A mutanase and its use

Abstract

The present application relates to the field of enzyme engineering, and in particular to a mutanase and application thereof. At present, mutan is mostly prepared by bacterial extraction, and there are problems such as low product yield and many impurities. The present application provides a mutanase, a MutI gene fragment of the mutanase is obtained by cloning a genome of Leuconostoc pseudomesenteroides G496, and a genetically engineered bacterium is constructed. The mutanase provided by the present application is subjected to enzymatic property analysis, and it is found that the mutanase has good stability under acidic conditions, and the optimal induction expression temperature and reaction temperature are close to room temperature, and the production energy consumption is low and the raw material conversion rate is high. The catalytic product of the mutant enzyme is linear mutan with a high polymerization degree, the molecular weight of the linear mutan is as high as 650 kDa, the linear mutan contains 90% of alpha-1,3 glycosidic bonds, and the structure is relatively dense, and the linear mutan has a good application prospect in the field of biological base materials.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, specifically to a polysaccharide sucrase and its applications. Background Technology

[0002] Glycosyltransferases are a type of glucan sucrase belonging to the glycoside hydrolase 70 family. They can transfer glucose groups from sucrose to extended sugar chains, forming polysaccharide polymers. Early research on glycosyltransferases focused primarily on methods for preventing tooth decay. However, with further research, glycosyltransferases were discovered to be excellent bio-based materials, further expanding research into glycosyltransferases. Currently, glycosyltransferases from Streptococcus salivarius, Streptococcus mutans, Penicillium, Penicillium, and Aspergillus are extensively studied. Among them, the glycosyltransferase GTFJ from Streptococcus mutans is used for glycosyltransferase production, capable of converting 1M of sucrose, but it requires oligosaccharide chains as primers for production.

[0003] Mutagenic polysaccharides are insoluble linear polysaccharides primarily composed of glucose monomers linked by α-1,3-glycosidic bonds. Due to their safety, non-toxicity, immunomodulatory activity, and heat resistance, they have wide applications in medicine, fine chemicals, and food, such as in the preparation of polysaccharide-loaded drug-eluting gel dressings. As a novel bio-based material, mutagenic polysaccharides have high molecular weights of 20-70 kDa, exhibiting advantages such as low density, small particle diameter, and large specific surface area. They can be used as dispersants in latex agents, additives in natural rubber and plastic products, and as alternatives to fossil resources. Furthermore, as a biopolymer, they possess excellent biodegradability. In nature, mutagenic polysaccharides mostly exist as branched heteropolysaccharides (α-1,3 / α-1,6-glucan) or mixtures thereof, making it difficult to obtain large quantities of homogeneous and linear mutagenic polysaccharides. Streptococcus mutans 6715 can produce α-1,3-glycosidic bonds with 67% content, and Leuconostoc mesenteroides NRRL B-1118's DSR-I enzyme can catalyze the production of α-1,3-glycosidic bonds with 50% content. However, the bond structure of current polysaccharide products is difficult to achieve a uniform bond structure, and there are many branches, which seriously affects the thermoplasticity of the products.

[0004] *Leuconostoc* species produce mutagensulase in the presence of sucrose and secrete it into the culture medium. Mutagensulase can synthesize mutagens from sucrose, catalyzing the transfer of glucosyl residues from sucrose to mutagens polymers and releasing fructose. Early methods for producing mutagens mainly involved fermentation from strains or cell extraction, which faced problems such as low yield and poor separation efficiency. Enzyme engineering technology is currently a more advanced method for producing mutagens. To prepare highly polymerized linear mutagens, it is necessary to construct recombinant mutagensulase and leverage the advantages of enzyme engineering to produce high-purity mutagens, thereby further expanding the application areas of this biopolysaccharide. Summary of the Invention

[0005] Objective of the Invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a polysaccharide sucrase and its applications.

[0006] To address the aforementioned technical problems, this invention discloses a polysaccharide sucrase and its applications. The technical solution is as follows:

[0007] In a first aspect, the present invention provides a polysaccharide sucrase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] In a second aspect, the present invention provides a gene encoding the polysaccharide sucrase described in the first aspect, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] Thirdly, the present invention provides a recombinant expression vector comprising the gene described in the second aspect.

