Sucrase mutant, expression strain of sucrase mutant and application of sucrase mutant in hydrolysis of carbohydrates in soybean milk

Abstract

The invention discloses a sucrase mutant, an expression strain of the sucrase mutant and application of the sucrase mutant in hydrolysis of carbohydrates in soybean milk, and belongs to the technical field of biology. The sucrase mutant disclosed by the invention is simply recorded as InvDz13-TBT-01, and the amino acid sequence of the sucrase mutant is shown as SEQ ID NO: 2. The sucrase mutant InvDz13-TBT-01 is subjected to heterologous expression in bacillus subtilis, a recombinant strain is obtained, and the specific enzyme activity and the stability of protein produced by fermentation of the recombinant strain are remarkably improved. When sucrose is used as a substrate, the specific enzyme activity of the mutant enzyme is improved by 1.94 times, and when raffinose is used as a substrate, the specific enzyme activity of the mutant enzyme is improved by 1.7 times. Under the condition of 35 DEG C, the stability of the mutant enzyme is improved by 4 times. Compared with the starting enzyme, the thermal stability of the mutant enzyme is greatly improved while the specific enzyme activity is maintained. The sucrase mutant disclosed by the invention has a potential value in the application of hydrolyzing carbohydrates such as raffinose in soybean milk.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a sucrase mutant, its expression strain, and its application in the hydrolysis of sugars in soy milk. Background Technology

[0002] Soy milk, soy products, and other soybean derivatives are important sources of high-nutritional-value protein in daily life due to their affordability, availability, and high isoflavone content. However, soy products contain a large amount of raffinose, which, when utilized by intestinal bacteria, can cause bloating and negatively impact health. Traditional methods for reducing raffinose content in soy products involve soaking and cooking, but this method is inefficient and leads to nutrient loss. Currently, various processing techniques for removing raffinose from soybeans and soy milk have been researched. Among these techniques, the method of using α-galactosidase to hydrolyze raffinose into sucrose and galactose has been extensively studied because raffinose is an α-galactosyl derivative of sucrose. In contrast to the strategy using α-galactosidase, using sucrase to hydrolyze raffinose into melibiose under mild conditions can effectively increase the nutritional value of soy products.

[0003] Invertase (EC 3.2.1.26), also known as β-D-fructofuranoside hydrolase, catalyzes the hydrolysis of β-D-fructofuranoside bonds in non-reducing sugars with relative specificity. It can catalyze the hydrolysis of sucrose to glucose and fructose, as well as raffinose to melibiose and fructose. However, the lack of high-quality enzyme resources with high catalytic activity for raffinose and the low production level of invertase limit the practical application of invertase-based enzyme catalysis processes in the soybean product processing industry.

[0004] Previously, our laboratory obtained a sucrase enzyme, InvDz13, with high catalytic activity for raffinose from *Microbacterium trichothecenolyticum* (its amino acid sequence is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 3). InvDz13 can effectively hydrolyze raffinose into melibiose under mild conditions, significantly improving the nutritional value of soy products. However, the enzyme expression level is low, and its half-life is short, only about 10 hours at room temperature, which greatly limits the practical application of sucrase-based enzyme catalysis in the soy product processing industry. Therefore, obtaining a sucrase mutant with good enzyme activity and stability using protein engineering technology has potential value for the application of hydrolyzing sugars in soy milk. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a sucrase mutant, InvDz13-TBT-01, with enhanced specific activity and stability, its encoding gene, expression strain, and applications. Based on InvDz13 derived from *Trichoderma unicinophila*, this invention utilizes PROSS to design mutation sites and synthesizes the entire mutant gene. After induction expression in engineered bacteria containing the mutant gene's recombinant expression vector, a mutant with significantly improved specific activity and stability is obtained. With sucrose as a substrate, the mutant enzyme exhibits a specific activity of 424.26 U / mg, 1.94 times that of the original enzyme. With raffinose as a substrate, the specific activity is 382 U / mg, 1.7 times that of the original enzyme. Its thermal stability is also significantly improved; at 35 °C and pH 7.5, the mutant enzyme's stability increases fourfold, greatly extending its stability. Therefore, this mutant enzyme has application value in the hydrolysis of sugars in soy milk.

