L-arabinose isomerase mutant and application thereof in preparation of D-tagatose

Abstract

The invention discloses an L-arabinose isomerase mutant and an application of the L-arabinose isomerase mutant in preparation of D-tagatose. On the basis of an amino acid sequence SEQ ID NO.1, the L-arabinose isomerase mutant is subjected to any one or a combination of at least two of the following mutations: F119M, M186A, M186Q, F280I, F280N, F280W, M350L, I371F, I371Y or P421G. The activity of the obtained optimal mutant F119M / M186A / F280I / I371F is 3.1 times of that of a wild type, 260 g / L of D-tagatose can be accumulated through enzyme catalysis of the mutant for 10 h, the conversion rate reaches 52%, and 305 g / L of D-tagatose can be accumulated through whole-cell catalysis of escherichia coli expressing the optimal mutant.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to an L-arabinose isomerase mutant and its application in the preparation of D-tagatose. Background Technology

[0002] D-tagatose is a rare sugar with low calories and low absorption, and its molecular formula is C6H. 12 O6, with a molecular weight of 180.16, has a taste similar to sucrose, with 92% of its sweetness but only one-third of its calories, making it an ideal sucrose substitute. It also possesses functions such as lowering blood sugar, improving gut microbiota, anti-oxidation, and anti-caries properties, making it an ideal functional sweetener. Currently, D-tagatose is mainly synthesized through chemical and biological methods. However, chemical synthesis requires high temperature and pressure conditions and easily generates impurities such as sorbitol and mannose, which are difficult to separate and purify. To overcome these drawbacks, the bioconversion method for producing D-tagatose has received widespread attention and research.

[0003] D-Tagatose, an isomer of D-galactose, can be synthesized by isomerase catalysis. Currently, various L-arabinose isomerases can catalyze the conversion of D-galactose to D-tagatose; however, due to substrate preference, these enzymes exhibit low catalytic activity for D-galactose. Therefore, it is necessary to modify these enzymes using enzyme engineering techniques to further enhance their activity and thermostability, improve substrate utilization, and achieve efficient and stable synthesis of D-tagatose. Summary of the Invention

[0004] To address the shortcomings of existing technologies and practical needs, this invention provides an L-arabinose isomerase mutant and its application in the preparation of D-tagatose. The optimal mutant F119M / M186A / F280I / I371F exhibits an activity 3.1 times that of the wild type. This mutant enzyme can accumulate 260 g / L of D-tagatose after 10 h of catalysis, with a conversion rate of 52%. Whole-cell catalysis by E. coli expressing the optimal mutant can accumulate 305 g / L of D-tagatose.

[0005] To achieve this objective, the present invention employs the following technical solution:

[0006] In a first aspect, the present invention provides an L-arabinose isomerase mutant, wherein the L-arabinose isomerase mutant is based on the amino acid sequence SEQ ID NO.1 and undergoes any one or a combination of at least two of the following mutations: F119M, M186A, M186Q, F280I, F280N, F280W, M350L, I371F, I371Y or P421G.

[0007] This invention provides an L-arabinose isomerase mutant capable of catalyzing the synthesis of D-tagatose from D-galactose. The optimal mutant F119M / M186A / F280I / I371F exhibits 3.1 times the activity of the wild type. This mutant enzyme can accumulate 260 g / L of D-tagatose after 10 h of catalysis, with a conversion rate of 52%. In whole-cell catalysis of E. coli expressing the optimal mutant, 305 g / L of D-tagatose can be accumulated.

[0008] Preferably, the L-arabinose isomerase mutant includes any one of the following:

[0009] (1) Single-point mutants: F119M, M186A, M186Q, F280I, F280N, F280W, M350L, I371F, I371Y or P421G;

[0010] (2) Two-point combination mutants: F119M / M186A, F119M / F280I, F119M / I371F, F119M / P421G, M186A / F280I, M186A / I371F, M186A / P421G, F280I / I371F, F280I / P421G or I371F / P421G;

[0011] (3) Three-point combination mutants: F119M / M186A / F280I, F119M / M186A / I371F, F119M / M186A / P421G, F119M / F280I / I371F, F119M / F280I / P421G, F119M / I371F / P421G, M186A / F280I / I371F, M186A / F280I / P421G, M186A / I371F / P421G or F280I / I371F / P421G;

[0012] (4) Four-site combination mutations: F119M / M186A / F280I / I371F, F119M / M186A / F280I / P421G, F119M / M186A / I371F / P421G, F119M / F280I / I371F / P421G or M186A / F280I / I371F / P421G;

[0013] (5) Five-site combination mutation: F119M / M186A / F280I / I371F / P421G.

