implementation

[0040] The original strain *Escherichia coli* BL21 and the plasmid vector pET-28a(+) used in the embodiments of this invention were obtained from Shandong Ruiheying Biotechnology Co., Ltd., and the wild-type L-rhamnosyl isomerase was synthesized by Nanjing Novizan Biotechnology Co., Ltd. (the L-Rhi amino acid sequence was obtained according to PDB:2i57, and the codons of the nucleotide sequence from *Pseudomonas stutzeri* were optimized to obtain the target gene sequence (i.e., the wild-type plasmid template, corresponding to control example 1), which was artificially synthesized by Nanjing Novizan Biotechnology Co., Ltd.).

[0041] Note: Unless otherwise specified, the solvents used in the solutions involved in this invention are all water; all concentrations mentioned are mass concentrations; and all raw materials used are commercially available.

[0042] Example Example 1 This embodiment provides an L-rhamnose isomerase mutant, recombinant plasmid, mutant strain and its application, wherein the L-rhamnose isomerase mutant is H281R.

[0043] The amino acid sequence of the mutant is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3.

[0044] The L-rhamnosyl isomerase mutant was obtained by designing mutant primers based on wild-type plasmid templates (see Table 1) and performing site-directed mutagenesis PCR amplification.

[0045] The recombinant plasmid contains the coding gene of the mutant as described above. The preparation method of the recombinant plasmid includes: ligating the H281R nucleotide sequence between the restriction sites BamHI and HindIII of the plasmid vector pET-28a(+) to complete the construction of the recombinant plasmid pET-28a(+)-L-Rhi. The mutant strain contains the recombinant plasmid as described above, and the method for preparing the mutant strain includes: The recombinant plasmid was transformed into the original strain *Escherichia coli* BL21 using a chemical transformation method to construct a mutant strain. E. coli BL21-pET28a(+)-L-Rhi); the specific operation is as follows: S1. Thaw E. coli BL21 competent cells on ice, add the recombinant plasmid into the competent cells, gently tap the tube wall to mix, and place on ice for 30 min; S2. Heat shock at 42℃, then incubate on ice for 2 min; S3. Add antibiotic-free LB medium and resuscitate at 37°C; S4. After centrifugation, discard part of the supernatant, resuspend, plate, and incubate overnight for 12 hours to obtain the mutant strain.

[0046] The mutant strain was cultured at 37℃ and 120 rpm with shaking. When the OD value reached 0.7±0.1, IPTG inducer was added to a final concentration of 0.2 mM. The culture was induced at 18℃ for 14 h. After centrifugation, the bacterial cells were collected and resuspended in PBS buffer. The cells were then sonicated, centrifuged, and the supernatant was collected. The supernatant was purified and eluted using a nickel column to obtain the heterologous expression of L-rhamnosyl isomerase mutant enzyme solution.

[0047] The L-rhamnose isomerase mutant enzyme solution is used to prepare D-allose, and the preparation steps of D-allose include: The L-rhamnosyl isomerase mutant enzyme solution with a final concentration of 2 mg / mL was added to an alkaline buffer solution, followed by the addition of a metal salt solution (final concentration of 1 mM) and D-allulose (final concentration of 10 g / L) to catalyze the reaction (final volume of the reaction system was 10 mL). The reaction was then terminated by boiling in water for 9 ± 1 min to obtain a reaction solution containing D-allose.

[0048] The alkaline buffer solution is a glycine-sodium hydroxide buffer solution with a pH of 9.0.

[0049] The metal salt is zinc chloride.

[0050] The conditions for the catalytic reaction are: catalytic reaction at 60℃ for 30 min.

[0051] Example 2 This embodiment provides an L-rhamnose isomerase mutant, recombinant plasmid, mutant strain, and application, with the specific implementation method being the same as in Embodiment 1; the difference is that the L-rhamnose isomerase mutant is H257R; the amino acid sequence of the mutant is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0052] Example 3 This embodiment provides an L-rhamnose isomerase mutant, recombinant plasmid, mutant strain, and application. The specific implementation method is the same as in Embodiment 1; the difference is that the L-rhamnose isomerase mutant is H257K; the amino acid sequence of the mutant is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5.

[0053] Example 4 This embodiment provides an L-rhamnose isomerase mutant, recombinant plasmid, mutant strain, and application, with the specific implementation method being the same as in Embodiment 1; the difference is that the L-rhamnose isomerase mutant is H281K; the amino acid sequence of the mutant is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7.

[0054] Example 5 This embodiment provides an L-rhamnose isomerase mutant, recombinant plasmid, mutant strain, and application, with the specific implementation method being the same as in Embodiment 1; the difference is that the L-rhamnose isomerase mutant is H257R / H281R; the amino acid sequence of the mutant is shown in SEQ ID NO.10, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.9.

