Heat-resistant arabinose isomerase gene araA and application thereof

Abstract

The invention discloses a heat-resistant arabinose isomerase gene araA and application of the heat-resistant arabinose isomerase gene araA. The nucleotide sequence of the heat-resistant arabinose isomerase gene araA is as shown in SEQ ID NO.1, and the amino acid sequence of enzyme obtained from the heat-resistant arabinose isomerase gene araA is as shown in SEQ ID NO.2. Through a specific modification method, the heat resistance and the optimum temperature of the enzyme are improved, the optimum pH is reduced, and the enzyme activity is improved. Compared with the prior art, the specific enzyme activity is improved by about 32%, the heat resistance is remarkably improved, and the high enzyme activity is achieved within the temperature range of 70-90 DEG C and the pH value range of 4.5-5.5. Especially, under the conditions that the temperature is 80 DEG C and the pH value is 5.0, the highest enzyme activity is achieved, and the specific enzyme activity reaches 49.7 micromol / (mg.min). The D-galactose can be effectively converted into the D-tagatose. The enzyme has a wide industrial application prospect, is especially suitable for producing D-tagatose, and has huge economic benefits.

Description

Technical Field

[0001] The invention relates to a heat-resistant arabinose isomerase gene araA and an expression protein, a recombinant vector, a recombinant bacterium and an application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] As a natural ketohexose with high sweetness (92% of sucrose), low calories (only 1 / 3 of sucrose) and no bad flavor and aftertaste, tagatose has broad application prospects in the food industry. It is safe to use due to its non-toxic and carcinogenic effects, and has the advantages of anti-caries, hypoglycemia, improvement of intestinal flora and good physical and chemical properties. However, the content of D-tagatose in nature is extremely low, and its synthesis methods mainly include chemical catalysis and biological (enzyme) conversion. The chemical catalysis method has the disadvantages of environmental pollution, many by-products, and high requirements for reaction vessels and conditions, while the biological conversion method has the advantages of mild reaction conditions, few by-products, safety and high efficiency. L-arabinose isomerase from microorganisms can convert D-galactose into D-tagatose. At present, biological conversion mainly revolves around this enzyme, because galactose can be obtained by decomposing cheap whey, which is suitable for the production of D-tagatose.

[0003] Although L-arabinose isomerase has a wide range of microbial sources, the catalytic efficiency of existing enzymes in high temperature and acidic environments is mostly unsatisfactory. For example, the optimum temperature of arabinose isomerase from Lactobacillus reuteri SDMCC 050455 is 60°C and the optimum pH is 6.0-7.5; the optimum temperature of L-arabinose isomerase from Lactobacillus plantarum WCFS1 is 55°C and the optimum pH is 7.0; the optimum temperature of L-arabinose isomerase from lactic acid bacteria SK1.002 is 60°C and the optimum pH is 7.0. In actual industrial production, high temperature and acidic conditions are more common. The characteristics of existing enzymes lead to limited activity under these conditions, which prolongs the production cycle of D-tagatose, increases costs, and makes it difficult for product purity and output to reach ideal levels, which seriously restricts the large-scale application of arabinose isomerase in industries such as food additives and cannot give full play to its application potential. Currently, there are few studies on the commonalities between L-arabinose isomerases from different sources. Therefore, it is of great significance to determine the key areas that affect the properties based on their structure and to optimize their optimal pH, optimal temperature and improve substrate specificity through molecular biological methods. Summary of the invention

[0004] Objectives of the invention: In view of the problem that the arabinose isomerase in the prior art has low catalytic efficiency under high temperature and acidic environment, which leads to limitations in industrial applications, the first objective of the present invention is to provide a thermostable arabinose isomerase gene araA, the second objective of the present invention is to provide an expression protein of the thermostable arabinose isomerase gene araA, the third objective of the present invention is to provide a derivative protein containing the expression protein of the thermostable arabinose isomerase gene araA, the fourth objective of the present invention is to provide a recombinant vector containing the thermostable arabinose isomerase gene araA of the present invention, the fifth objective of the present invention is to provide a recombinant organism containing the thermostable arabinose isomerase gene araA of the present invention or the recombinant vector of the present invention, and the sixth objective of the present invention is to provide the application of the thermostable arabinose isomerase gene araA of the present invention, the expression protein of the thermostable arabinose isomerase gene araA of the present invention, the derivative protein of the expression protein of the present invention, the recombinant vector of the present invention or the recombinant organism of the present invention in the food industry for enzyme-catalyzed substrate D-galactose.