[0006] This invention relates to a sucrase mutant, abbreviated as InvDz13-TBT-01, whose amino acid sequence is shown in SEQ ID NO: 2. Specifically, in the amino acid sequence of sucrase InvDz13 shown in SEQ ID NO: 1, aspartic acid at position 35 is mutated to proline, alanine at position 41 is mutated to proline, alanine at position 90 is mutated to proline, serine at position 106 is mutated to threonine, glutamine at position 122 is mutated to tryptophan, valine at position 141 is mutated to tryptophan, aspartic acid at position 210 is mutated to alanine, lysine at position 244 is mutated to tryptophan, glutamic acid at position 314 is mutated to proline, glycine at position 335 is mutated to alanine, asparagine at position 391 is mutated to proline, isoleucine at position 407 is mutated to leucine, phenylalanine at position 440 is mutated to proline, and isoleucine at position 444 is mutated to proline.

[0007] The coding gene for the sucrase mutant of this invention has the nucleotide sequence shown in SEQ ID NO: 4.

[0008] The recombinant vector of the present invention contains the coding gene of the sucrase mutant as described in SEQ ID NO: 4.

[0009] The strain expressing the sucrase mutant of the present invention contains the recombinant vector described above.

[0010] The engineered strain expressing the sucrase mutant of this invention, classified as Bacillus subtilis WB600 / pBHSS4-SPyomL-sppA-InvDz13-TBT-01, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026019, deposited on January 6, 2026, at Wuhan University, Wuhan, China.

[0011] The method for constructing an engineered strain expressing a sucrase mutant according to the present invention includes the following steps:

[0012] Sequence alignment revealed a high degree of similarity between InvDz13 and 3wpu.1.A Beta-fructofuranosidase (PDB code: 3WPU) from Saccharomyces cerevisiae, with an amino acid sequence similarity of 85.10%. Therefore, using the structure of 3wpu.1.A Beta-fructofuranosidase as a template, the structure of sucrase InvDz13-TBT-01 was homologously modeled using Swiss-Model.

[0013] PROSS (https: / / pross.weizmann.ac.il) is an automated algorithm based on atomic Rosetta modeling and phylogenetic sequence information. Without altering the conformation and catalytic properties of the enzyme's active site, PROSS efficiently enhances the thermal stability and heterologous expression levels of proteins by introducing surface mutations. In PROSS, small molecule ligands and interacting strands are specified; mutations do not occur around these ligands and at the amino acid positions in contact with the strand, and their conformations remain fixed. Furthermore, completely conserved amino acids can be specified, which also remain fixed during mutation. Nine mutant designs were ultimately generated and ranked according to mutation rate. Their solubility in *E. coli* was predicted, with a result greater than 0.5 considered soluble. The target amino acid for the mutation was determined, the entire gene was synthesized, and ligated into the expression vector pET-28a(+). The mutant gene was then digested with enzymes and ligated into the *Bacillus subtilis* expression vector pBHSS4-SP. yomL- sppA was expressed in Bacillus subtilis WB600 as the host, with NdeI and XhoI restriction sites, to obtain the engineered strain Bacillus subtilis WB600 / pBHSS4-SP containing the mutant gene of this invention. yomL- sppA-InvDz13-TBT-01.

[0014] The expression plasmid vector described in the above construction method includes pBHSS4.

[0015] The host bacteria mentioned in the above construction method include Bacillus subtilis WB600.

[0016] The sucrase mutant of this invention can be obtained by fermentation using the engineered strain described above. The fermentation conditions are as follows: The obtained engineered strain Bacillus subtilis WB600 / pBHSS4-SP is used... yomL- sppA-InvDz13-TBT-01 was inoculated into 200 mL of YT liquid medium containing kanamycin (the composition and concentration of YT liquid medium are as follows: yeast extract 10 g / L, peptone 16 g / L, sodium chloride 5 g / L), and cultured at 30℃ and 200 rpm for 48 h; the supernatant was collected by centrifugation at 4℃ and 8000 g to obtain the crude enzyme solution of the sucrase mutant InvDz13-TBT-01.

[0017] The application of the sucrase mutant of this invention in the carbohydrates of hydrolyzed soy milk.

