Glycosan sucrase and application thereof

Abstract

The invention relates to the field of enzyme engineering, in particular to a transglycanase and application thereof. At present, the preparation of the glycan mostly adopts thallus extraction, so that the problems of low product yield, more impurities and the like exist. The invention provides a transglycanase, which is characterized in that a gene segment of the transglycanase MutI is obtained by cloning a genome of leuconostoc pseudomesenteroides G496, and a genetically engineered bacterium is constructed. The enzyme property analysis of the transglycanase provided by the invention shows that the transglycanase has better stability under an acidic condition, the optimal induction expression temperature and reaction temperature are close to room temperature, the production energy consumption is low, and the raw material conversion rate is high. A catalytic product of the mutant enzyme is high-polymerization-degree linear variable glycan, the molecular weight reaches up to 650 kDa, 90% of alpha-1, 3 glucosidic bonds are contained, the structure is compact, and the mutant enzyme has good application prospects in the field of bio-based materials.

Description

Technical Field

[0001] The invention relates to the field of enzyme engineering, and in particular to a mutanesucrase and an application thereof. Background Art

[0002] Mutansucrase is a type of glucansucrase, belonging to the glycoside hydrolase 70 family, which can transfer the glucose group in sucrose to the extended sugar chain to form a polysaccharide polymer. In the early days, the research on mutansucrase focused on the study of methods to prevent dental caries; with the deepening of research, mutans was found to be an excellent bio-based material, which further increased the research on mutansucrase. At present, glycosyltransferases from Streptococcus salivarius, Streptococcus mutans, Penicillium, Penicillium and Aspergillus have been widely studied. Among them, mutansucrase GTFJ from Streptococcus mutans is used for the production of mutans, which can convert 1M sucrose, but it requires oligosaccharide chains as primers for production.

[0003] Mutans are insoluble linear polysaccharides, mainly composed of glucose monomers connected by α-1,3-glycosidic bonds. Due to their safety, non-toxicity, immunological activity and heat resistance, they are widely used in the fields of medicine, fine chemicals, food, etc., such as the preparation of polysaccharide-loaded gel dressings. As a new type of bio-based material, mutans have a high molecular weight of 20-70kDa, and have the advantages of light density, small particle diameter, and large specific surface area. They can be used as dispersants for latex agents, additives for natural rubber and plastic products, and can be used to replace fossil resources; and as a biological polysaccharide, they have excellent biodegradability. In nature, mutans mostly exist in the form of branched heteropolysaccharides (α-1,3 / α-1,6-glucans) or their mixtures, and it is difficult to obtain a large amount of homogeneous and linear mutans. Streptococcus mutans 6715 can produce 67% α-1,3-glycosidic bonds, and the DSR-I enzyme of Leuconostoc mesenteroides NRRL B-1118 can catalyze the production of 50% α-1,3-glycosidic bonds. However, the bond type of current mutans products is difficult to achieve a uniform bond type, and most of them have a large number of branches, which seriously affects the thermoplasticity of the product.

[0004] Leuconostoc produces mutansucrose in the presence of sucrose and secretes it into the culture medium. Mutansucrose can synthesize mutans from sucrose, catalyzing the transfer of glucosyl residues from sucrose to mutans polymers and releasing fructose. In the early days, mutans were mainly prepared by fermentation from strains or extraction from bacteria, which faced problems such as low yield and poor separation effect. The use of enzyme engineering technology to produce mutans is currently a more advanced method. In order to prepare linear mutans with a high degree of polymerization, it is necessary to construct recombinant mutansucrose and use the advantages of enzyme engineering to prepare high-purity mutans, thereby further expanding the application field of mutans as a biopolysaccharide. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a polysaccharide sucrase and its application in view of the shortcomings of the prior art.

[0006] In order to solve the above technical problems, the present invention discloses a polysaccharase and its application. The technical solution is as follows:

[0007] In a first aspect, the present invention provides a mutansucrose enzyme, 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 mutanesucrase described in the first aspect, and the nucleotide sequence thereof is shown in SEQ ID NO.2.

