Aldehyde sugar isomerase mutant and use thereof

Abstract

The application belongs to the technical field of biology, and discloses an aldose isomerase mutant and application thereof. The aldose isomerase mutant with significantly improved stability is obtained by enzyme design and modification, accurate identification of amino acid residues related to improved thermal stability, and substitution mutation at positions of D296 and A128 corresponding to the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. The mutation sites in the three sequences with a sequence similarity of less than 60% all show similar effects and have certain universality. The enzyme mutant can be applied to convert glucose into fructose, and the conversion rate is effectively improved compared with the wild type.

Description

Technical Field

[0001] This invention relates to aldose isomerase variants with significantly enhanced thermal stability and catalytic activity, and their application in the biomanufacturing of starch sugars, belonging to the field of biotechnology. Background Technology

[0002] High-fructose corn syrup, the world's largest-produced starch-based sugar, has been widely used to replace sucrose in beverages, baking, and food processing due to its high sweetness, good processing performance, and cost advantages. Its core production step involves the conversion of glucose in starch saccharification liquid into fructose under the catalysis of aldose isomerase (glucose isomerase). Therefore, the catalytic efficiency and operational stability of glucose isomerase directly determine the sugar conversion rate, energy consumption, and overall cost of the production line, making it a key target for industrial technology upgrading.

[0003] Industrial-scale fructose syrup production typically takes place under continuous reaction conditions at high temperatures (60-65°C) and slightly alkaline conditions (pH 7.5-8.5). This process is designed to reduce system viscosity, inhibit microbial growth, and shift the reaction equilibrium towards fructose production. However, this harsh operating environment poses a significant challenge to enzyme molecules: high temperatures can easily lead to irreversible denaturation and inactivation of enzyme proteins, while alkaline conditions may disrupt the fine structure of their active sites. Currently, industrially used natural or early-modified glucose isomerase variants often suffer from short high-temperature half-lives, insufficient catalytic specific activity, or require expensive metal ions (such as Co). 2+ Issues such as the need for enzymes to maintain stability as cofactors force production to adopt methods such as increasing enzyme addition, shortening catalyst replacement cycles, or sacrificing reaction rates to maintain operation, severely restricting the improvement of production efficiency and economic benefits.

[0004] While protein engineering has enabled the directed evolution of enzymes, existing research on modifying glucose isomerases has largely focused on optimizing single properties. For example, some mutants improve thermal stability by introducing rigid structures or adding salt bridges, but this often comes at the cost of decreased substrate binding capacity or a lower turnover number. Other modifications aimed at broadening the pH adaptation range or improving activity may come at the expense of overall structural rigidity. This trade-off makes it exceptionally difficult to develop variants that synergistically possess both excellent stability and high catalytic activity in real-world industrial environments. Therefore, breaking this performance balance constraint and creating a glucose isomerase mutant that can adapt to extreme process conditions while significantly improving stability and catalytic activity is of crucial practical significance for driving the development of fructose syrup and downstream technologies toward greater efficiency, energy conservation, and environmental friendliness. Summary of the Invention

[0005] This invention relates to a thermostability-enhanced aldose isomerase mutant site and / or mutants and combinations thereof. The invention utilizes rational design, site-directed saturation mutagenesis, and combined mutagenesis to screen for mutants with increased enzyme activity. The objective of this invention is to provide mutants capable of enhancing aldose isomerase activity.

[0006] This invention is achieved through the following technical solution:

[0007] The present invention provides an aldose isomerase mutant, which is obtained by substitution mutation at one or both positions of D296 and A128 corresponding to the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0008] Preferably, the substituted mutation is D296 mutated to Q or K, A128 mutated to V, L or F, or D296 mutated to Q or K and A128 mutated to V, L or F.

[0009] The present invention also provides the encoding gene of the aldose isomerase mutant.

[0010] The present invention also provides a recombinant expression vector containing the aforementioned coding gene.

[0011] The present invention further provides a recombinant bacterium containing the aforementioned coding gene and the aforementioned recombinant expression vector.