[0018] The application steps are as follows: Dissolve crude soybean powder in water at a total ratio of 1:7. Filter the crude slurry through a fine cotton cloth to obtain crude soybean milk. Boil the crude soybean milk for 15 minutes to obtain boiled soybean milk. Add InvDz13 and InvDz13-TBT-01 (final concentration 10 U / mL) to 100 mL of both crude soybean milk and boiled soybean milk. React in a shaking incubator at 30°C and 100 rpm / min for 1 hour. Boil the crude soybean milk treated with InvDz13 and InvDz13-TBT-01 for another 15 minutes. Take 5 mL of each soybean milk sample and mix it with 70% ethanol (1:1, v / v) for 5 minutes. Then centrifuge at 12000 × g for 30 minutes, recover the supernatant, filter through a 0.22 µm filter, and determine the sugar content using HPLC after sample preparation.

[0019] Under conditions of 35℃ and pH 7.5, the specific activity of the mutant enzyme increased by 1.94 times when using sucrose as a substrate, and by 1.7 times when using raffinose as a substrate. The stability of the mutant enzyme also increased by 4 times. In the application of hydrolyzing sugars such as raffinose in soy milk under mild conditions, after treating the sample with the mutant enzyme for 1 hour, the degradation rate of sucrose, stachyose, and raffinose approached 100%, and the content of melibiose increased by 2.6 times. This mutant enzyme has potential value in the application of hydrolyzing sugars in soy milk.

[0020] This invention measured and compared the specific activity, optimal pH, optimal temperature, and stability of sucrase InvDz13 and its mutant, InvDz13-TBT-01. The results showed that the mutant enzyme exhibited a 4-fold increase in stability at 35℃ and pH 7.5. With sucrose as a substrate, the specific activity of the mutant enzyme increased by 1.94 times; with raffinose as a substrate, the specific activity increased by 1.7 times. Compared to sucrase InvDz13, the optimal pH of the mutant changed from 7.0 to 7.5; and the optimal temperature changed from 45℃ to 55℃. Attached Figure Description

[0021] Figure 1 SDS-PAGE images of the purified mutant protein and the starting enzyme InvDz13: Figure 1 In the table, 1 is the pure InvDz13 enzyme, 2 is the InvDz13 fermentation supernatant, 3 is the InvDz13 cell precipitate, 4 is the InvDz13-TBT-01 cell precipitate, 5 is the InvDz13-TBT-01 fermentation supernatant, 6 is the pure InvDz13-TBT-01 enzyme, and M is the protein marker.

[0022] Figure 2 In this context, a represents the optimal pH, and b represents the optimal temperature.

[0023] Figure 3 The half-life at 35°C is the starting enzyme InvDz13 and the mutant InvDz13-TBT-01.

[0024] Figure 4 The application of the starting enzyme InvDz13 and the mutant InvDz13-TBT-01 in the hydrolysis of sugars in soy milk. Detailed Implementation

[0025] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.

[0026] (I) Construction of the expression strain of the sucrase mutant gene of the present invention

[0027] 1. Selection of mutation sites in sucrase genes

[0028] Sequence alignment revealed a high degree of identity between InvDz13 and 3wpu.1.A Beta-fructofuranosidase (PDB code: 3WPU) from *Saccharomyces cerevisiae*, with an amino acid sequence identity of 85.10%. Therefore, using the structure of 3wpu.1.A Beta-fructofuranosidase as a template, homology modeling of the sucrase InvDz13-TBT-01 was performed using Swiss-Model. Literature review confirmed that the catalytic sites of 3wpu.1.A Beta-fructofuranosidase are 76 Asp, 246 Asp, and 338 Glu. Using PyMOL software, structural alignment of the amino acid sequence data of this enzyme with that of InvDz13 was performed, determining the active sites of InvDz13 to be 105 Asp, 275 Asp, and 367 Glu.