[0005] Technical solution: The present invention discloses a thermostable arabinose isomerase gene araA, and the nucleotide sequence of the thermostable arabinose isomerase gene araA is shown in SEQ ID NO.1.

[0006] Furthermore, the thermostable arabinose isomerase gene araA is amplified by PCR using a primer pair with amino acid sequences such as SEQ ID NO.3 and SEQ ID NO.4.

[0007] An expression protein of the thermostable arabinose isomerase gene araA of the present invention, wherein the amino acid sequence of the expression protein is shown in SEQ ID NO.2.

[0008] A derivative protein of an expressed protein, wherein the derivative protein of the expressed protein is a derivative protein formed by replacing, deleting or adding one or more amino acid residues of the amino acid of the expressed protein of the thermostable arabinose isomerase gene araA of the present invention.

[0009] A recombinant vector containing the thermostable arabinose isomerase gene araA of the invention.

[0010] A recombinant organism containing the thermostable arabinose isomerase gene araA of the invention or the recombinant vector of the invention.

[0011] Furthermore, the recombinant organism is a strain.

[0012] Application of the thermostable arabinose isomerase gene araA of the present invention, the expression protein of the thermostable arabinose isomerase gene araA of the present invention or the derivative protein of the expression protein of the present invention in the enzyme catalysis of substrate D-galactose in the food industry.

[0013] The recombinant vector or the recombinant organism of the present invention is used in the food industry to enzymatically catalyze the substrate D-galactose.

[0014] Further, D-galactose is converted into D-tagatose at 70-90°C and pH 4.5-5.5, preferably 80°C and pH 5.0.

[0015] The expression protein of the thermostable arabinose isomerase gene araA of the present invention is arabinose isomerase, and its amino acid sequence is shown in SEQ ID NO. 2. The derivative protein is obtained by a specific modification method, and on the basis of the sequence of SEQ ID NO. 5, lysine (K) is mutated to cysteine ​​(C), histidine (H) is mutated to threonine (T), tyrosine (Y) is mutated to glutamine (Q), methionine (M) is mutated to alanine (A), phenylalanine (F) is mutated to isoleucine (I), histidine (H) is mutated to cysteine ​​(C), and asparagine (N) is mutated to cysteine ​​(C) at position 5, and the activity of arabinose isomerase is maintained.

[0016] The transformation method of the present invention:

[0017] 1) Database comparison: By comparing the arabinose isomerase sequences from various sources, the conserved region in the Pseudothermotogat hermarum DSM5069 arabinose isomerase sequence was determined as the skeleton.

[0018] 2) Potential modification sites: The irregular curled parts of the non-conservative sequences in the amino acid sequence were selected as potential modification sites. These regions may affect the activity and stability of the enzyme. After database comparison, molecular simulation technology analysis and research on enzyme structure and function, specific amino acid sites were selected for mutation modification. In the amino acid sequence, positions 5, 17, 19, 183, 273, 344 and 386 are located in areas that may affect enzyme activity and stability. Among them, the lysine at position 5 mutated to cysteine, aiming to introduce the heat-resistant amino acid cysteine ​​to form a disulfide bond, enhance the interaction within the enzyme molecule, and thus improve the stability of the enzyme; the histidine at position 17 mutated to threonine, the tyrosine at position 19 mutated to glutamine, and the asparagine at position 386 mutated to cysteine, which is to change the properties of amino acids, optimize the internal charge interaction of the enzyme, and further stabilize the enzyme structure; the methionine at position 183 mutated to alanine and the phenylalanine at position 273 mutated to isoleucine, which can modify the amino acids near the active center of the enzyme, rigidify the structure of the active center, and improve the catalytic efficiency of the enzyme; the histidine at position 344 mutated to cysteine, which also helps to form a disulfide bond and enhance the stability of the enzyme. After determining these mutation sites, artificial synthesis was used to obtain them.