[0055] Example 6 This embodiment provides an L-rhamnose isomerase mutant, recombinant plasmid, mutant strain, and application, with the specific implementation method being the same as in Embodiment 1; the difference is that the L-rhamnose isomerase mutant is A259V; the amino acid sequence of the mutant is shown in SEQ ID NO.12, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.11.

[0056] Example 7 This embodiment provides an L-rhamnose isomerase mutant, recombinant plasmid, mutant strain, and application, with the specific implementation method being the same as in Embodiment 1; the difference is that the L-rhamnose isomerase mutant is A259G; the amino acid sequence of the mutant is shown in SEQ ID NO.14, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.13.

[0057] Example 8 This embodiment provides an L-rhamnose isomerase mutant, recombinant plasmid, mutant strain, and application, with the specific implementation method being the same as in Embodiment 1; the difference is that the L-rhamnose isomerase mutant is A259Y; the amino acid sequence of the mutant is shown in SEQ ID NO.16, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.15.

[0058] Example 9 This embodiment provides an L-rhamnose isomerase mutant, recombinant plasmid, mutant strain, and application, with the specific implementation method being the same as in Embodiment 1; the difference is that the L-rhamnose isomerase mutant is H220K; the amino acid sequence of the mutant is shown in SEQ ID NO.18, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.17.

[0059] Example 10 This embodiment provides an L-rhamnose isomerase mutant, recombinant plasmid, mutant strain, and application, with the specific implementation method being the same as in Embodiment 1; the difference is that the L-rhamnose isomerase mutant is H220R; the amino acid sequence of the mutant is shown in SEQ ID NO.20, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.19.

[0060] The information on the mutant primers used for each mutant in Examples 1-10 is shown in Table 1. The PCR amplification conditions were as follows: pre-denaturation at 95°C for 1 minute, denaturation at 95°C for 15 seconds, annealing at 57°C for 15 seconds, extension at 72°C for 60 seconds, and final extension for 10 minutes, with each of the denaturation, annealing, and extension cycles performed 30 times.

[0061] Table 1

[0062] Table 2 summarizes the mutation sites of the amino acid sequences of each mutant in Examples 1-10.

[0063] Table 2

[0064] Compare with Example 1 This comparative example provides a wild-type L-rhamnose isomerase and applies it to the preparation of D-allose; the amino acid sequence of the wild-type L-rhamnose isomerase is shown in SEQ ID NO.22, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.21; the steps for preparing D-allose are the same as in Example 1.

[0065] Compare with Example 2 This comparative example provides a wild-type L-rhamnose isomerase and applies it to the preparation of D-allose; the gene sequence of the wild-type L-rhamnose isomerase is the same as that of Comparative Example 1, and the steps for preparing D-allose are the same as those of Example 1; the difference is that the alkaline buffer solution is a potassium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with pH=9.0.

[0066] Compare with Example 3 This comparative example provides a wild-type L-rhamnose isomerase and applies it to the preparation of D-allose; the gene sequence of the wild-type L-rhamnose isomerase is the same as that of Comparative Example 1, and the steps for preparing D-allose are the same as those of Example 1; the difference is that the metal salt is ferrous sulfate.

[0067] Compare with Example 4 This comparative example provides a wild-type L-rhamnose isomerase and applies it to the preparation of D-allose; the gene sequence of the wild-type L-rhamnose isomerase is the same as that of Comparative Example 1, and the steps for preparing D-allose are the same as those of Example 1; the difference is that the metal salt is nickel chloride.

[0068] Compare with Example 5 This comparative example provides a wild-type L-rhamnose isomerase and applies it to the preparation of D-allose; the gene sequence of the wild-type L-rhamnose isomerase is the same as that of Comparative Example 1, and the steps for preparing D-allose are the same as those of Example 1; the difference is that the metal salt is cobalt chloride.

[0069] Compare with Example 6 This comparative example provides a wild-type L-rhamnose isomerase and applies it to the preparation of D-allose; the gene sequence of the wild-type L-rhamnose isomerase is the same as that of Comparative Example 1, and the steps for preparing D-allose are the same as those of Example 1; the difference is that the metal salt is copper sulfate.

[0070] Compare with Example 7 This comparative example provides a wild-type L-rhamnose isomerase and applies it to the preparation of D-allose; the gene sequence of the wild-type L-rhamnose isomerase is the same as that of Comparative Example 1, and the steps for preparing D-allose are the same as those of Example 1; the difference is that the catalytic reaction conditions are: catalytic reaction at 50°C for 30 min.

[0071] Compare with Example 8 This comparative example provides a wild-type L-rhamnose isomerase and applies it to the preparation of D-allose; the gene sequence of the wild-type L-rhamnose isomerase is the same as that of Comparative Example 1, and the steps for preparing D-allose are the same as those of Example 1; the difference is that the catalytic reaction conditions are: catalytic reaction at 55°C for 30 min.

[0072] Compare with Example 9 This comparative example provides a wild-type L-rhamnose isomerase and applies it to the preparation of D-allose; the gene sequence of the wild-type L-rhamnose isomerase is the same as that of Comparative Example 1, and the steps for preparing D-allose are the same as those of Example 1; the difference is that the catalytic reaction conditions are: catalytic reaction at 65°C for 30 min.

