A starch sucrolase mutant and its use in the preparation of medium polymeric degree amylopectin

Abstract

This invention discloses a starch sucrase mutant and its application in the preparation of medium-polymerized amylopectin, belonging to the fields of genetic engineering and enzyme engineering technology. The mutant is H422A obtained by mutating histidine at position 422 of the starch sucrase enzyme (as shown in SEQ ID No. 1) to alanine, or E373A obtained by mutating glutamic acid at position 373 to alanine. The mutant provided by this invention achieves a moderate reduction in starch chain length and a significant enrichment of medium and short chains, solving the technical problems of excessively long or unevenly distributed chains in existing modified starch sucrase products. The resulting medium-polymerized amylopectin has good application prospects in food processing and materials fields.

Description

Technical Field

[0001] This invention relates to a starch sucrase mutant and its application in the preparation of amylopectin with moderate degree of polymerization, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] Starch is a natural high-molecular-weight polysaccharide composed of glucose units linked by α-1,4-glycosidic bonds and α-1,6-glycosidic bonds. Its molecular structure includes amylose and amylopectin. The chain length distribution of amylopectin directly affects its physicochemical properties and functional characteristics, such as gelatinization characteristics, viscosity, digestibility, and processing adaptability. Amylopectin with a moderate degree of polymerization (DP 18~25) has unique application value. After gelatinization, it has moderate viscosity and good transparency, and can be used to improve product texture in the food industry and to prepare biodegradable films in the materials field.

[0003] Amylosucrase (AS) is a type of enzyme that catalyzes the transglycosylation reaction between starch and sucrose, belonging to the GH13 family of glycosidases. This enzyme uses sucrose as a glycosyl donor, transferring glucose units to the non-reducing ends of starch acceptor molecules, thereby elongating the branched chain. (Source: [Original Source Name]) Neisseria polysaccharea The starch sucrase has strong transglycosylation activity and is an important biocatalyst for the preparation of modified starch.

[0004] However, modified products of wild-type starch sucrase suffer from excessively long chain lengths, with a Peak-DP of approximately 30. The high proportion of long-chain molecules and enhanced intermolecular entanglement limit processing performance. To address this issue, researchers have modified starch sucrase through protein engineering. However, existing modified starch products still suffer from uneven chain length distribution, and the insufficient thermal stability of current starch sucrase restricts their application in industrial production. Extreme differentiation in chain length distribution affects product uniformity and application adaptability. The chain length distribution (CLD) of starch is a key molecular structural parameter determining its digestibility and processing performance. Zhu et al. (2024) conducted a systematic study on seven waxy rice amylopectins, showing that the proportion of short chains (DP 6–24) in amylopectin was significantly positively correlated with the disintegration value (BDV) (r = 0.801), which is beneficial for improving processing adaptability; while the proportion of long chains (DP 68–78) was significantly negatively correlated with BDV (r = -0.911), and accelerated the second stage of digestion, reducing the content of slowly digestible starch. Specifically, the higher the proportion of shorter chains (DP 28–33) in the long-chain region, the faster the second-stage digestion rate (r = 0.751). These results confirm that uneven chain length distribution, an excessively high proportion of long chains, or a large peak chain length (e.g., Peak-DP ≈ 30) can lead to increased molecular entanglement, limited processing performance, and decreased product uniformity. Therefore, optimizing chain length distribution through protein engineering and other methods is an effective strategy for improving the application performance of modified starch.

[0005] Therefore, there is an urgent need in this field to develop a starch sucrase mutant that can moderately regulate the starch chain length to a medium degree of polymerization and achieve enrichment and concentrated distribution of medium and short chains, so as to obtain modified starch products with moderate chain length, uniform distribution and excellent processing performance. Summary of the Invention

[0006] To address the technical problems of excessively elongated chain length, insufficient content of medium and short chains, and poor thermal stability in the modified starch sucrase products of the prior art, this invention provides a starch sucrase mutant that can moderately reduce the Peak-DP of modified starch from 30 in the wild type to 19, while significantly enriching short chain B1 (DP 13-24) and inhibiting the synthesis of medium and long chain B2 (DP 25-36) and ultra-long chain B3 (DP>36).