[0019] 3) Three-dimensional structure simulation: Use molecular simulation technology to simulate the three-dimensional structure of arabinose isomerase, and select appropriate sites for modification based on the structural information.

[0020] 4) Introduction of heat-resistant amino acids: Introducing heat-resistant amino acids (such as cysteine) at selected sites to form disulfide bonds and enhance the stability of the enzyme.

[0021] 5) Optimize charge interactions: Change the amino acid sequence to optimize the internal charge interactions of the enzyme and further stabilize the enzyme structure.

[0022] 6) Rigidizing the active center: Modify the amino acids near the active center, rigidify the structure of the active center, and improve the catalytic efficiency of the enzyme.

[0023] Through the above transformation, 1) the enzyme activity is improved: compared with the original enzyme, the specific enzyme activity of the modified arabinose isomerase is increased by about 32% to 49.7μmol / (mg·min). This is mainly attributed to the amino acid mutations at positions 183 and 273, which have optimized the binding ability and catalytic efficiency of the enzyme and substrate by rigidifying the active center structure, thereby improving the enzyme activity.

[0024] 2) Improved heat resistance: The optimum temperature was raised from 55°C to 80°C. The disulfide bonds formed by the introduction of cysteine ​​at positions 4, 344, and 386, as well as the optimization of charge interactions by amino acid mutations at positions 17 and 19, jointly enhanced the stability of the enzyme molecule at high temperatures, enabling it to maintain higher activity at higher temperatures, thereby raising the optimum temperature.

[0025] 3) Improved temperature stability: Under the condition of 70-80℃, the enzyme activity retention rate is above 80%. The synergistic effect of amino acid mutations at multiple sites, especially the optimization of disulfide bond formation and charge interaction, effectively reduces the damage of high temperature to the enzyme molecular structure, enables the enzyme to maintain a high level of activity within this temperature range, and improves temperature stability.

[0026] The protein expressed by it has significantly improved heat resistance and can catalyze the isomerization of arabinose to L-ribulose under extreme conditions. At the same time, the recombinant vector of the gene, the recombinant bacteria and the application of its expressed protein in catalyzing the substrate D-galactose are provided. It is applied to the isomerization of D-galactose to D-tagatose and has potential industrial application value.

[0027] The present invention contains a recombinant vector of the above-mentioned heat-resistant arabinose isomerase gene araA. When constructing a recombinant vector, it is necessary to accurately control the conditions of enzyme cutting and ligation reaction. For example, use specific restriction endonucleases (such as NcoⅠ and EcoRⅠ) for enzyme cutting to ensure that the proportion of each component in the enzyme cutting reaction system is appropriate, and the reaction temperature and time are accurate, so as to obtain accurate enzyme cutting fragments. During the ligation reaction, pay attention to the molar ratio of the target gene to the vector, which is generally controlled at about 3:1-5:1. At the same time, ensure that the activity of the ligase is normal, the composition of the ligation buffer is appropriate, and connect overnight at 16°C to improve the connection efficiency and avoid problems such as vector self-ligation or wrong insertion direction of the target fragment.

[0028] The present invention contains the recombinant bacteria of the thermostable arabinose isomerase gene araA or the recombinant vector. When constructing the recombinant bacteria, the selection of the host bacteria is very important. The present invention uses E. coli BL21 (DE3) as the host bacteria because it has good protein expression ability and is easy to culture. During the transformation process, the optimization of heat shock conditions has a great impact on the transformation efficiency.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0030] 1) Excellent heat resistance: After incubation at 70-80°C for 40 minutes, the enzyme activity still remains above 80%, which is about 32% higher than the enzyme activity, and the heat resistance is significantly improved.

[0031] 2) High activity under acidic conditions: It still has high enzyme activity under acidic conditions, especially suitable for the isomerization of D-galactose to D-tagatose under high temperature above 70°C and acidic pH conditions.

[0032] 3) High catalytic efficiency: It can more effectively convert D-galactose into D-tagatose.

