A starch sucrolase mutant capable of changing starch chain length and its use in the preparation of long chain amylopectin

Abstract

The application discloses a starch sucrolase mutant capable of changing starch chain length and application thereof in preparation of long-chain amylose, and belongs to the technical field of genetic engineering. The application provides a starch sucrolase mutant, wherein the mutant is obtained by mutating the amino acid at the 340th position of the amino acid sequence of the starch sucrolase shown in SEQ ID No. 1. The starch sucrolase mutant of the application has changed starch chain length distribution relative to modified wild type, and has equivalent enzyme activity, so that the application of the enzyme in the actual industrial application can be widened, and the starch sucrolase mutant has good industrial application prospect.

Description

Technical Field

[0001] This invention relates to a starch sucrase mutant capable of altering starch chain length and its application in the preparation of long amylopectin, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] Long-chain amylopectin (LCA) is a type of starch in which the longer branched chains predominate. It is characterized by low calories and high dietary fiber content, making it a natural, safe, and dual-purpose food resource. LCA offers numerous health benefits. Because it is not digested and absorbed in the small intestine, it can lower postprandial blood glucose levels. It ferments in the large intestine, increasing intestinal contents volume, altering gut microbiota structure, and preventing constipation and colorectal cancer. Furthermore, LCA can lower serum cholesterol levels and help control weight. In recent years, the synthesis mechanism and molecular regulation of LCA have become research hotspots, possessing significant industrial application value and broad market development prospects. With increasing consumer demand for health foods, LCA is expected to play a greater role in the functional food sector.

[0003] Starch sucrase is a class of enzymes that catalyze the transglycosylation reaction between starch and sucrose. When both starch and sucrose are used as substrates, it hydrolyzes the glycosidic bonds of glucose and fructose in sucrose, simultaneously acting on the non-reducing ends of amylopectin to elongate the branched portion of the starch. It is an important transglycosylation tool. In the main reaction system, after the reaction is fully completed and the starch branches have elongated to a certain extent, starch precipitates, and the product separates from the substrate. This has significant research value in the food and pharmaceutical industries.

[0004] This invention selects materials from... Neisseria polysaccharea The wild-type starch sucrase possesses strong transglycosylation capabilities, generating long amylopectin with a different branching ratio than the original starch. However, this wild-type starch sucrase can only produce medium-length amylopectin, and medium-length chains are a "weak region" sensitive to the enzyme, limiting research on long amylopectin and restricting its practical industrial application. Short chains with a DP < 21 can rapidly form perfect crystals during retrogradation, increasing their orderliness, while long chains with a DP > 40 can act as a "skeleton" to stabilize the gel network, forming a thicker, denser crystalline layer that significantly inhibits amylase diffusion and binding. Increasing their proportion significantly improves starch's resistance content, thermal stability, and processing adaptability, representing an important molecular design strategy for developing functional starch foods and high-performance starch materials. Therefore, regulating the chain length distribution of starch modified by starch sucrase through genetic engineering and enzyme engineering is crucial for the molecularly directed modification of starch sucrase and the realization of high-efficiency, low-cost long amylopectin. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a starch sucrase mutant capable of regulating starch chain length distribution and its applications.

[0006] The first technical solution provided by this invention is a starch sucrase mutant capable of regulating starch chain length distribution. The starch sucrase mutant is obtained by mutating the starch sucrase parent with the amino acid sequence shown in SEQ ID No. 1 using any of the following methods: (1) Histidine at position 340 is mutated to alanine; (2) Alanine at position 178 is mutated to arginine; (3) The glycine at position 356 is mutated to alanine or serine; (4) The phenylalanine at position 606 is mutated to alanine.

[0007] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0008] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0009] In one embodiment of the present invention, the recombinant vector is pET-20b(+) as the expression vector.

[0010] The present invention also provides a recombinant cell carrying the above-mentioned gene or the above-mentioned recombinant vector.

[0011] In one embodiment of the present invention, the recombinant cells use bacteria or fungi as host cells.

[0012] 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.

[0013] 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.

[0014] The present invention also provides a method for regulating starch chain length distribution, which utilizes the starch sucrase mutant to regulate starch chain length distribution using starch and sucrose as substrates.

[0015] 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 4-8 h.

[0016] The present invention also provides the application of the starch sucrase mutant, the gene sequence, the recombinant expression vector, the recombinant cells, or the starch sucrase mutant prepared by the method in food, medicine, biology, or materials.

[0017] The beneficial effects of this invention are as follows: 1) This invention utilizes the source... Neisseria polysaccharea By mutating starch sucrase, the key active sites of the enzyme are changed, transforming the starch sucrase, which originally could not directionally regulate the distribution of starch chain length, into one that can directionally regulate the distribution of starch chain length. Therefore, it can meet the need to regulate the distribution of starch chain length and expand its application range in the preparation of long amylopectin or resistant starch.

[0018] 2) The starch sucrase mutant provided by this invention has higher enzyme activity than the wild type with lower enzyme activity, increasing by 103.9~111.2%, providing a reference solution for the production of long amylopectin.

[0019] 3) This invention provides a mutant capable of regulating starch chain length distribution. Compared with the wild type, H340A modified starch has a 44.6% higher proportion of DP8~21 segments, an 80.6% higher proportion of DP>40 segments, and a 21.3% lower proportion of DP21~39 segments. The starch synthesized by this mutant exhibits a distribution characteristic of simultaneous enrichment of short-chain and ultra-long-chain regions and a reduction in intermediate-chain regions. Attached Figure Description

[0020] Figure 1 Agarose gel electrophoresis images of wild-type amylose sucrase and its mutant plasmids; where M: standard molecular weight of DNA, 1: wild-type plasmid, 2-6: mutant plasmids, namely H340A, A178R, G356A, G356S, and F606A.