[0012] This invention also provides the application of the aldose isomerase mutant, the encoding gene, the recombinant expression vector, or the recombinant bacteria of the recombinant expression vector in the catalytic production of fructose from glucose.

[0013] The present invention provides a method for catalyzing the production of fructose from glucose, wherein the aldose isomerase mutant is contacted with the substrate glucose to catalyze the reaction to produce fructose.

[0014] Specifically, the reaction system is as follows: 300-800 g / L glucose, 40-60 mM PBS buffer at pH 7.5, and 30-80 mg / L aldose isomerase mutant;

[0015] The reaction conditions are 30-70℃; after the reaction is completed, the reaction is terminated by heating in a boiling water bath at 100℃.

[0016] Preferably, the reaction system is as follows: 400-600 g / L glucose, 45-55 mM PBS buffer at pH 7.5, and 40-60 mg / L aldose isomerase mutant;

[0017] The reaction conditions are 40-65℃; after the reaction is completed, the reaction is terminated by heating in a boiling water bath at 100℃ for 10 minutes.

[0018] Particularly preferred is the following reaction system: 500 g / L glucose, 50 mM PBS buffer at pH 7.5, and 50 mg / L aldose isomerase mutant; the reaction condition is 60°C.

[0019] This invention, through enzyme design and modification, precisely identifies amino acid residues associated with improved thermostability, obtaining an aldose isomerase mutant with significantly enhanced stability. Experiments show that the enzyme's stability at 70℃ is increased by 4-6 times, and the catalytic efficiency for the isomerization of glucose to fructose is increased by 2 times or more. The mutation site exhibits similar effects in three sequences with less than 60% sequence similarity, demonstrating a certain degree of versatility. Applying this enzyme mutant can convert glucose into fructose, effectively improving the conversion rate compared to the wild type. Detailed Implementation

[0020] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0021] Example 1: Expression and activity evaluation of aldose isomerase (glucose isomerase)

[0022] 1. Glucose isomerase sequences from thermophilic species were selected from gene and protein databases such as KEGG, UniProt, and NCBI. Six amino acid sequences were selected as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. E. coli codon optimization and gene synthesis were performed (Nanjing Genscript Biotech Co., Ltd.). The sequences were then constructed into the pET21 vector to obtain recombinant expression vectors pET21-GI1, pET21-GI2, pET21-GI3, pET21-GI4, pET21-GI5, and pET21-GI6, respectively.

[0023] 2. The above positive recombinant plasmid was transformed into the expression host BL21(DE3) of Escherichia coli to obtain the recombinant expression strain expressing GI.

[0024] 3. The GI wild-type and its mutant recombinant expression strains were inoculated into 3 mL of LB medium and activated overnight to obtain seed culture. The seed culture was then transferred to LB medium at a 1% inoculation rate and cultured until OD (Organization Time). 600=0.6-0.8, add IPTG to a final concentration of 0.1mM to induce the expression of heterologous proteins. Collect the strain by centrifugation, suspend the bacterial cells in 50mM PBS (pH=6.5) buffer, then sonicate to disrupt the protein, centrifuge at 14000rpm for 30min to obtain the supernatant, purify it by Ni column affinity chromatography, and further ultrafilter it with a 30 kDa ultrafiltration tube to obtain concentrated and purified wild-type and mutant proteins.

[0025] Example 2. Design and Construction of GI Mutants

[0026] (1) Select 200 items from KEGG, UniProt, NCBI and other databases including Dissulfuribactersp., Candidatus Thermofonsia, Thermosporothrix sp., Thermasporomyces sp., Sulfobacillus thermotolerans, Thermomicrobiales, Georgenia thermotolerans, Thermus thermophilus, Thermoflexia bacterium, Thermoleophilaceae bacterium, Moorella thermoacetica, Thermoactinospora sp, Streptomycesthermocyaneoviolaceus, Acetivibrio saccincola, Thermobacillus sp., Thermoproteiarchaeon, Dictyoglomus thermophilum, Geobacillus thermopakistaniensis, Capillibacterium thermochitinicola, Acidothermus sp., Rhodothermus Glucose isomerase sequences from thermophilic species such as *Marinus* were analyzed using computational virtual software including ProteinMPNN, HotSpot, Rosetta Design, and Caver for sequence analysis, structural analysis, molecular docking, channel analysis, and free energy mutation detection. Amino acid residues D296 and A128 were identified as exhibiting strong interactions with glucose at both substrate entry and exit channels in the protein structure. Virtual saturation mutagenesis revealed that mutating D at position 296 to Q or K, and mutating A at position 128 to V, L, or F, lowered the binding free energy and improved substrate binding efficiency. Therefore, these two sites will be used to validate the results in glucose isomerases such as SEQ ID NO:1.