[0029] PROSS is an automated design method based on sequence and structure. In PROSS, small molecule ligands and interacting strands are specified, ensuring that no mutations occur around these ligands and at the amino acid positions in contact with the strand, and that their conformations remain fixed. Furthermore, completely conserved amino acids can be specified, which also remain fixed during mutation. An automated algorithm based on atomic Rosetta modeling and phylogenetic sequence information yielded nine mutation designs with progressively increasing mutation rates. These designs were then used to predict the solubility in *E. coli*, with a result greater than 0.5 considered soluble. Selecting soluble mutation designs based on predicted results, the target amino acids for mutation were ultimately determined as follows: aspartic acid at position 35 was mutated to proline, alanine at position 41 was mutated to proline, alanine at position 90 was mutated to proline, serine at position 106 was mutated to threonine, glutamine at position 122 was mutated to tryptophan, valine at position 141 was mutated to tryptophan, aspartic acid at position 210 was mutated to alanine, lysine at position 244 was mutated to tryptophan, glutamic acid at position 314 was mutated to proline, glycine at position 335 was mutated to alanine, asparagine at position 391 was mutated to proline, isoleucine at position 407 was mutated to leucine, phenylalanine at position 440 was mutated to proline, and isoleucine at position 444 was mutated to proline.

[0030] 2. Construction of the InvDz13 mutant genetically engineered strain of sucrase

[0031] The InvDz13 mutant gene of sucrase from step 1 was synthesized in its entirety and ligated into the expression vector pET-28a(+). The mutant gene was then digested and ligated into the Bacillus subtilis expression vector pBHSS4-SP. yomL-sppA was expressed in Bacillus subtilis WB600 as the host, with NdeI and XhoI restriction sites, to obtain the engineered strain Bacillus subtilis WB600 / pBHSS4-SP containing the mutant gene of this invention. yomL- sppA-InvDz13-TBT-01.

[0032] The engineered strain expressing the sucrase mutant of this invention, classified as Bacillus subtilis WB600 / pBHSS4-SPyomL-sppA-InvDz13-TBT-01, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026019, deposited on January 6, 2026, at Wuhan University, Wuhan, China.

[0033] (II) Expression and protein purification of the InvDz13 mutant genetically engineered strain of sucrase in this invention

[0034] The engineered strain Bacillus subtilis WB600 / pBHSS4-SP obtained in (I) yomL- sppA-InvDz13-TBT01 was inoculated into 200 mL of YT liquid medium containing kanamycin and cultured at 30℃ and 200 rpm for 48 h. The supernatant was collected by centrifugation at 4℃ and 8000 g to obtain the crude enzyme solution of the sucrase mutant InvDz13-TBT-01. The crude enzyme solution was purified by Ni-NTA column chromatography. The elution buffer contained 90 mM imidazole, and elution was performed for 3 column volumes. The purity of the obtained protein was determined by SDS-PAGE.

[0035] When sucrose and raffinose are used as substrates, the mutant enzyme has an optimal pH of 7.5 and exhibits over 50% catalytic activity within the pH range of 5.0-8.5. The mutant has an optimal temperature of 55℃, and the enzyme shows over 50% activity within the temperature range of 40℃-65℃.

[0036] (III) Detection of specific enzyme activity of the InvDz13 mutant of the present invention (DNS method)

[0037] 1. Definition of enzyme activity

[0038] 1 U is the amount of enzyme required to produce 1 µM sucrose per minute.

[0039] 2. Enzyme activity assay

[0040] The reaction system consisted of 900 µL of a mixture of 430 µL of 50 mM, pH 6.5 citrate-disodium hydrogen phosphate buffer and 1 M sucrose solution, and preheated at 35 °C for 10 min. 50 µL of crude enzyme solution was added, and the mixture was shaken and allowed to react for 10 min. 300 µL of DNS was added, and the mixture was shaken and boiled for 15 min, then cooled on ice. 300 µL of the supernatant was transferred to a 96-well plate, and the absorbance was read at A540 nm. The reducing sugar content was calculated based on the DNS standard curve, and the enzyme activity was calculated according to the definition of enzyme activity.

[0041] Protein content was determined using the BCA assay: First, solution A and solution B (4:1 ratio) were mixed in an EP tube. A 96-well plate was then added, along with 200 μL of the A / B mixture and 25 μL of concentrated protein. The protein concentration buffer was used as a control. After shaking, the plate was incubated at 37 °C for 30 min. The absorbance was measured at 562 nm using a microplate reader. The absorbance value was then substituted into the BCA standard curve to calculate the protein content.