[0014] SEQ ID NO.1:

[0015] MLKNNNNYKFWFITGSQALYGPEALQQVEADAKKMVSALNAAHNLPYPIEFKLVATTSENITQLMKDANYDNSVAGVITWMHTFSPAKNWIRGTKLLQKPLLHLATQVLDYIPYDTIDF DYMNLNQSAHGDREYAFINARLRKNNKIIFGFWGDKAIQKQIAQWMDVAVAYNESFNIKIVTFADKMRNVAVTDGDKIEAQIKFGWTVDYWGVGDLVSYVNAVDEADIDQLYEDLHDKY LFVEGENSKEKFDHNVKYQLREYLGLKKFLTEKGYSGFTTNFEDLVGLEQLPGLAVQMLMAEGYGFAGEGDWKTAALDRLLKILGHNRATAFMEDYTLDLRQGHEAILGSHMLEVDPTI ASDKPRVEVHLMDIGGKEDPARLVFTGRTGDAVDVTMADFGDEFKLISYDVTGNKPEKETPHLPVAKQLWTPKVGLKAGAEGWLTVGGGHHTVLSFSVNSEQLADLSKMFDLKFVDIK.

[0016] In a second aspect, the present invention provides a nucleic acid molecule that encodes the L-arabinose isomerase mutant described in the first aspect.

[0017] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecules described in the second aspect.

[0018] Fourthly, the present invention provides a genetically engineered strain that expresses the L-arabinose isomerase mutant described in the first aspect, or contains the nucleic acid molecule described in the second aspect, or contains the recombinant vector described in the third aspect.

[0019] Fifthly, the present invention provides a genetically engineered preparation containing any one or a combination of at least two of the following: a culture of the genetically engineered strain described in the fourth aspect, a culture extract, cell fragments, bacterial cells, fermentation broth, fermentation broth precipitate, or lyophilized powder.

[0020] Sixthly, the present invention provides a method for preparing the L-arabinose isomerase mutant described in the first aspect, the method comprising the following steps:

[0021] (1) Insert the nucleic acid sequence SEQ ID NO.2 of L-arabinose isomerase into a plasmid to obtain a recombinant plasmid. Using the recombinant plasmid as a template, use primers to perform site-directed mutagenesis on any one or at least two combinations of the 119th, 186th, 280th, 350th, 371st or 421st sites of the L-arabinose isomerase. The nucleic acid sequence of the primers includes the sequences shown in SEQ ID NO.4-SEQ ID NO.23.

[0022] (2) The mutant plasmid obtained in step (1) is transformed into the host bacteria, cultured and purified to obtain the L-arabinose isomerase mutant.

[0023] SEQ ID NO.2:

[0024]

[0025] In a seventh aspect, the present invention provides the application of the L-arabinose isomerase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, the genetically engineered strain described in the fourth aspect, the genetically engineered preparation described in the fifth aspect, or the method described in the sixth aspect in the preparation of D-tagatose.

[0026] Eighthly, the present invention provides a method for preparing D-tagatose, the method comprising: mixing D-galactose and the L-arabinose isomerase mutant described in the first aspect in a reaction system, reacting, and purifying the product after the reaction to obtain the D-tagatose.

[0027] Preferably, the reaction system contains divalent manganese ions.

[0028] Preferably, the concentration of divalent manganese ions in the reaction system is 2-10 mM, for example, 2 mM, 5 mM or 10 mM.

[0029] Preferably, the concentration of D-galactose in the reaction system is 300-600 g / L, for example, 400 g / L, 500 g / L or 600 g / L.

[0030] Preferably, the pH of the reaction system is 6.0-8.5, for example 6.0, 7.5 or 8.5.

[0031] Preferably, the temperature of the reaction system is 50-70°C, for example, 50°C, 60°C or 70°C.