[0010] Fourthly, the present invention provides a genetically engineered bacterial strain comprising the gene described in the second aspect. Preferably, the method for constructing the genetically engineered bacterial strain includes primer design, recombinant plasmid construction, and host bacterial transformation: the primer design is based on the polysaccharide sucrase gene sequence and the sequence of the vector pET28a(+), and mutant primers are designed using SnapGene software as follows:

[0011] Upstream mutation primer (SEQ ID NO.3):

[0012] 5'-ATGGGTCGCGGATCCGAATTCATGTTAGTAACAGCTGGTATTTTTTCTG-3'

[0013] Downstream mutant primer (SEQ ID NO.4):

[0014] 5'-TGCGGCCGCAAGCTTGTCGACATTATGCAGGTAAGCCATATTCATTGATTG-3';

[0015] The recombinant plasmid was constructed using the genome of Leuconostoc pseudomesenteroides G496 as a template. The polysaccharide sucrase gene fragment was obtained by PCR amplification. The gene fragment was then ligated into the expression vector pET28a(+) to obtain the recombinant expression plasmid pET28a(+)-MutI.

[0016] The host bacteria transformation involved transforming the recombinant expression plasmid pET28a(+)-MutI into Escherichia coli competent cells BL21(DE3). After kanamycin resistance screening, enzyme digestion, colony PCR, and DNA sequencing verification, the polysaccharide sucrase engineered strain BL21(DE3) / MutI was obtained.

[0017] Fifthly, the present invention provides a method for preparing the polysaccharide sucrase described in the first aspect, comprising the following steps:

[0018] (1) The gene encoding polysaccharide sucrase was cloned into an expression vector to obtain a recombinant expression vector, which was then introduced into the starting bacteria to obtain a genetically engineered strain.

[0019] (2) The genetically engineered strain is inoculated into a culture medium for fermentation to obtain a fermentation broth, which is then broken and centrifuged to obtain polysaccharide sucrase.

[0020] In step (1), the expression vector is pET28a, preferably pET28a(+); the starting bacterium is Escherichia coli BL21.

[0021] In step (2), the fermentation culture involves inoculating the genetically engineered strain into a culture medium and culturing it at 35–40°C until the OD reaches 100°C. 600 When the OD value is 0.60–0.80, add 0.1–0.5 mM IPTG and induce culture at 18–24°C for 20–26 h to obtain the fermentation broth. Preferably, the fermentation culture method is as follows: inoculate the genetically engineered strain into LB medium, rotate at 180 rpm, and culture at 37°C for 12 h; take 1 mL of the above culture broth and add it to 100 mL of fermentation medium, place it in a shaker at 37°C, and culture until the OD value of the enriched culture broth reaches 0.60–0.80. 600 When the pH reaches 0.60–0.80, 0.2 mM IPTG can be added to induce enzyme production. Fermentation is induced at 18°C ​​for 20–26 hours. The inducing bacterial suspension is then centrifuged at 8000 rpm for 10 minutes at 0°C, with one centrifuge tube per suspension. Distilled water is added and the suspension is washed, followed by another centrifugation. 20 mL of pH 5.5 PBS buffer is added to each centrifuge tube, vortexed, and then placed in an ice-water bath. The suspension is sonicated for 20 minutes, centrifuged, and the supernatant is the crude enzyme solution. More preferably, the fermentation medium has the following formula: peptone 12 g / L, yeast extract 24 g / L, KH₂PO₄ 2.31 g / L, K₂HPO₄ 16.43 g / L, and glucose 10 g / L.

[0022] Sixthly, this invention provides the application of the polysaccharide sucrase described in the first aspect, the recombinant expression vector described in the third aspect, or the genetically engineered strain described in the fourth aspect in the preparation of polysaccharides.

[0023] The variable polysaccharide has a weight-average molecular weight of 600–700 kDa and contains >90% α-1,3-glycosidic bonds, where >90% refers to the proportion of α-1,3-glycosidic bonds in the bond structure composition of the variable polysaccharide. Preferably, the variable polysaccharide has a bond structure composition containing 91% α-1,3-glycosidic bonds and 9% α-1,6-glycosidic bonds.

[0024] The method involves preparing polysaccharides from sucrose using polysaccharide sucrase. The amount of polysaccharide sucrase added is 1–10 U / L, the amount of sucrose added is 100–250 g / L, the reaction pH is 4.5–7.0, the reaction temperature is 20–40℃, and the reaction time is 4–6 h. Preferably, the amount of enzyme added is 5 U / L, the amount of sucrose added is 200 g / L, the reaction pH is 5.5, the reaction temperature is 30℃, and the reaction time is 4 h.