[0042] The results showed that, using sucrose as a substrate, the specific activity of the mutant enzyme obtained in this invention was 424.26 U / mg, which was 1.94 times that of the starting enzyme. Using raffinose as a substrate, the specific activity of the mutant enzyme obtained in this invention was 382 U / mg, which was 1.7 times that of sucrase InvDz13.

[0043] (iv) Detection of the stability of the InvDz13 mutant of the present invention

[0044] Sucrosease InvDz13 and sucrosease mutant InvDz13-TBT-01 were heat-treated at 35℃ and pH 7.5. The enzyme activity was initially 100%, and the enzyme activity remaining rate after heat treatment for a certain period of time was calculated using the following formula: Enzyme activity remaining rate = Enzyme activity after heat treatment / Enzyme activity before heat treatment × 100%.

[0045] The results showed that at 35℃, the half-life of the sucrase mutant InvDz13-TBT-01 was 125 h, which was 4 times that of sucrase InvDz13.

[0046] (iv) Application of the InvDz13 mutant of the present invention in hydrolyzing raffinose and other sugars in soybean milk

[0047] Crude soybean powder and water were dissolved in a 1:7 ratio. The crude slurry was filtered through a fine cotton cloth to obtain crude soybean milk. The crude soybean milk was boiled for 15 min to obtain boiled soybean milk. InvDz13 and InvDz13-TBT-01 with a final concentration of 10 U / mL were added to 100 mL of crude soybean milk and boiled soybean milk, respectively. The mixture was reacted in a shaking incubator at 30℃ and 100 rpm / min for 1 h. The crude soybean milk treated with InvDz13 and InvDz13-TBT-01 was boiled again for 15 min. 5 mL of each soybean milk sample was mixed with 70% ethanol (1:1, v / v) for 5 min, then centrifuged at 12000 × g for 30 min. The supernatant was collected, filtered through a 0.22 µm filter, and the sugar content was determined by HPLC after sample preparation.

[0048] The sugar content in soy milk was determined by HPLC. Experimental results showed that sucrose, melibiose, raffinose, and stachyose were present in both crude and boiled soy milk. Boiling can partially remove raffinose from soy milk. Therefore, traditional processes mainly reduce raffinose through soaking and boiling, but these methods only partially remove raffinose (2.61 mM → 2.47 mM); they also increase the content of stachyose (1.7 mM → 1.97 mM) and sucrose (6.89 mM → 10.05 mM), failing to completely solve the problem of bloating. Adding InvDz13 and InvDz13-TBT-01 to crude or boiled soy milk resulted in the complete hydrolysis of sucrose, raffinose, and stachyose within one hour. After treatment with InvDz13, the melibiose concentration in crude soy milk increased from 1.29 mM to 2.09 mM, while the melibiose concentration in boiled soy milk increased from 1.30 mM to 2.42 mM. After treatment with InvDz13-TBT-01, the melibiose concentration in crude soy milk increased from 1.29 mM to 2.44 mM, while the melibiose concentration in boiled soy milk increased from 1.30 mM to 3.4 mM.

[0049] Both sucrase InvDz13 and its mutant InvDz13-TBT-01 can successfully convert raffinose, which causes bloating, into melibiose. However, the melibiose concentration in soy milk increased by approximately 2.6 times after treatment with InvDz13-TBT-01. Therefore, using InvDz13-TBT-01 to enzymatically hydrolyze raffinose in soy milk may be an alternative method to improve the nutritional value of soy milk.

Claims

1. A sucrase mutant, abbreviated as InvDz13-TBT-01, characterized in that... Its amino acid sequence is shown in SEQ ID NO:

2.

2. The coding gene of the sucrase mutant according to claim 1, characterized in that... Its nucleotide sequence is shown in SEQ ID NO:

4.

3. An engineered strain expressing the sucrase mutant of claim 1, characterized in that: The engineered strain, classified as Bacillus subtilis WB600 / pBHSS4-SPyomL-sppA-InvDz13-TBT-01, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2026019, on January 6, 2026, at Wuhan University, Wuhan, China.

4. The application of the sucrase mutant of claim 1 in the carbohydrates of hydrolyzed soy milk.

5. The application according to claim 4, characterized in that: The pH value of the hydrolysis system is 7.0-7.5, and the temperature is 30-45℃.

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