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

[0010] In a fourth aspect, the present invention provides a genetically engineered strain comprising the gene described in the second aspect. Preferably, the construction method of the genetically engineered bacteria comprises the unit processes of primer design, recombinant plasmid construction and host bacteria transformation: the primer design is based on the sequence of the mutanesucrase gene and the sequence of the vector pET28a(+), and the mutation primers are designed with the help of SnapGene software as follows:

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

[0012] 5'-ATGGGTCGCGGATCCGAATTCATGTTAGTAACAGCTGGTATTTTTTCTG-3'

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

[0014] 5'-TGCGGCCGCAAGCTTGTCGACATTATGCAGGTAAGCCATATTCATTGATTG-3';

[0015] The recombinant plasmid is constructed by using the genome of Leuconostoc pseudomesenteroides G496 as a template and using PCR amplification technology to obtain a mutanesucrase gene fragment; the gene fragment is connected to the expression vector pET28a(+) to obtain a recombinant expression plasmid pET28a(+)-MutI;

[0016] The host bacteria are transformed by transforming the recombinant expression plasmid pET28a(+)-MutI into the competent Escherichia coli cell BL21(DE3), and after kanamycin resistance screening, enzyme digestion, colony PCR and DNA sequencing verification, the mutansucrose enzyme engineering strain BL21(DE3) / MutI is obtained.

[0017] In a fifth aspect, the present invention provides a method for preparing the mutansucrase according to the first aspect, comprising the following steps:

[0018] (1) cloning a gene encoding mutansucrase into an expression vector to obtain a recombinant expression vector, and introducing the vector into a starting bacterium to obtain a genetically engineered strain;

[0019] (2) inoculating the genetically engineered strain into a culture medium for fermentation to obtain a fermentation broth, crushing and centrifuging to obtain the mutansucrose enzyme.

[0020] Wherein, in step (1), the expression vector is pET28a, preferably pET28a(+); and the starting bacteria is Escherichia coli BL21.

[0021] Wherein, in step (2), the fermentation culture is to inoculate the genetically engineered strain into the culture medium and culture at 35-40°C until the OD 600 When the concentration of IPTG 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 hours to obtain a fermentation broth. Preferably, the fermentation culture method is as follows: inoculate the genetically engineered strain into LB culture medium, culture at a speed of 180 rpm and 37°C for 12 hours; draw 1 mL from the culture broth and add it to 100 mL of fermentation medium, place it in a shaking table at 37°C for culture, and when the OD of the enriched culture broth reaches 0. 600 At 0.60-0.80, 0.2 mM IPTG can be added to start inducing enzyme production, and the fermentation is induced at 18°C ​​for 20-26 hours. The bacterial suspension after induced fermentation is centrifuged at 8000 rpm for 10 minutes at 0°C. One centrifuge tube corresponds to one bottle of bacterial suspension, and then distilled water is added for oscillation and washing, and centrifuged again; 20 mL of PBS buffer with a pH value of 5.5 is added to each centrifuge tube for shaking and evenly shaking, an ice water bath is added, ultrasonically broken for 20 minutes, and centrifuged. The supernatant is the crude enzyme solution. Further preferably, the fermentation medium has a formula of: peptone 12 g / L, yeast powder 24 g / L, KH2PO4 2.31 g / L, K2HPO4 16.43 g / L, and glucose 10 g / L.

[0022] In a sixth aspect, the present invention provides use of the mutansucrase 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 mutans.

[0023] The weight average molecular weight of the mutans is 600-700 kDa, and it contains >90% α-1,3-glycosidic bonds, wherein >90% refers to the proportion of α-1,3-glycosidic bonds in the bond composition of the mutans. Preferably, the bond composition of the mutans contains 91% α-1,3 glycosidic bonds and 9% α-1,6 glycosidic bonds.

[0024] Wherein, the preparation of mutans is carried out by catalyzing sucrose with mutansucrose, the amount of mutansucrose 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°C, and the reaction time is 4-6 hours. Preferably, the enzyme addition amount is 5 U / L, the sucrose addition amount is 200 g / L, the reaction pH is 5.5, the reaction temperature is 30°C, and the reaction time is 4 hours.

[0025] Beneficial effects:

[0026] The effective technical effect of the present invention is that compared with the general mutans preparation method, mutansucrose can be used to directly synthesize mutans of high molecular weight in one step enzymatically, and the catalytic conditions are mild, the separation steps are simple, the product molecular weight distribution is concentrated, and it contains up to 90% of α-1,3-glycosidic bonds, which expands its application range. Through the study of the enzymatic properties of the enzyme, it is found that it has great potential for industrial production and application. The present invention lays a foundation for the production of mutans by enzyme engineering and the application of products. The molecular weight of the mutans prepared by the present invention is about 650 kDa, and it can maintain a stable structure at a high temperature of 300 ° C. As a polysaccharide of biological origin, it is used as a bio-based material, and it naturally has the advantages of biocompatibility and biodegradability. It has good application prospects in the field of bio-based materials, such as the preparation of drug nanocarriers, drug gel dressings, and highly absorbent materials for sanitary products. The present invention lays a foundation for the production of mutans by enzyme engineering and the application of products. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0028] Figure 1 The invention discloses a preparation process and application of mutated sucrase MUT-I.

[0029] Figure 2 The optimum pH, optimum temperature, pH stability and temperature stability of the mutansucrose enzyme MUT-I.