[0032] Preferably, the reaction time is 5-10 h, for example 5 h, 8 h or 10 h.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention provides an L-arabinose isomerase mutant capable of catalyzing the synthesis of D-tagatose from D-galactose. The optimal mutants F119M / M186A / F280I / I371F exhibit 3.1 times the activity of the wild type. By optimizing the reaction conditions, in a system with 55°C, pH 6.5, D-galactose concentration of 500 g / L, and the addition of 6 mM divalent manganese ions, this mutant enzyme can accumulate 260 g / L of D-tagatose after 10 h of catalysis, achieving a conversion rate of 52%. Whole-cell catalysis by E. coli expressing the optimal mutant can accumulate 305 g / L of D-tagatose. Attached Figure Description

[0035] Figure 1 Electrophoresis image of purified OsAI protein;

[0036] Figure 2 HPLC chromatograms of D-galactose and D-tagatose;

[0037] Figure 3 The standard curve for D-galactose;

[0038] Figure 4 The standard curve of D-tagatose;

[0039] Figure 5 A bar chart showing the relative yield of D-tagatose at different temperatures;

[0040] Figure 6 Line graph showing the relative yield of D-tagatose at different pH values;

[0041] Figure 7 Line plots showing the thermal stability of OsAI and the optimal mutant;

[0042] Figure 8 The bar chart shows the yield of D-tagatose at different substrate concentrations. Detailed Implementation

[0043] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0045] Example 1

[0046] This embodiment describes the construction of an L-arabinose isomerase mutant.

[0047] The pET15b-OsAI recombinant plasmid was synthesized by Nanjing Genscript Biotech Co., Ltd., and chemically transformed into E. coli BL21(DE3) competent cells. 1 μL of the recombinant plasmid was added to 50 μL of competent cells, gently resuspended and mixed, incubated on ice for 30 min, heat-shocked at 42℃ for 30 s, incubated on ice for 2 min, and then 550 μL of LB liquid was added. After recovery at 37℃ and 220 rpm for 1 h, 100 μL of the bacterial culture was plated on an Amp-resistant plate. The L-arabinose isomerase OsAI has the amino acid sequence shown in SEQ ID NO.1 and can stably catalyze the conversion of D-galactose to D-tagatose at 60℃. OsAI has the nucleotide sequence shown in SEQ ID NO.2, and the nucleotide sequence of the plasmid vector pET15b is shown in SEQ ID NO.3.

[0048] SEQ ID NO.3:

[0049]

[0050] Using the recombinant plasmid pET15b-OsAI as a template, primers as shown in Table 1 were designed and synthesized, where F represents the forward primer and R represents the reverse primer. The specified mutation was introduced using PCR. The PCR reaction program was as follows: 94℃ for 3 min; 94℃ for 30 s, 69℃ for 30 s, 72℃ for 2 min 30 s, repeated for 34 cycles; extension at 72℃ for 5 min. DpnI was added to the PCR product and treated at 37℃ for 2 h before transformation into E. coli BL21(DE3). The transformed product was plated on LB agar plates containing Amp resistance and incubated at 37℃ for 12 h. Randomly selected single colonies were sequenced and analyzed to obtain the mutants F119M, M186A, M186Q, F280I, F280N, F280W, M350L, I371F, I371Y, P421G, F119M / M186A, F119M / F280I, F119M / I371F, F119M / P421G, M186A / F280I, M186A / I371F, M186A / P421G, F280I / I371F, F280I / P421G, and I371F / P4. 21G, F119M / M186A / F280I, F119M / M186A / I371F, F119M / M186A / P421G, F119M / F280I / I371F, F119M / F280I / P421G, F11 9M / I371F / P421G, M186A / F280I / I371F, M186A / F280I / P421G, M186A / I371F / P421G, F280I / I371F / P421G, F119M / M186A / F280I / I371F、F119M / M186A / F280I / P421G、F119M / M186A / I371F / P421G、F119M / F280I / I371F / P421G, M186A / F280I / I371F / P421G, F119M / M186A / F280I / I371F / P421G.

[0051] Table 1

[0052]

[0053] Example 2

[0054] This example demonstrates the expression and purification of an L-arabinose isomerase mutant.