[0021] Figure 2 SDS-PAGE images of wild-type amylose sucrase and its mutants are shown; where M: standard molecular weight of protein, 1: wild type, 2-6: mutants, namely H340A, A178R, G356A, G356S, and F606A.

[0022] Figure 3 The results show the enzyme activity of wild-type starch sucrase and its mutants.

[0023] Figure 4 Chain length distribution of wild-type starch sucrase and its mutant H340A modified starch.

[0024] Figure 5 Chain length distribution of wild-type starch sucrase and its mutant G356S modified starch.

[0025] Figure 6 Chain length distribution of wild-type starch sucrase and its mutant F606A modified starch. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] (2) Methods for determining enzyme activity The Bradford method was used to detect the specific enzyme activity of amylase. A standard curve was plotted with the average A595 concentration of each tube in the standard group as the ordinate and the corresponding protein concentration as the abscissa. The protein sample was diluted 10-fold, and 10 μL was added to each centrifuge tube. 300 μL of G250 staining solution was added to each tube, and the mixture was shaken thoroughly. 200 μL of the developed solution was added to a 96-well plate, and the absorbance was measured at 595 nm using a microplate reader. The protein concentration of the sample was calculated from the standard curve. The specific enzyme activity was defined as the enzyme activity corresponding to a unit mass of protein.

[0029] (3) 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: Method for preparing mutants 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. 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 according to... E. coli The JM109 competent state conversion method was transferred to... E. coli Transformants were prepared from JM109 and spread on LB solid medium containing ampicillin (20 μg / mL). The medium was incubated at 37 °C for 12 h in an inverted position. Positive clones were picked and transferred to LB liquid medium containing ampicillin (20 μg / mL) and incubated at 37 °C 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 mutants (1) The wild-type and mutant recombinant plasmids obtained in Example 1 were transformed into E. coli BL21 to prepare wild-type recombinant strains and mutant recombinant strains, respectively; the recombinant strains were streaked on LB solid medium containing ampicillin (20 ug / mL) and incubated upside down in a constant temperature incubator at 37 ℃ for 12 h. Positive single clones were picked and cultured in LB liquid medium containing ampicillin (20 ug / mL) at 37 ℃ and 200 rpm for 8-10 h to prepare seed liquid; (2) The seed culture was inoculated into fermentation broth containing ampicillin at an inoculation rate of 2% (v / v) in shake flasks and cultured at 37 °C until OD. 600When the pH value 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 figure, the results indicate that both the wild-type enzyme and the mutant enzyme were expressed.

[0038] Example 3 Enzyme activity assay The enzyme activities of the wild-type and mutant enzymes obtained in Example 2 were determined, as shown in Table 2. The wild-type enzyme activity was set as 100%, and the relative enzyme activity of the mutant enzyme was detected and calculated. The results are as follows: Figure 3 As shown in the figure. Among them, mutants H340A and G356S showed slightly increased enzyme activity, while the remaining mutants showed varying degrees of decrease. Mutants with enzyme activity comparable to or higher than that of the wild type were screened for subsequent experiments.

[0039] Table 2. Enzyme activity of wild-type and mutant starch sucrase

[0040] Example 4: Determination of product chain length distribution 2% ordinary corn starch (CS) was prepared with distilled water and gelatinized at 60 °C or above for 30 min. After the gelatinized starch cooled to 30-40 °C, 5% sucrose was added and stirred to dissolve. Wild-type starch sucrase and its mutant were added to a final concentration of 10%, with gelatinized ordinary corn starch without enzyme as a control. The mixture was reacted in a 40 °C constant temperature shaking water bath for 5 h, and then samples were taken. The mixture was centrifuged at 10000 r / min for 10 min, the supernatant was discarded, and the precipitate was washed twice with water, once with anhydrous ethanol, and once with water. After centrifugation, the product was detected by HPAEX-PAD using the chain length distribution method.

[0041] The relative peak areas in ion chromatography showed that different mutants had varying proportions across different degrees of polymerization, as shown in Table 3 and Figures 4-6. Specifically, mutant H340A exhibited a higher proportion of short chains compared to the wild type in the DP 8–21 range, increasing by 44.6%. Furthermore, H340A produced significantly more long chains than the wild type, with an 80.6% increase in DP values ​​>40, indicating the generation of more ultra-long chains. However, in the medium-chain range (DP 21–39), the wild type produced the majority of the product, while the proportion of mutant H340A decreased by 21.3%. The starch chain length distribution after enzymatic hydrolysis of mutants G356S and F606A was similar to that of the wild-type enzyme. In conclusion, mutant H340A can synthesize longer branched starch chains than the wild type.

[0042] Table 3. Determination of product chain length distribution

[0043] 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, The starch sucrase mutant was obtained by performing any of the following mutations on the starch sucrase parent with the amino acid sequence shown in SEQ ID No. 1: (1) Histidine at position 340 is mutated to alanine; (2) Alanine at position 178 is mutated to arginine; (3) The glycine at position 356 is mutated to alanine or serine; (4) The phenylalanine at position 606 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 cells carrying the gene of claim 2 or the recombinant vector of claim 3.

5. The recombinant 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 adjusting starch chain length distribution, characterized in that, The starch sucrase mutant of claim 1 is added to a starch-containing reaction system for transformation.

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 4-8 h.

9. The method according to claim 8, characterized in that, The reaction system contains gelatinized starch and sucrose.

10. The application of the starch sucrase mutant of claim 1, or the gene of claim 2, or the recombinant vector of claim 3, or the recombinant cell of claim 4 or 5, or the recombinant Escherichia coli of claim 6, or the method of any one of claims 7 to 9 in the fields of food, medicine, biology or materials.

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