[0033] 4) Broad application prospects: Especially suitable for the production of D-tagatose, with huge economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The SDS-PAGE protein electrophoresis diagram of the thermostable arabinose isomerase mutant TaraA;

[0035] Figure 2 This is a diagram of the enzymatic properties of the thermostable arabinose isomerase mutant TaraA. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below in conjunction with the embodiments and drawings.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] Example 1 Preparation of arabinose isomerase gene araA

[0039] The amino acid sequence of Pseudothermotoga thermarum DSM5069 arabinose isomerase (SEQ ID NO.5) (NCBI No.: CP002351.1) was used as a template, and its base sequence was SEQ ID NO.6. Non-conserved sites of arabinose isomerase were obtained as potential modification sites by database comparison; at the same time, according to the amino acid preference of thermophilic arabinose isomerase and the strategy of optimizing intramolecular interactions was introduced, mutations were introduced at specific positions, that is, position 5 was mutated from lysine (K) to cysteine ​​(C), position 17 was mutated from histidine (H) to threonine (T), position 19 was mutated from tyrosine (Y) to glutamine (Q), position 183 was mutated from methionine (M) to alanine (A), position 273 was mutated from phenylalanine (F) to isoleucine (I), position 344 was mutated from histidine (H) to cysteine ​​(C), and position 386 was mutated from asparagine (N) to cysteine ​​(C). After introducing these mutations, the araA gene was prepared by artificial synthesis through codon optimization, and its gene nucleotide sequence is shown in SEQ ID NO.1. The protein encoded by this gene is arabinose isomerase containing the above mutations.

[0040] SEQ ID NO.5:

[0041] MIDLKKYEFWLLVGSQHLYGSETLKKVEQQARKIVEELSKDLPSPLLFKGVLTTPEEILRTFEQANAQTNCAGVITWMHTFSPSKMWIKGLLANKKPLLHLHTQFNREIPWDTIDMDYMNLNQSAHGDREHGYIHARLRLPRKVVVGHWQDSEVKREISKWMRVACAIADGRSGQIVRFGDNMREVASTEADKVEAQIKIGWSINTWGVGELAERVKSVSENLVEDLIKHYAEKYVLPSGEYELKAIKEQARIEIALREFLKEKNAIAFTTTFEDLHDLPQLPGLAVQRLMEEGYGFGAEGDWKVAGLVRALKVMGVGLKGGTSFMEDYTYHLPEGNELVLGAHMLEICPSIAKEKPRIEVHPLSIGGKADPARLVFEAQVGEALNASIVDLGNRFRLVVNKVISVPLVKPMPKLPVARVLWKPLPNFKAAATAWILAGGSHHTAFSTAVDPSYLIDWAEMLDIECVVIDEKLDLERFKAELRANEVYWGFFKK;

[0042] SEQ ID NO.6:

[0043]

[0044] Example 2 Subcloning of the arabinose isomerase gene araA

[0045] The arabinose isomerase gene araA in Example 1 was amplified by PCR using primer pairs, and the primer sequences were as follows: araA-F: 5'-GGGCCATGGATGATTGATCTGT (as shown in SEQ ID NO.3, with the introduction of NcoⅠ restriction site); araA-R: 5'-GGGGAATTCTCACTTTTTGAAAAAGC (as shown in SEQ ID NO.4, with the introduction of EcoRI restriction site). The PCR reaction system was: 1μL artificial synthetic araA gene template, 1μL araA-F, 1μL araA-R, 9.5μL dd H2O, 12.5μL primeSTARHSDNA Polymerase. The reaction conditions were: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30sec, 50℃ annealing for 30sec, 72℃ extension for 1.5min, 30 cycles; 72℃ extension for 10min; 12℃ insulation. The PCR product was purified using a PCR product recovery kit after the yield and specificity were detected by 1% agarose gel electrophoresis.