[0055] (1) Protein-induced expression

[0056] Recombinant strain E. coli BL21(DE3) / pET15b-OsAI and its mutants were inoculated into 20 mL LB liquid medium containing ampicillin resistance at a final concentration of 100 μg / mL and cultured at 37°C and 220 rpm for 12 h. Then, at a 2% inoculum, the culture was transferred to 100 mL LB medium containing ampicillin resistance at a final concentration of 100 μg / mL and cultured in a shaker at 37°C and 220 rpm until OD (digestion / degradation). 600 Once the protein content reached 0.6-0.8, IPTG was added to a final concentration of 0.1 mM, and expression was induced at 16°C for 18 h. Wet cells were collected by centrifugation at 8000 rpm for 6 min, and the cells were resuspended and washed with 5 mL of 50 mM phosphate-buffered saline (pH 7.4). After centrifugation, the supernatant was discarded, and the cells were resuspended in buffer and sonicated on ice. The lysate was centrifuged at 10000 rpm for 20 min, and the supernatant was collected. The precipitate was resuspended in an equal volume of buffer, and protein expression was verified by SDS-PAGE. The supernatant was used as crude enzyme solution for subsequent purification.

[0057] (2) Protein purification

[0058] The crude enzyme solution was purified by nickel affinity chromatography. First, the nickel column was equilibrated with a low concentration of imidazole phosphate buffer. Then, the crude enzyme solution was slowly and uniformly added to the nickel column. The nickel column was washed with wash buffer (50 mM Na₂HPO₄-NaH₂PO₄, 500 mM NaCl, 20 mM imidazole, pH 8.0) to remove contaminating proteins. The target protein was then eluted with elution buffer (50 mM Na₂HPO₄-NaH₂PO₄, 500 mM NaCl, 300 mM imidazole, pH 8.0). Finally, the target protein eluent was concentrated by centrifugation using a 30 kDa ultrafiltration tube to obtain the purified enzyme solution. The electrophoresis image of the pure enzyme is shown below. Figure 1 As shown. Enzyme concentration was determined using a BCA kit.

[0059] Example 3

[0060] This embodiment compares the catalytic activity of the mutant and the wild type.

[0061] The OsAI and its mutants F119M, M186A, M186Q, F280I, F280N, F280W, M350L, I371F, I371Y, P421G, F119M / M186A, F119M / F280I, F119M / I371F, F119M / P421G, M186A / F280I, and M186A / I37 obtained in Examples 1 and 2 were compared. 1F, M186A / P421G, F280I / I371F, F280I / P421G, I371F / P421G, F119M / M186A / F280I, F119M / M 186A / I371F, F119M / M186A / P421G, F119M / F280I / I371F, F119M / F280I / P421G, F119M / I371F The pure enzymes / P421G, M186A / F280I / I371F, M186A / F280I / P421G, M186A / I371F / P421G, F280I / I371F / P421G, F119M / M186A / F280I / I371F, F119M / M186A / F280I / P421G, F119M / M186A / I371F / P421G, F119M / F280I / I371F / P421G, M186A / F280I / I371F / P421G, F119M / M186A / F280I / I371F / P421G are used to catalyze the substrate D-galactose. The catalytic system and reaction conditions are as follows: the final concentration of D-galactose is 300 g / L, the concentration of divalent manganese ions is 5 mM, and the enzyme dosage for both wild-type and mutant OsAI is 1.0 mg / mL. A 1 mL reaction system is prepared using 50 mM Tris-HCl (pH 7.0). D-tagatose is prepared by reacting at 200 rpm and 60℃ for 10 h in a water bath shaker. The yield of D-tagatose is detected by high-performance liquid chromatography (HPLC), and then the relative activity is calculated.

[0062] The HPLC detection method is as follows: Agilent 1260, equipped with a RID detector, Waters Sugar-PakI column (6.5×300 mm), pure water as the mobile phase, flow rate of 0.6 mL / min, column temperature of 90℃, detector temperature of 50℃, and injection volume of 10 μL.

[0063] A schematic diagram of HPLC detection of substrate D-galactose and product D-tagatose is shown below. Figure 2 As shown, the corresponding standard curve is as follows: Figure 3 and Figure 4 As shown in Table 2, the relative activities of OsAI and its mutants are shown in Table 2.