[0027] (2) Using recombinant plasmid pET21-GI1 as a template, primers with mutation sites were used to perform full plasmid PCR amplification with high-fidelity enzymes. The amplification products were digested with DpnI enzyme at 37°C for 2 hours to degrade the initial template. The digestion products were transformed into E. coli DH5α, plated on LB agar plates containing 100 μg / mL ampicillin, and cultured overnight at 37°C. Sequencing was used to verify whether the mutation site was successful, thereby obtaining the recombinant plasmid with the specified mutation site.

[0028] The catalytic activity of the mutant was determined using the protein expression and purification method shown in Example 1. The glucose isomerase activity assay system was as follows: 100 mM glucose, PBS buffer (pH=7.5, 50 mM), 50 μg / mL purified protein, reacted at 60 ℃ and 850 rpm for 10 minutes. After the reaction, the reaction was immediately terminated by placing the mixture in a boiling water bath for 10 minutes, and the activity was determined by high-performance liquid chromatography (HPLC). The HPLC analysis was performed under the following conditions: Agilent HPLC 1200 instrument, Sugar-Pak column, column temperature: 80 ℃, mobile phase: ddH2O, flow rate: 0.4 mL / min, and sample loading volume: 10 μL.

[0029] To test the thermostability of the mutants, the purified enzyme was placed in a 70 °C water bath for different times (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h). Samples were then taken and enzyme activity was measured according to the enzyme activity assay method described above, thus comparing the thermostability and catalytic activity of different mutants. Table 1 lists the dominant single-point mutants with improved thermostability and catalytic activity, with wild type 1.

[0030] Table 1. Single-point mutants with improved stability and catalytic activity.

[0031] ;

[0032] Experimental results showed that adding D296Q, D296K, A128V, A128L, and A128F to the GI1 sequence can improve the catalytic activity and stability of the mutant, with stability increased by 1.9-3.2 times and catalytic activity increased by 1.3-2.1 times compared to the wild type.

[0033] Example 3. Combination mutations at dominant sites in GI

[0034] The single-point mutants exhibiting significantly enhanced thermal stability and catalytic activity were combined in pairs, and their catalytic activity and enzyme stability at 70°C were measured to calculate the fold increase compared to the wild type. Table 2 lists the relative proportions of the combined mutants with significantly enhanced thermal stability and catalytic activity, with the wild type represented by 1.

[0035] Table 2. Combined mutants with enhanced stability and catalytic activity

[0036] ;

[0037] As shown in Table 2, six combined mutants were obtained by combining the above-mentioned advantageous single-site mutants with GI1. Using the stability and catalytic activity determination methods shown in Example 2, the stability and catalytic activity of the combined mutants were further improved compared with the single-site mutants shown in Table 1. The stability was increased by 3.9-5.4 times compared with the wild type, and the catalytic activity was increased by 1.8-2.6 times compared with the wild type. Among them, the mutant GI1-D296K-A128L showed a 5.4-fold increase in stability and a 2.3-fold increase in catalytic activity compared with the wild type, showing a good synergistic effect between the mutant residues at the two sites.

[0038] Example 4. Application testing of combinatorial mutations in other GIs

[0039] Considering the excellent effects of combined mutations of D296K, A128L, and A128F in GI1, these mutations were introduced into GI2, GI3, GI4, GI5, and GI6, respectively, to test the effects of these mutations on enzyme activity and stability.