[0007] The first technical solution provided by this invention is a starch sucrase mutant, which is obtained by performing any one of the following mutations on the starch sucrase parent with the amino acid sequence shown in SEQ ID No. 1: (1) Histidine at position 422 is mutated to alanine to obtain H422A; (2) The glutamic acid at position 373 is mutated to alanine, resulting in E372A.

[0008] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0009] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0010] In one embodiment of the present invention, the recombinant vector is pET-20b(+) as the expression vector.

[0011] The present invention also provides a recombinant cell carrying the above-mentioned gene or the above-mentioned recombinant vector.

[0012] In one embodiment of the present invention, the recombinant cells use bacteria or fungi as host cells.

[0013] The present invention also provides a recombinant Escherichia coli expressing the above-mentioned mutant, to... Escherichia coli BL21(DE3) was used as the host cell and pET-20b(+) was used as the expression vector.

[0014] The present invention also provides a method for preparing the above-mentioned mutant, comprising the steps of constructing the expression vector and fermenting the recombinant cells for production.

[0015] This invention also provides a method for preparing amylopectin with a medium degree of polymerization, using the starch sucrase H422A and E372A mutants, with starch and sucrose as substrates, controlling the peak-DP of the modified starch in the range of 18 to 22, controlling the proportion of medium and short chain B1 to be above 45%, and controlling the proportion of long chain B3 to be below 10%.

[0016] In one embodiment, the method involves gelatinizing the ordinary corn starch, dissolving it in sucrose, adding the starch sucrase mutant containing the substrate, and reacting at 40-50 °C for 4-10 h.

[0017] The present invention also provides the application of the starch sucrase mutant, the gene sequence, the recombinant expression vector, or the recombinant cell in the preparation of amylopectin with moderate degree of polymerization in the food, pharmaceutical, biological, or materials fields.

[0018] The beneficial effects of this invention are as follows: 1) This invention achieves moderate and precise regulation of starch chain length by site-directed mutagenesis at positions 422 and 373 of the starch sucrase enzyme. Compared with the wild type, the Peak-DP of starch modified by the H422A and E373A mutants decreased from 30 to 19, a reduction of 36.7%, and the product chain length was within the range of moderate polymerization.

[0019] 2) The mutants provided by this invention significantly enriched short- and medium-chain proteins and inhibited the synthesis of very long-chain proteins. Compared with the wild type (Peak-DP 30, A chain 5.23%, B1 chain 22.82%, B2 chain 41.74%, B3 chain 28.32%), the Peak-DP of H422A modified starch decreased to 19, the proportion of short- and medium-chain B1 proteins significantly increased from 22.82% to 53.46%, an increase of 134.3%; the proportion of medium- and long-chain B2 proteins decreased from 41.74% to 28.14%, a decrease of 32.6%; and the proportion of long-chain B3 proteins decreased from 28.32% to 9.97%, a decrease of 64.8%. The Peak-DP of E373A modified starch also decreased to 19, the proportion of B1 proteins further increased to 54.79%, an increase of 140.1% compared with the wild type; the proportion of B2 proteins decreased to 28.75%, a decrease of 31.1%; and the proportion of B3 proteins decreased to 8.61%, a decrease of 69.6%. E373A has a 1.33% higher proportion of B1 chain and a 1.36% lower proportion of B3 chain compared to H422A, resulting in a more concentrated chain length distribution. This achieves improvements in the centralization and uniformity of chain length distribution.

[0020] 3) Unlike the modified starch products in the prior art that have excessively elongated chain lengths or uneven distribution, the H422A and E373A mutants of this invention produce a distribution pattern with significant enrichment of medium and short chains and moderate inhibition of long chains. They have both appropriate chain lengths and good uniformity, which can effectively change the thermal stability and energy absorption characteristics of starch granules, enabling them to complete the gelatinization process at lower temperatures. This reduces processing energy consumption, improves processing adaptability, and endows the product with controllable thermal processing properties and application potential.