[0046] Example 3 Recombinant cloning, construction and verification of expression vector pET-22b-araA

[0047] The purified PCR product (prepared in Example 2) and pET-22b (Novagen) were double-digested with NcoⅠ and EcoRⅠ, respectively, and the digested PCR and vector large fragments were recovered by agarose electrophoresis. After gel tapping, the target fragment and vector were added with 1μL 10XLigase Buffer and 1μL Ligase, and connected at 16°C overnight. The ligation reaction product was transformed into Escherichia coli DH 5α (purchased from China Center for Type Culture Collection CCTCC, item number AB 2014207), applied to a culture dish containing 100μg / mL Amp (ampicillin), and cultured at 37°C for 10-15h. Multiple single colonies were picked from the transformation plate, and the plasmid was extracted using ThermoFisher's plasmid mini-extraction kit. The obtained plasmid was double-digested and verified and sequenced. The sequencing results showed that the cloned target fragment (nucleotide length of 1485bp) was inserted into the pET-22b vector, and the recombinant clone and expression vector pET-22b-araA was obtained. The DNA sequence of araA is shown in SEQ ID NO.1, and the amino acid sequence of the mutant protein (heat-resistant and acid-resistant arabinose isomerase) expressed by it is shown in SEQ ID NO.2. The mutant was named TaraA. In the process of constructing the recombinant vector, the amount of enzyme, reaction temperature and time of the double enzyme digestion reaction must be strictly controlled. For example, the amount of NcoⅠ and EcoRI must be accurately calculated according to the amount of the vector and the target fragment. Generally, 1-2U of enzyme is used per μg of DNA, the reaction temperature is 37℃, and the time is recommended according to the enzyme manual, usually 1-3 hours, to ensure complete enzyme digestion. During the ligation reaction, ligation at 16℃ overnight can make the ligation reaction fully proceed, while avoiding enzyme inactivation or non-specific ligation caused by excessive temperature.

[0048] SEQ ID NO.1:

[0049]

[0050] SEQ ID NO.2 (underlined amino acids after mutation):

[0051] MIDL C KYEFWLLVGSQ T L Q GSETLKKVEQQARKIVEELSKDLPSPLLFKGVLTTPEEILRTFEQANAQTNCAGVITWMHTFSPSKMWIKGLLANKKPLLHLHTQFNREIPWDTIDMDYMNLNQSAHGDREHGYIHARLRLPRKVVVGHWQDSEVKREISKWMRVACAIADGRSGQIVRFGDN A REVASTEADKVEAQIKIGWSINTWGVGELAERVKSVSENLVEDLIKHYAEKYVLPSGEYELKAIKEQARIEIALREFLKEKNAIAFTTT I EDLHDLPQLPGLAVQRLMEEGYGFGAEGDWKVAGLVRALKVMGVGLKGGTSFMEDYTYHLPEGNELVLGA C MLEICPSIAKEKPRIEVHPLSIGGKADPARLVFEAQVGEAL C ASIVDLGNRFRLVVNKVISVPVVKPMPKLPVARVLWKPLPNFKAAATAWILAGGSHHTAFSTAVDPSYLIDWAEMLDIECVVIDEKLDLERFKAELRANEVYWGFFKK.

[0052] Example 4 Construction of recombinant bacteria and expression and purification of mutant TaraA protein

[0053] The recombinant clone, expression vector pET-22b-araA (prepared in Example 3) was heat-shocked and transformed into the host bacteria E.coliBL21 (DE3) (purchased from the American Type Culture Collection Center ATCC, Article No. AC0001), and the recombinant bacteria containing the recombinant plasmid were obtained. In the recombinant bacteria culture process, 37°C was selected as the initial culture temperature because this temperature is suitable for the growth of E.coli BL21 (DE3), which can make it rapidly propagate to a certain cell density. When the cell grows to the logarithmic growth phase (absorbance 0.4-0.6), IPTG was added to induce protein expression, and the final concentration of IPTG was 0.1mM (800μL of 0.1M IPTG was added to 800mL culture medium), and 16°C was induced to express for 15h. The lower temperature induction can reduce the formation of inclusion bodies and increase the expression of soluble proteins, which is particularly important for the expression of recombinant proteins containing mutation sites, because mutations may affect the folding and stability of proteins, and low temperature induction helps to fold correctly. When disrupting bacterial cells with ultrasound, doing it in an ice water bath can avoid protein denaturation caused by ultrasound heat generation, which can affect enzyme activity. In particular, for proteins that have undergone mutation and whose structure and activity may have changed, maintaining a low temperature environment can better maintain their activity.