[0040] Using the methods described in Examples 2 and 3, and with pET21-GI2, pET21-GI3, pET-GI4, pET21-GI5, and pET-GI6 as templates, respectively, combined mutants D296K / A128L and D296K / A128F were introduced to obtain 12 mutants, as shown in Table 3. These mutants were further transformed into E. coli BL21 for expression, and the stability and catalytic activity of the purified enzymes were tested. Table 3 lists the relative proportions of combined mutants with significantly improved thermostability and catalytic activity, with wild-type represented by 1.

[0041] Table 3. Effects of different GI mutants on stability and catalytic activity

[0042] ;

[0043] The results above show that the combined mutations of D296K / A128L and D296K / A128F in GI2, GI3, GI4, GI5 and GI6 can improve the enzyme's catalytic activity and stability, with the highest catalytic activity increase being 5.4 times and the highest stability increase being 3.2 times.

[0044] Example 5. Using GI and its mutants to convert glucose into fructose

[0045] The above-mentioned GI and its mutants were applied to the conversion of glucose into fructose. The reaction system was as follows: 500 g / L glucose, 50 mM PBS buffer (pH=7.5), 50 mg / L glucose isomerase GI, and reaction was carried out at 60℃ for 12 h. Samples were taken at different time points during the reaction. After the reaction was completed, the reaction was terminated by heating in a boiling water bath at 100℃ for 10 min, and the results were determined by high performance liquid chromatography.

[0046] Table 4 Comparison of glucose-to-fructose synthesis efficiency of different mutants

[0047] ;

[0048] As shown in Table 4, in the reaction system catalyzing the conversion of glucose to fructose, the fructose conversion rate of the mutant was higher than that of the corresponding wild type after 4 hours of reaction, with a maximum increase of 2.6 times. Therefore, the GI mutant obtained by this invention has a superior fructose synthesis efficiency compared to the wild type. Furthermore, when the glucose obtained by this invention reaches reaction equilibrium after 12 hours, both the wild-type and mutant catalyzed reaction systems achieve an equilibrium conversion rate of approximately 42%, thus proving that the GI and its mutant can be applied to the fructose biomanufacturing field in the starch sugar industry.

[0049] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. 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 aldose isomerase mutant, characterized in that, The mutant, based on the amino acid sequence corresponding to SEQ ID NO:3, has only the following mutations: D296 is mutated to K and A128 is mutated to L or F.

2. The encoding gene of the aldose isomerase mutant as described in claim 1.

3. A recombinant expression vector containing the encoding gene as described in claim 2.

4. Recombinant bacteria containing the coding gene as described in claim 2 and the recombinant expression vector as described in claim 3.

5. The application of the aldose isomerase mutant as described in claim 1, the encoding gene as described in claim 2, the recombinant expression vector as described in claim 3, or the recombinant bacteria as described in claim 4 in catalyzing the production of fructose from glucose.

6. A method for catalyzing the conversion of glucose into fructose, characterized in that, The aldose isomerase mutant as described in claim 1 is contacted with the substrate glucose to catalyze a reaction to produce fructose.

7. The method as described in claim 6, characterized in that, The reaction system is as follows: 300-800 g / L glucose, 40-60 mM PBS buffer at pH 7.5, and 30-80 mg / L aldose isomerase mutant.

8. The method as described in claim 7, characterized in that, The reaction conditions are 30-70℃; after the reaction is completed, the reaction is terminated by heating in a boiling water bath.

9. The method as described in claim 8, characterized in that, The reaction system is as follows: 400-600 g / L glucose, 45-55 mM PBS buffer at pH 7.5, and 40-60 mg / L aldose isomerase mutant; The reaction conditions are 40-65℃; after the reaction is completed, the reaction is terminated by heating in a boiling water bath at 100℃ for 10 minutes.

10. The method as described in claim 9, characterized in that, The reaction system was as follows: 500 g / L glucose, 50 mM PBS buffer (pH 7.5), and 50 mg / L aldose isomerase mutant; the reaction temperature was 60 °C.