[0021] 4) The preparation method provided by this invention has mild conditions and is easy to operate, providing a feasible technical solution for the large-scale production of amylopectin with medium degree of polymerization. Attached Figure Description

[0022] Figure 1 Agarose gel electrophoresis images of wild-type amylose sucrase and its H422A mutant plasmid; where M: standard molecular weight of DNA, 1: wild-type plasmid, 2: H422A mutant plasmid, 3: D421A mutant plasmid, 4: E373A mutant plasmid.

[0023] Figure 2 SDS-PAGE images of wild-type amylose sucrase and its H422A mutant; where M: standard molecular weight of protein, 1: D421A mutant, 2: wild type, 3: H422A mutant, 4: E373A mutant.

[0024] Figure 3 The results show the enzyme activity of wild-type starch sucrase and its mutants.

[0025] Figure 4 The results show the thermostability of wild-type amylase and various mutants.

[0026] Figure 5 This is a comparison diagram of the chain length distribution of wild-type starch sucrase and modified starches from various mutants. Detailed Implementation

[0027] The embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0028] The detection method used in the example: (1) Method for determining starch sucrase activity Starch sucrase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method. 0.1 mL of appropriately diluted enzyme solution was added to a centrifuge tube containing 0.9 mL of a mixture of pre-gelatinized and cooled starch solution and sucrose. The reaction was carried out at 40 °C for 10 min. 1.0 mL of DNS was added to terminate the reaction. After vortexing and mixing, the mixture was boiled in a water bath for 5 min, followed by cooling in an ice-water bath. 2.0 mL of deionized water was added, and the absorbance was measured at 540 nm. An inactivated enzyme solution was used as a blank control. One unit of enzyme activity was defined as the release of 1 μmol of fructose per minute.

[0029] (2) Analytical methods for reaction products The chain length distribution of starch was determined using high-performance anion chromatography-pulse amperometric detector (HPAEC-PAD). 10 mg of sample was accurately weighed into a 10 mL centrifuge tube, then placed in a rotor and 2.0 mL of sodium acetate buffer (50 mmol / L, pH 3.5) was added. The tube was then placed in a 500 or 1000 mL beaker and subjected to a boiling water bath with continuous stirring for 30 min to gelatinize. The sample was then placed in a 40 °C water bath shaker for 15 min to equilibrate, followed by the addition of 100 μL of isoamylase (10000 U / mL). The reaction was carried out at 40 °C and 160 r / min for 24 h to completely debranch the starch. The reaction was terminated by boiling in a water bath for 30 min. After the sample solution cooled to room temperature, it was transferred to a 5 mL centrifuge tube and centrifuged at 10000 r / min for 10 min. A suitable amount of the supernatant was filtered through a 0.22 μm aqueous filter membrane to ensure the sample was free of turbidity. The product was then analyzed using ion chromatography chain length distribution detection.

[0030] Raw materials used in the examples: LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0031] LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0, 1.5% (w / v) agar.

[0032] pET20b(+) vector Escheerichia coli JM109 competent cells, Escherichia coli BL21(DE3) is a commercial plasmid and a commercial strain.

[0033] Raw material sources: tryptone and yeast extract were purchased from Oxoid, UK; sodium chloride, glycerol, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0034] Example 1: Preparation method of H422A mutant The artificially synthesized wild-type starch sucrase gene sequence (shown in SEQ ID No. 2) was inserted between the NcoⅠ and XhoⅠ restriction sites of the vector pET-20b(+) to obtain the wild-type recombinant plasmid pET-Np.