[0054] The crude extract was purified by Ni-NTA affinity chromatography (see His-Band Kits, Novagen). The purity of the purified enzyme and the molecular weight were determined by SDS-PAGE. Figure 1 , where 1 represents the purified TaraA protein eluted with 200 mM imidazole (used as pure enzyme solution in subsequent experiments), with a molecular weight of about 55 kDa, which is close to the theoretical value, indicating that the mutation has no significant effect on the protein molecular weight. The mutant protein was subsequently subjected to enzymatic property analysis (as described in Example 5) to comprehensively evaluate the effects of the mutation on enzyme activity, optimal temperature, optimal pH, thermal stability, metal ion stability, and enzyme reaction kinetic parameters.

[0055] Example 5 Analysis of the Enzymatic Properties of the Recombinant Arabinose Isomerase Mutant TaraA

[0056] 1) Definition of enzyme activity

[0057] Under optimal conditions, the amount of enzyme required to produce 1 μg of tagatose per minute using D-galactose as substrate is one enzyme activity unit (U / mL).

[0058] 2) Optimum temperature

[0059] The pure enzyme solution obtained in Example 4 was diluted with a 20mM phosphate buffer having a pH value of 6.8, and the enzyme solution diluted 10 times was taken for enzyme activity determination. The enzyme activity determination reaction system was 1500 μL, including 500 μL enzyme solution, 100 μL MnCl2 solution with a concentration of 10mM, 900 μL 0.5M D-galactose solution prepared with 20mM, pH 6.8 phosphate buffer, and the reaction system pH was 6.8. After the reaction system was reacted in a water bath at 40-100°C (with an interval gradient of 10°C) for 1h, 500 μL 0.1M hydrochloric acid was added to terminate the reaction. 1 mL of the diluted reaction product was taken, and 0.2 mL of cysteine ​​hydrochloride solution was immediately added after adding 6 mL of sulfuric acid solution, and after vigorous shaking, 0.2 mL of carbazole alcohol solution was added, shaken, kept warm at 60°C for 10min, and the absorbance value was measured at 560nm after cooling in an ice bath. The enzymatic reaction without adding diluted enzyme solution was performed, and the mixed solution under the same treatment conditions was used as the blank control. The experiment was repeated 3 times, and the average value was taken for plotting. The results showed that the activity of arabinose isomerase was the highest at 80°C. The absorbance value of the enzyme activity reaction system at this temperature was set as 100% relative activity, and the absorbance value of the enzyme activity reaction system at other temperatures was compared with it to obtain the relative activity, such as Figure 2 As shown in (a), the enzyme activity is higher in the reaction system at 70-90°C.

[0060] 3) Optimum pH

[0061] The pure enzyme solution was diluted with 20mM phosphate buffer with a pH value of 6.8, and the enzyme solution diluted 10 times was taken for enzyme activity determination. The enzyme activity determination reaction system is 1500μL, including 500μL enzyme solution, 100μL 10mM MnCl2 solution, and 900μL 0.5M galactose solution prepared with 20mM, pH 4.0-7.0 (interval gradient is 0.5) buffer. After the reaction system reacts in an optimal temperature water bath for 1h, 500μL 0.1M hydrochloric acid is added to terminate the reaction. The subsequent steps for determining the absorbance value are the same as the optimal temperature determination. The results show that the enzyme activity is highest at pH 5. The absorbance value of the enzyme activity reaction system at this pH value is set to the relative activity of 100%, and the absorbance value of the enzyme activity reaction system at other pH values ​​is compared with it to obtain the relative activity, such as Figure 2 As shown in (b), the enzyme activity is higher at pH 4.5-5.5.

[0062] 4) Thermal stability of recombinant enzyme

[0063] Dilute the pure enzyme solution with 20mM phosphate buffer with a pH value of 6.8, and take the enzyme solution diluted 10 times for enzyme activity determination. Set the temperature range to 70-100℃, and set a temperature gradient every 10℃. The enzyme activity reaction system is 1500μL, add 500μL of purified enzyme solution diluted 10 times into the centrifuge tube respectively, and place it in a constant temperature metal bath with different temperature gradients for insulation for 0.5-2.0h (a time gradient is set every 0.5h), and then take it out according to the insulation time, add 100μL of 10mM MnCl2 solution and 900μL 0.5M galactose solution, and react in the optimal temperature water bath for 10min, and measure the absorbance at 560nm. Set up three parallels for each group, and use the enzymatic reaction without adding diluted enzyme solution and the mixed solution under the same treatment conditions as the blank control, and the enzyme activity of the diluted enzyme solution without insulation at the optimal temperature and optimal pH conditions for 10min as the relative activity of 100%. Figure 2 As shown in (c), the arabinose isomerase has good thermal stability at 70-80° C. At the same time, the above method was used to measure the arabinose isomerase before modification (the template sequence was prepared according to the method of Example 2-4), and the results are shown in Table 1.