[0064] Table 2

[0065]

[0066]

[0067] As shown in Table 2, the L-arabinose isomerase mutants of this invention exhibit higher activity than the wild type, with the optimal mutant F119M / M186A / F280I / I371F showing 3.1 times the activity of the wild type.

[0068] Example 4

[0069] This embodiment investigates the effect of temperature on the preparation of D-tagatose from the optimal mutants F119M / M186A / F280I / I371F.

[0070] D-galactose was added to the buffer solution as a substrate to a final concentration of 300 g / L. 6 mM of divalent manganese ions were added, followed by the purified enzyme of the optimal mutants F119M / M186A / F280I / I371F, to a final concentration of 0.5 mg / mL. The buffer solution used was 50 mM Tris-HCl (pH 7.0), and the reaction temperatures were 50℃, 55℃, 60℃, 65℃, and 70℃. The reaction was terminated by boiling in a water bath for 10 min after completion. The samples were analyzed by HPLC under the same conditions as in Example 3.

[0071] The results are as follows Figure 5 As shown, the mutant maintained good catalytic activity at temperatures ranging from 50 to 60°C, exhibiting the highest activity at 55°C, indicating that OsAI has good tolerance to high temperatures, which is advantageous for promoting the forward reaction of this reversible isomerization.

[0072] Example 5

[0073] This embodiment investigates the effect of pH on the preparation of D-tagatose from the optimal mutant F119M / M186A / F280I / I371F.

[0074] D-galactose was added to the buffer solution as a substrate to a final concentration of 300 g / L. 6 mM of divalent manganese ions were added, followed by the purified enzyme of the optimal mutants F119M / M186A / F280I / I371F, to a final concentration of 0.2 mg / mL. The reaction was conducted at 55°C. The pH values ​​of the phosphate buffer were 6, 6.5, and 7, and the pH values ​​of the Tris-HCl buffer were 6.5, 7.0, 7.5, 8, and 8.5, respectively. The reaction was terminated by boiling in a water bath for 10 min. The samples were analyzed by HPLC under the same conditions as in Example 3.

[0075] The results are as follows Figure 6 As shown, the enzyme activity is optimal in Tris-HCl buffer at pH 6.5. When the pH is below 6.5 or above 8, the activity decreases significantly.

[0076] Example 6

[0077] This embodiment investigates the thermal stability of wild-type OsAI and the optimal mutants F119M / M186A / F280I / I371F.

[0078] The purified enzymes of wild-type and optimal mutant were heat-treated at their optimal temperatures of 60°C and 55°C, respectively. The substrate D-galactose was added to initiate the reaction after hot water bath treatment for 0, 12, 24, 36, and 48 h. The reaction was terminated by boiling in a water bath for 10 min after completion. Samples were analyzed by HPLC under the same conditions as in Basic Example 3.

[0079] The results are as follows Figure 7 As shown, the optimal mutant retained 80% of its activity after 48 h of heat treatment, which is a significant advantage for enzyme reuse. Compared to the wild type, the mutant exhibited improved thermostability.

[0080] Example 7

[0081] This embodiment investigates the effect of substrate concentration on the preparation of D-tagatose from the optimal mutant F119M / M186A / F280I / I371F.

[0082] To achieve high substrate utilization and maximize D-tagatose yield, a D-galactose concentration gradient of 50-500 g / L was established, with the optimal enzyme concentration for the mutants F119M / M186A / F280I / I371F being 10 mg / mL. The reaction was carried out in Tris-HCl buffer at the optimal temperature of 55℃ and the optimal pH of 6.5, with the addition of 6 mM divalent manganese ions. The reaction was terminated by boiling in a water bath for 10 min after completion. Samples were analyzed by HPLC under the same conditions as in Example 3.

[0083] The results are as follows Figure 8 As shown, a conversion rate of 52% can still be achieved when the D-galactose concentration reaches 500 g / L, and the D-tagatose concentration reaches 260 g / L.

[0084] Example 8

[0085] In this embodiment, D-tagatose was prepared by whole-cell catalysis using Escherichia coli expressing the optimal mutant F119M / M186A / F280I / I371F.

[0086] The reaction system contained 130 g / L of wet Escherichia coli cells and 670 g / L of D-galactose substrate. The reaction was carried out in 50 mM Tris-HCl (pH 6.5) at 55°C for 48 h, which accumulated 305 g / L of D-tagatose.