[0035] Using the recombinant vector pET-Np as a template, site-directed mutagenesis was performed using the primers listed in Table 1. The primers were synthesized by Genewiz Biotechnology Co., Ltd. A one-step PCR method was used for synthesis. The PCR system consisted of: 25 μL of 2×phanta Max Master Mix (Dye plus), 2 μL of forward primer (10 μM), 2 μL of reverse primer (10 μM), 1 μL of template DNA, and double-distilled water to a final volume of 50 μL. PCR amplification conditions were: 95 °C pre-denaturation for 3 min; followed by 30 cycles (95 °C for 15 s, 60 °C for 15 s, 72 °C for 5 min); and finally, incubation at 72 °C for 5 min. The PCR product was digested with DpnI enzyme to obtain the processed PCR product. E. coli The JM109 competent state conversion method was transferred to... E. coli In JM109, transformants were prepared and plated on LB solid medium containing ampicillin (20 ug / mL). The medium was incubated at 37 ℃ inverted for 12 h. Positive clones were picked and transferred to LB liquid medium containing ampicillin (20 ug / mL), and incubated at 37 ℃ and 200 rpm for 10–12 h. Plasmids were extracted and sent to the company for sequencing, yielding recombinant plasmids containing the mutant gene. The plasmid extraction verification results are as follows: Figure 1 As shown.

[0036] Table 1. Mutant Primer Sequences

[0037] Example 2: Construction of genetically engineered bacteria and expression of the H422A mutant (1) The wild-type and H422A, D421A, and E373A mutant recombinant plasmids obtained in Example 1 were transformed into [the following] respectively. E. coli Wild-type recombinant strains and H422A, D421A, and E373A mutant recombinant strains were prepared from BL21. The recombinant strains were streaked on LB solid medium containing ampicillin (20 ug / mL) and incubated upside down in a 37 ℃ incubator for 12 h. Positive single clones were picked and transferred to LB liquid medium containing ampicillin (20 ug / mL) and incubated at 37 ℃ and 200 rpm for 8-10 h to prepare seed culture. (2) The seed culture was inoculated into LB liquid medium containing ampicillin at an inoculation rate of 2% (v / v) and fermented in shake flasks at 37 °C until OD. 600 When the pH reaches 0.6-0.8, add IPTG (100 μL / 50 mL, final concentration 0.05 mM) and incubate in shake flasks at 20℃ and 200 rpm for 12-16 h. The resulting fermentation broth is centrifuged at 4℃ and 10000 rpm for 20 min. The cells are then collected, and lysis buffer (20 mM Tris-HCl, pH 8.0) is added in equal proportions to resuspend the cells. After sonication, the supernatant is collected by high-speed centrifugation to obtain the crude intracellular enzyme solution. The SDS-PAGE gel electrophoresis image is shown below. Figure 2 As shown in the results, the wild-type enzyme and the H422A, D421A, and E373A mutant enzymes were all expressed.

[0038] Example 3: Mutants alter enzyme activity and thermal stability The enzyme activities of the crude enzyme solutions of the wild-type enzyme and each mutant enzyme obtained in Example 2 were determined, as shown in Table 2. The wild-type enzyme activity was set as 100%, and the relative enzyme activities of the mutant enzymes were detected and calculated. The results are as follows: Figure 3 As shown in the figure, each mutant exhibited varying degrees of reduction. Further modifications of ordinary corn starch using wild-type and mutants will be conducted to identify the product chain length distribution.

[0039] Wild-type and D421A and E373A mutant enzymes were heat-treated at 50℃ for 2, 5, 8, 10, 15, 20, 30, and 60 min, respectively. The remaining enzyme activity was measured, and the half-life was calculated accordingly. The results showed that the half-life of wild-type starch sucrase at 50℃ was 8–10 min; the half-life of the D421A mutant was 10–15 min; and the half-life of the E373A mutant was approximately 20 min. Figure 4 The above results indicate that although the enzyme activity of the mutant of this invention is reduced, its thermal stability is improved.

[0040] Table 2. Enzyme activity of wild-type and mutant starch sucrase

[0041] Example 4: Mutants alter the chain length distribution of products Prepare a 2% (w / v) solution of ordinary corn starch (NCS) using distilled water and gelatinize it at above 60 °C for 30 min. After the gelatinized starch cools to 30-40 °C, add 5% (w / v) sucrose based on the volume of the gelatinized starch solution, stir to dissolve, and then add wild-type or mutant enzymes at an enzyme activity of 30 U / g (based on starch dry weight). Use gelatinized ordinary corn starch without enzyme as a control. After reacting in a 40 °C constant temperature shaking water bath for 10 h, take samples, centrifuge at 10000 r / min for 10 min, discard the supernatant, wash the precipitate once with anhydrous ethanol, then wash three times with water, and centrifuge again. Detect the product using HPAEX-PAD method for chain length distribution determination after centrifugation.