[0064] Table 1 Comparison results between mutant TaraA and arabinose isomerase before modification

[0065]

[0066] As shown in Table 1, compared with the arabinose isomerase before modification, the specific enzyme activity of the arabinose isomerase prepared by the present invention is increased by about 32%, the heat resistance is significantly improved from 55°C to 80°C, and the temperature stability is improved to a certain extent, which has better industrial application value.

[0067] 5) Metal ion stability

[0068] The pure enzyme solution was diluted with 20 mM phosphate buffer at pH 6.8, and the enzyme solution diluted 10 times was used for enzyme activity determination. The enzyme activity reaction system was 1500 μL, including 500 μL enzyme solution, 100 μL metal ion solution with a concentration of 10 mM (Mn 2+ 、Cd 2 + , Ca 2+ 、Zn 2+ , Ba 2+), 900μL of 0.5M galactose solution prepared with optimal pH buffer. After the reaction system reacted in a water bath at the optimal temperature for 1h, 500μL of 0.1M hydrochloric acid was added to terminate the reaction. The subsequent steps for determining the absorbance value were the same as before. Each group was set up with three parallels, and the enzymatic reaction of the diluted enzyme solution without adding any metal ions was used. The mixed solution under the same treatment conditions was used as the blank control, and the enzyme activity of the diluted enzyme solution that was not treated with metal ions at the optimal temperature and optimal pH for 10 minutes was taken as the relative activity of 100%. The results showed that the enzyme activity of all metal ions involved in the reaction was not significantly decreased after 1h in the water bath, among which Mn 2+ 、Cd 2 + 、Zn 2+ Ions promote enzyme activity, such as Figure 2 As shown in (d), the arabinose isomerase in Mn 2+ 、Cd 2+ 、Zn 2+ The enzyme activity is higher under the conditions of the reaction. In the determination of metal ion stability, these common metal ions are selected because they may exist in biological systems and industrial production. Studying their effects on enzyme activity helps to evaluate the stability and activity changes of enzymes in practical applications.

Claims

1. A thermostable arabinose isomerase gene araA, characterized in that The nucleotide sequence of the thermostable arabinose isomerase gene is shown in SEQ ID NO.

1.

2. The thermostable arabinose isomerase gene araA according to claim 1, characterized in that The thermostable arabinose isomerase gene araA is obtained by amplifying the amino acid sequence of the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4 using the PCR method.

3. An expression protein of the thermostable arabinose isomerase gene araA according to claim 1, characterized in that: The amino acid sequence of the expressed protein is shown in SEQ ID NO.

2.

4. A derivative protein of an expressed protein, characterized in that The derivative protein of the expressed protein is a derivative protein formed by substitution, deletion or addition of one or more amino acid residues of the amino acid of the expressed protein of the thermostable arabinose isomerase gene araA according to claim 3.

5. A recombinant vector containing the thermostable arabinose isomerase gene araA according to claim 1.

6. A recombinant organism comprising the thermostable arabinose isomerase gene araA according to claim 1 or the recombinant vector according to claim 5.

7. The recombinant organism according to claim 6, characterized in that The recombinant organism is a strain.

8. Use of the thermostable arabinose isomerase gene araA according to claim 1 or 2, the expression protein of the thermostable arabinose isomerase gene araA according to claim 3, or the derivative protein of the expression protein according to claim 4 in the food industry for enzymatically catalyzing the substrate D-galactose.

9. Use of the recombinant vector according to claim 5 or the recombinant organism according to claim 6 or claim 7 in the food industry for enzymatically catalyzing the substrate D-galactose.

10. The use according to claim 8 or 9, characterized in that: D-galactose is converted into D-tagatose at 70-90°C and pH 4.5-5.5.

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