[0087] In summary, this invention provides an L-arabinose isomerase capable of catalyzing the synthesis of D-tagatose from D-galactose and its applications. The optimal mutant exhibits catalytic activity 3.1 times that of the wild type. The expressed enzyme can stably catalyze the conversion of D-galactose to D-tagatose. This mutant enzyme can accumulate 260 g / L of D-tagatose after 10 h of catalysis, achieving a conversion rate of 52%. Whole-cell catalysis by E. coli expressing the optimal mutant can accumulate 305 g / L of D-tagatose.

[0088] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An L-arabinose isomerase mutant, characterized in that, The L-arabinose isomerase mutant is based on the amino acid sequence SEQ ID NO.1 and undergoes any one or a combination of at least two of the following mutations: F119M, M186A, M186Q, F280I, F280N, F280W, M350L, I371F, I371Y or P421G.

2. The L-arabinose isomerase mutant according to claim 1, characterized in that, The L-arabinose isomerase mutant includes any one of the following: (1) Single-point mutants: F119M, M186A, M186Q, F280I, F280N, F280W, M350L, I371F, I371Y or P421G; (2) Two-point combination mutants: F119M / M186A, F119M / F280I, F119M / I371F, F119M / P421G, M186A / F280I, M186A / I371F, M186A / P421G, F280I / I371F, F280I / P421G or I371F / P421G; (3) Three-point combination mutants: F119M / M186A / F280I, F119M / M186A / I371F, F119M / M186A / P421G, F119M / F280I / I371F, F119M / F280I / P421G, F119M / I371F / P421G, M186A / F280I / I371F, M186A / F280I / P421G, M186A / I371F / P421G or F280I / I371F / P421G; (4) Four-site combination mutations: F119M / M186A / F280I / I371F, F119M / M186A / F280I / P421G, F119M / M186A / I371F / P421G, F119M / F280I / I371F / P421G or M186A / F280I / I371F / P421G; (5) Five-site combination mutation: F119M / M186A / F280I / I371F / P421G.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the L-arabinose isomerase mutant as described in claim 1 or 2.

4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 3.

5. A genetically engineered strain, characterized in that, The genetically engineered strain expresses the L-arabinose isomerase mutant of claim 1 or 2, or contains the nucleic acid molecule of claim 3, or contains the recombinant vector of claim 4.

6. A genetically engineered preparation, characterized in that, The genetically engineered preparation contains any one or a combination of at least two of the following: culture of the genetically engineered strain described in claim 5, culture extract, cell fragments, bacterial cells, fermentation broth, fermentation broth precipitate, or lyophilized powder.

7. A method for preparing the L-arabinose isomerase mutant according to claim 1 or 2, characterized in that, The method includes the following steps: (1) Insert the nucleic acid sequence SEQ ID NO.2 of L-arabinose isomerase into a plasmid to obtain a recombinant plasmid. Using the recombinant plasmid as a template, use primers to perform site-directed mutagenesis on any one or at least two combinations of the 119th, 186th, 280th, 350th, 371st or 421st sites of the L-arabinose isomerase. The nucleic acid sequence of the primers includes the sequences shown in SEQ ID NO.4-SEQ ID NO.

23. (2) The mutant plasmid obtained in step (1) is transformed into the host bacteria, cultured and purified to obtain the L-arabinose isomerase mutant.

8. The use of the L-arabinose isomerase mutant of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant vector of claim 4, the genetically engineered strain of claim 5, the genetically engineered preparation of claim 6, or the method of claim 7 in the preparation of D-tagatose.

9. A method for preparing D-tagatose, characterized in that, The method includes: mixing D-galactose and the L-arabinose isomerase mutant according to claim 1 or 2 in a reaction system, reacting, and purifying the product after the reaction to obtain the D-tagatose.

10. The method for preparing D-tagatose according to claim 9, characterized in that, The reaction system contains divalent manganese ions; Preferably, the concentration of divalent manganese ions in the reaction system is 2-10 mM; Preferably, the concentration of D-galactose in the reaction system is 300-600 g / L; Preferably, the pH of the reaction system is 6.0-8.5; Preferably, the temperature of the reaction system is 50-70°C; Preferably, the reaction time is 5-10 h.

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