[0025] Beneficial effects:

[0026] The effective technical advantages of this invention are: compared with general methods for preparing polysaccharides, the use of polysaccharide sucrase allows for the direct one-step enzymatic synthesis of high molecular weight polysaccharides, with mild catalytic conditions, simple separation steps, and a concentrated molecular weight distribution of the product, containing up to 90% α-1,3-glycosidic bonds, thus expanding its application range. Enzymatic property studies of this enzyme have revealed its significant potential for industrial production applications. This invention lays the foundation for the enzyme-engineered production of polysaccharides and the application of its products. The polysaccharides prepared by this invention have a molecular weight of approximately 650 kDa and can maintain a stable structure at 300°C. As a biologically derived polysaccharide, it is used as a bio-based material, naturally possessing advantages such as biocompatibility and biodegradability, and has promising application prospects in the field of bio-based materials, such as the preparation of drug nanocarriers, drug gel dressings, and highly absorbent materials for hygiene products. This invention lays the foundation for the enzyme-engineered production of polysaccharides and the application of its products. Attached Figure Description

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0028] Figure 1 Preparation process and application of polysaccharide sucrase MUT-I.

[0029] Figure 2 The optimal pH, optimal temperature, pH stability, and temperature stability of polysaccharide sucrase MUT-I were determined.

[0030] Figure 3 Optimized catalytic conditions for polysaccharide sucrase MUT-I. Figure 3 A represents the optimization of sucrose concentration. Figure 3B represents reaction time optimization. Figure 3 C represents the optimal pH for the reaction. Figure 3 D represents the optimal reaction temperature. Figure 3 E represents the optimal amount of enzyme added.

[0031] Figure 4 This is a scanning electron microscope image of the catalytic product of polysaccharide sucrase MUT-I. Figure 4 A, Figure 4 B Figure 4 C shows scanning electron microscope images at different resolutions.

[0032] Figure 5 The catalytic product of polysaccharide sucrase MUT-I 1 H and 13 C-spectrum.

[0033] Figure 6 This is the TG-DSC characterization diagram of the polysaccharide. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be fully described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0035] A schematic diagram illustrating the preparation process and application of the polysaccharide sucrase described in this invention is shown below. Figure 1 As shown.

[0036] Example 1: Construction of recombinant expression plasmid pET28a(+)-MutI and genetically engineered bacteria BL21(DE3) / MutI

[0037] Based on the genome of Leuconostoc pseudomesenteroides G496 and the sequence of vector pET28a(+), mutation primers were designed using SnapGene software as follows:

[0038] Upstream mutation primer (SEQ ID NO.3):

[0039] 5'-ATGGGTCGCGGATCCGAATTCATGTTAGTAACAGCTGGTATTTTTTCTG-3'; Downstream mutant primer (SEQ ID NO.4):

[0040] 5'-TGCGGCCGCAAGCTTGTCGACATTATGCAGGTAAGCCATATTCATTGATTG-3';

[0041] Using the genome of *Leuconostoc pseudomesenteroides* G496 as a template, the mutagenin sucrase gene MutI (its nucleotide sequence is shown in SEQ ID NO. 2) was amplified by PCR using upstream and downstream mutant primers with nucleotide sequences as shown in SEQ ID NO. 3-4. After 1% agarose gel electrophoresis to verify the PCR amplification product, DpnI was added to remove the methylated original template. After 4 hours of enzyme digestion, the digested PCR reaction solution was purified and recovered by PCR for later use.

[0042] The pET28a(+) plasmid was digested with EcoRI and SalRI, and the digestion products were recovered by gel extraction and used as linearized cloning vectors.

[0043] The purified MutI gene and linearized cloning vector obtained by PCR were transformed into E. coli competent cells BL21(DE3) using a one-step cloning kit. The preparation method for E. coli BL21(DE3) competent cells was as follows: 50 μL of E. coli BL21(DE3) bacterial culture was added to a 5 mL LB tube and cultured at 37°C, 180 rpm for 12 h on a shaker. Then, 1 mL of the bacterial culture from the test tube was added to a shake flask containing 100 mL of LB medium and cultured at 37°C, 180 rpm on a shaker until the bacterial culture reached OD. 600 When the concentration is 0.5–0.6, remove the shake flask, place it on ice for 10 min, centrifuge at 4100 rpm for 10 min at 4℃, discard the supernatant, resuspend it in 2 mL of 0.05 M CaCl2 solution containing 15% glycerol, aliquot and store in a -80℃ refrigerator for later use.