[0073] Compare with Example 10 This comparative example provides a wild-type L-rhamnose isomerase and applies it to the preparation of D-allose; the gene sequence of the wild-type L-rhamnose isomerase is the same as that of Comparative Example 1, and the steps for preparing D-allose are the same as those of Example 1; the difference is that the catalytic reaction conditions are: catalytic reaction at 70°C for 30 min.

[0074] Compare with Example 11 This comparative example provides a wild-type L-rhamnose isomerase and applies it to the preparation of D-allose; the gene sequence of the wild-type L-rhamnose isomerase is the same as that of Comparative Example 1, and the steps for preparing D-allose are the same as those of Example 1; the difference is that the catalytic reaction conditions are: catalytic reaction at 75°C for 30 min.

[0075] Performance testing 1. Conversion rate The reaction solutions finally prepared in Examples 1-10 and Control Examples 1-11 were used to determine the contents of D-allulose and D-allose in the reaction solutions and to calculate the conversion rate. The results are shown in Table 3.

[0076] The conversion rate is calculated as follows: (D-allose content in the reaction solution / D-allulose content in the initial substrate) × 100%.

[0077] 2. Thermal stability The L-rhamnose isomerase mutant enzyme solutions from Examples 1-5 and the wild-type L-rhamnose isomerase from Control Example 1 (sample labeled as wild-type) were incubated at 65°C or 70°C for different times (2, 4, 6, 8, 10, 12 h). The residual enzyme activity of each mutant and wild-type control sample in the reaction system after incubation for different times was measured (see...). Figure 1 and Figure 2 The half-lives at 65°C and 70°C were obtained and the data are recorded in Table 3. The conversion rates of the samples in Examples 6-10 and the control were low, so the half-lives were not determined.

[0078] Table 3

[0079] As can be seen from the data in Table 3, the mutants obtained by site-directed mutagenesis design in this invention can improve the catalytic conversion efficiency of L-rhamnosyl isomerase for D-allulose. In particular, the conversion rate of mutant H281R is as high as 45.32%, which is significantly higher than that of wild-type isomerase or other mutants. Moreover, the mutants have reliable thermal stability and effectively prolong the half-life of rhamnosyl isomerase. These results prove that this invention has successfully screened mutants with high catalytic activity and high heat resistance.

[0080] Further investigation revealed that, in addition to the mutant's effect on L-rhamnosyl isomerase expression, factors such as the choice of alkaline buffer solution, the choice of metal salt, and the reaction time all affected the final D-allose conversion rate. Comparative analysis showed that when the alkaline buffer solution was a glycine-sodium hydroxide buffer solution with pH=9, the metal salt was zinc chloride, and the reaction temperature was 60℃, the compatibility with L-rhamnosyl isomerase was better, resulting in the highest D-allose conversion rate.

Claims

1. An L-rhamnosyl isomerase mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

3.

2. A recombinant plasmid, characterized in that, A recombinant plasmid is obtained by inserting the coding gene of the mutant described in claim 1 into a plasmid vector.

3. A mutant strain, characterized in that, The recombinant plasmid described in claim 2 was transformed into the original bacteria to construct a mutant strain; The original bacterium was Escherichia coli BL21.

4. An enzyme solution containing an L-rhamnosyl isomerase mutant, characterized in that, The mutant strain described in claim 3 was induced to produce an L-rhamnosyl isomerase mutant enzyme solution.

5. The L-rhamnosyl isomerase mutant enzyme solution according to claim 4, characterized in that, The induction culture step specifically includes: placing the mutant strain in a shaking culture at 30-40℃ and 100-300rpm, adding an inducer when the OD value reaches 0.6-0.8, inducing culture at 15-25℃ for 10-20h, collecting the bacterial cells by centrifugation, resuspending them in buffer, then sonicating and centrifuging, collecting the supernatant, and purifying to obtain the L-rhamnosyl isomerase mutant.

6. The L-rhamnosyl isomerase mutant enzyme solution according to claim 5, characterized in that, The purification method is as follows: the supernatant is purified and eluted using a nickel column.

7. The application of an L-rhamnosyl isomerase mutant enzyme solution according to any one of claims 4-6 in the preparation of D-allose; characterized in that, The method for preparing the D-allose includes: The L-rhamnose isomerase mutant enzyme solution, D-allulose, and metal salt solution were sequentially added to an alkaline buffer solution to carry out a catalytic reaction. The reaction was then terminated by boiling in water to obtain a reaction solution containing D-allulose.

8. The application according to claim 7, characterized in that, The alkaline buffer solution is a glycine-sodium hydroxide buffer solution; the metal salt is zinc chloride.

9. The application according to claim 7, characterized in that, The conditions for the catalytic reaction are: catalytic reaction at 60℃ for 15-60 min.

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