[0042] The results showed that the H422A and E373A mutants of this invention successfully achieved a moderate reduction in starch chain length and a significant enrichment of medium and short chains, producing amylopectin with moderate polymerization and concentrated distribution. Compared with the wild type (Peak-DP 30, B3 chain 28.32%), the Peak-DP of H422A and E373A was reduced to 19, and the proportion of B3 chains decreased to 9.97% and 8.61%, respectively, with a significant reduction in long chain components; at the same time, the proportion of B1 chains increased to 53.46% and 54.79%, respectively, with a substantial enrichment of medium and short chains. In contrast, the D421A mutant (Peak-DP 28, B3 chain 26.34%) showed no significant difference from the wild type, indicating that this site has no significant effect on the regulation of chain length distribution, which in turn confirms the functional specificity of the H422 and E373 sites.

[0043] Table 3 Results of product chain length distribution determination

[0044] Note: The classification criteria for chain A (DP 6-12), B1 (DP 13-24), B2 (DP 25-36), and B3 (DP>36) are based on the HPAEC-PAD test results after isoamylase debranching.

[0045] Example 5: Analysis of the thermodynamic properties of modified starch Thermodynamic properties of wild-type and mutant modified starch samples obtained in Example 4 were analyzed using differential scanning calorimetry (DSC).

[0046] Accurately weigh 3-5 mg of starch sample into a dedicated aluminum crucible, add deionized water at a ratio of 1:2 (w / w), seal, and place in a 4℃ refrigerator overnight for equilibration for 12 h. Using an empty crucible as a reference, scan from 30℃ to 110℃ at a heating rate of 10℃ / min. Record the DSC heat flow curve of the sample, and analyze the initial gelatinization temperature (T0) using the instrument's built-in software. o Peak gelatinization temperature (T) p The analysis results are shown in Table 4. The lower melting temperature indicates that this degree of regeneration can usually achieve a certain structural strength, belonging to a moderately stable structure, and requires less processing energy compared to WT.

[0047] Table 4 Thermodynamic properties of the products

[0048] It is noteworthy that although the D421A mutant retained relatively high enzyme activity (16.84%), its chain length distribution was not significantly different from the wild type (Peak-DP 28, B3 chain 26.34%), indicating that the mutation at this site failed to achieve the chain length regulation target. This further illustrates that the effects of H422A and E373A on chain length distribution are site-specific and not a general phenomenon caused by reduced enzyme activity.

[0049] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A starch sucrase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID No. 1, perform any of the following mutations: (1) Histidine at position 422 is mutated to alanine; (2) Glutamic acid at position 373 is mutated to alanine.

2. The gene encoding the mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. Recombinant microbial cells carrying the gene of claim 2 or the recombinant vector of claim 3.

5. The recombinant microbial cell according to claim 4, characterized in that, Bacteria or fungi serve as host cells.

6. A recombinant Escherichia coli, characterized in that, Using Escherichia coli BL21(DE3) as the host and pET-20b(+) as the expression vector, the mutant described in claim 1 was expressed.

7. A method for preparing amylopectin with a moderate degree of polymerization, characterized in that, The starch sucrase mutant of claim 1 is added to a starch-containing reaction system for transformation; the medium degree of polymerization amylopectin refers to a peak-DP of 18-22, a medium-short chain ratio of DP 13-24 >45%, and a long chain ratio of DP>36 <10%.

8. The method according to claim 7, characterized in that, The conversion is carried out at 40-50 °C and pH 6.0-8.0 for 4-10 h.

9. The method according to claim 8, characterized in that, The reaction system contains gelatinized starch and sucrose; the starch includes ordinary corn starch.

10. The use of the starch sucrase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3, the recombinant microbial cell of claim 4 or 5, or the recombinant Escherichia coli of claim 6 in the preparation of amylopectin with moderate degree of polymerization in the food, pharmaceutical, biological, or materials fields.

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