[0044] The one-step cloning method is as follows: the above PCR amplification product and linearized plasmid pET-28a(+) are mixed, and a one-step cloning reaction is carried out under the catalysis of homologous recombinase Exnase II. The one-step cloning system (20 μL) is shown in Table 1:

[0045] Table 1 One-step cloning system

[0046]

[0047] Thaw 100 μL of prepared E. coli BL21(DE3) competent cells on an ice box for 5–10 min. Add 10 μL of cooled one-step cloning reaction mixture to the competent cells, gently tap to mix, and place on ice for 30 min. Then heat shock at 42°C for 90 s, followed by an immediate ice bath for 3 min. Add 900 μL of LB medium to the above reaction mixture and incubate at 37°C and 180 rpm for 1 h to revive the cells. Spread 100 μL of the mixture onto an antibiotic-resistant plate and incubate at 37°C for 12 h. Pick single colonies and perform colony PCR verification using the upstream and downstream mutant primers shown in SEQ ID NO. 3-4. Colonies that amplify the target gene fragment are the strains that successfully express the recombinant expression plasmid pET28a(+)-MutI, which are the genetically engineered bacteria BL21(DE3) / MutI.

[0048] Example 2: Preparation of polysaccharide sucrase by fermentation of engineered strain BL21(DE3) / MutI

[0049] Seed culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, sterilized at 121℃ for 15 min.

[0050] Fermentation medium: peptone 12 g / L, yeast extract 24 g / L, KH2PO4 2.31 g / L, K2HPO4 16.43 g / L, glucose 10 g / L, sterilized at 121℃ for 15 min.

[0051] Preparation method: A single colony of BL21(DE3) / MutI from an agar plate was inoculated into a 5 mL seed culture medium tube and incubated at 37°C and 180 rpm for 12 h. Then, 1 mL of the bacterial culture was inoculated into a 500 mL shake flask containing 100 mL of fermentation medium and incubated at 37°C and 180 rpm for 3 h. (OD) 600 The expression of MUT-I protein was induced by adding 0.2 mM IPTG (0.60-0.80 g) and culturing at 18°C ​​for 20-26 h. After stopping fermentation, the fermentation broth was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the cells were washed and resuspended with PBS buffer solution at pH 5.5. The cells were then disrupted by ultrasonic disruption at 300 W with a disruption temperature of 4°C and a disruption time of 20 min to obtain crude enzyme solution.

[0052] Example 3: Enzymatic Properties Study of Mutaglutamate Sucrase MUT-I

[0053] The enzyme activity of polysaccharide sucrase is defined as the amount of enzyme required to produce 1 μmol of fructose in 30 minutes under conditions of pH 5.5 and 30℃, which is one enzyme activity unit (U).

[0054] The crude enzyme solution of the polysaccharide sucrase MUT-I prepared in Example 2 was used to study its enzymatic properties. An enzyme activity assay system was prepared by dissolving 1.64 g / L sodium acetate and 0.05 g / L CaCl2 in distilled water. To prepare assay systems with different pH values, the pH was adjusted to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 6.5, 7.0, 7.5, and 8.0 by adding 0.1 M phosphate aqueous solution or 0.1 M NaOH aqueous solution. To determine the optimal pH of the enzyme, 1 mL of the crude enzyme solution was mixed with 2.5 mL of the different pH enzyme activity assay systems used for measuring enzyme activity. The mixture was reacted at 30°C for 30 min. After the reaction, 1.5 mL of DNS reagent was added, and the mixture was boiled for 5 min. The reaction was measured at 540 nm using a spectrophotometer. The fructose content was calculated by substituting the fructose standard curve to determine the enzyme activity. The results are as follows: Figure 2 As shown in Figure A, the pH value at which the highest enzyme activity was determined was 100%. To determine its thermal stability, the reaction temperature was controlled at 30°C, and MUT-I was reacted with the enzyme activity reaction system at three different pH values ​​(5.0, 5.5, and 6.0). Residual activity was measured at specific time intervals (1, 2, 3, and 4 hours). Figure 2 As shown in C, the pH value at which the highest enzyme activity is determined is 100%.

[0055] To determine the optimal temperature for MUT-I enzyme activity, MUT-I was reacted with the enzyme activity reaction system (pH 5.5) at different temperatures ranging from 20 to 45°C for 30 minutes. Figure 2 As shown in B, the temperature at which the highest enzyme activity was measured is 100%. To determine its thermal stability, MUT-I was reacted with the enzyme activity reaction system at four different temperatures (20, 25, and 30 °C), and residual activity was measured at specific time intervals (1, 2, 3, and 4 h). Figure 2 As shown in D, the temperature value for determining the highest enzyme activity is 100%.

[0056] Example 4: Preparation of polysaccharides catalyzed by polysaccharide sucrase MUT-I

[0057] A certain concentration of sucrose was added to the crude enzyme solution of the polysaccharide sucrase MUT-I obtained in Example 2, so that the final concentration of sucrose in the reaction system was 100-250 g / L, preferably 200 g / L (e.g., Figure 3 A); The reaction time is 4–6 hours, preferably 4 hours (e.g., ...). Figure 3 B), the pH of the reaction system is 5.0–7.0, preferably 5.5 (e.g., ...). Figure 3 C); The reaction temperature is 30–40°C, preferably 30°C (e.g., 40–40°C). Figure 3 D); The enzyme addition amount is 1-10 U / L, preferably 5 U / L (e.g., ...). Figure 3E). Through optimization of multiple catalytic conditions, the final catalytic system was determined as follows: temperature controlled at 30℃, system pH controlled at 5.5, enzyme addition amount at 5 U / L, substrate concentration at 200 g / L, and reaction duration at 4 h.

[0058] The catalytic reaction solution was filtered, the supernatant was discarded, and the solution was washed with water and dried under vacuum to obtain the polysaccharide. The scanning electron microscopy results are as follows: Figure 4 As shown. Figure 4 AC are electron microscope images at different resolutions. The images show that the product has a fine particle size, reaching the micron level, and the product has a high surface area, which can be widely used in solvent-based resin systems in the coating industry, effectively replacing the application of fossil resources in industrial production.

[0059] The GPC spectra of the polysaccharides prepared using the preferred polysaccharide sucrase MUT-I are shown in Table 2. The molecular weight is approximately 644,083 Da, and its NMR spectrum is shown in Table 2. Figure 5 As shown, the bond structure of this polysaccharide contains 91% α-1,3 glycosidic bonds and 9% α-1,6 glycosidic bonds. Thermogravimetric analysis was used to characterize the thermal stability of this polysaccharide product. The product has a Td5% of 287.65℃, exhibiting high thermal stability, which lays a good foundation for the subsequent preparation of derivatives to replace petrochemical products.

[0060] Table 2. Molecular weight determination results of polysaccharides

[0061]

[0062] Currently, in the one-step enzymatic synthesis process of polysaccharides of this invention, 200 g / L of sucrose substrate is added, and the final fructose concentration is detected to be 93.15 g / L, with a conversion rate of over 90%. The final polysaccharide product obtained is 86.15 g / L, with a final yield of over 85%.

[0063] This invention provides a method and approach for developing a polysaccharide sucrase and its applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A polysaccharide sucrase having the amino acid sequence shown in SEQ ID NO.

1.

2. A gene encoding the polysaccharide sucrase of claim 1, the nucleotide sequence of which is shown in SEQ ID NO.

2.

3. A recombinant expression vector comprising the gene of claim 2.

4. A genetically engineered strain comprising the gene of claim 2.

5. The method for preparing polysaccharide sucrase according to claim 1, characterized in that, Includes the following steps: (1) The gene encoding polysaccharide sucrase was cloned into the expression vector to obtain a recombinant expression vector, which was then introduced into the starting bacteria to obtain a genetically engineered strain; (2) The genetically engineered strain is inoculated into a culture medium for fermentation to obtain a fermentation broth, which is then broken and centrifuged to obtain polysaccharide sucrase.

6. The preparation method according to claim 5, characterized in that, In step (1), the expression vector is pET28a; the starting bacterium is Escherichia coli BL21.

7. The preparation method according to claim 5, characterized in that, In step (2), the fermentation culture involves inoculating the genetically engineered strain into LB medium and culturing it at 35-40 °C until OD. 600 When the concentration is 0.60–0.80, add IPTG at a concentration of 0.1–0.5 mM and induce culture at 18–24 °C for 20–26 h to obtain fermentation broth.

8. The application of the polysaccharide sucrase of claim 1, the recombinant expression vector of claim 3, or the genetically engineered strain of claim 4 in the preparation of polysaccharides, wherein the polysaccharide has a weight-average molecular weight of 600-700 kDa and contains >90% α-1,3-glycosidic bonds.

9. The application according to claim 8, characterized in that, The preparation of polysaccharides from sucrose was carried out using polysaccharide sucrase catalyzing polysaccharide sucrase, wherein the amount of polysaccharide sucrase added was 1~10 U / L, the amount of sucrose added was 100~250 g / L, the reaction pH was 4.5~7.0, the reaction temperature was 20~40 ℃, and the reaction time was 4~6 h.

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