[0030] Figure 3 To optimize the catalytic conditions of mutansucrase MUT-I. Figure 3 A is the optimized sucrose concentration, Figure 3B is the reaction time optimization, Figure 3 C is the reaction pH optimization, Figure 3 D is the reaction temperature optimization, Figure 3 E is the optimization of enzyme addition amount.

[0031] Figure 4 This is a scanning electron micrograph of the catalytic product of mutansucrase MUT-I. Figure 4 A. Figure 4 B. Figure 4 C is the scanning electron microscope image at different resolutions.

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

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

[0034] The following is a complete description of the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention.

[0035] The schematic diagram of the preparation process and application of the mutansucrase of the present invention is as follows: Figure 1 shown.

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

[0037] According to the sequence of Leuconostoc pseudomesenteroides G496 genome and vector pET28a(+), the mutation primers were designed with the help of SnapGene software as follows:

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

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

[0040] 5'-TGCGGCCGCAAGCTTGTCGACATTATGCAGGTAAGCCATATTCATTGATTG-3';

[0041] The genome of Leuconostoc pseudomesenteroides G496 was used as a template, and the upstream mutation primers and downstream mutation primers shown in SEQ ID NO.3-4 were used to amplify the mutant polysaccharase gene MutI (whose nucleotide sequence is shown in SEQ ID NO.2). After the PCR amplification product was tested by 1% agarose gel electrophoresis, DpnI was added to remove the original methylated template. After 4 hours of enzyme digestion, the PCR reaction solution after enzyme digestion was purified and recovered for later use.

[0042] The pET28a(+) plasmid was digested with EcoRI and SalI, and the digested product was recovered by gel and used as a linearized cloning vector.

[0043] The MutI gene and linearized cloning vector purified and recovered by PCR were transferred into the competent E. coli BL21 (DE3) cells using a one-step cloning kit. The preparation method of the competent E. coli BL21 (DE3) cells is as follows: 50 μL of E. coli BL21 (DE3) bacterial solution was added to a 5 mL LB test tube, and cultured on a shaking table at 37°C, 180 rpm for 12 hours; 1 mL of the bacterial solution in the test tube was added to a shaking flask containing 100 mL LB medium, and cultured on a shaking table at 37°C, 180 rpm, and the OD of the bacterial solution was 0. 600 When the pH value is 0.5-0.6, take out the shaking flask, place it on ice for 10 min, centrifuge it at 4°C, 4100 rpm for 10 min, discard the supernatant, resuspend it with 2 mL of 0.05 M CaCl2 solution containing 15% glycerol, divide it into portions and store it in a -80°C refrigerator for later use.

[0044] The one-step cloning method is as follows: the PCR amplification product and the linearized plasmid pET-28a(+) are mixed, and a one-step cloning reaction is performed under the catalysis of the 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 to 10 minutes; add 10 μL of cooled one-step cloning reaction system to the competent cells, flick with your fingers, mix well, and place on ice for 30 minutes; then heat shock at 42°C for 90 seconds, and quickly ice bath for 3 minutes. Add 900 μL of LB medium to the above reaction system, and culture on a shaker at 37°C and 180rpm for 1 hour to recover the cells. Take 100 μL and spread it on a resistant plate, culture it in a 37°C incubator for 12 hours, pick a single colony, and use the upstream mutation primer and downstream mutation primer shown in SEQ ID NO.3-4 for colony PCR verification. The colony that can amplify the target gene fragment is the strain that successfully expresses the recombinant expression plasmid pET28a (+) -MutI, that is, the genetically engineered bacteria BL21 (DE3) / MutI.

[0048] Example 2 Preparation of Mutansucrase by Fermentation with Genetically Engineered Bacteria BL21(DE3) / MutI

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

[0050] Fermentation medium: peptone 12 g / L, yeast powder 24 g / L, KH2PO4 2.31 g / L, K2HPO4 16.43 g / L, glucose 10 g / L, sterilization at 121°C for 15 min.

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

[0052] Example 3 Study on the enzymatic properties of mutansucrase MUT-I

[0053] The enzyme activity of sucrase is defined as: the amount of enzyme required to generate 1 μmol of fructose in 30 minutes at pH 5.5 and 30°C is one enzyme activity unit U.

[0054] The crude enzyme solution of the mutansucrose enzyme MUT-I prepared in Example 2 was used to study the enzymatic properties; the enzyme activity detection system was prepared: 1.64 g / L sodium acetate, 0.05 g / L CaCl2, dissolved in distilled water, and 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 phosphoric acid aqueous solution or 0.1 M NaOH aqueous solution. To detect the optimal pH of the enzyme, 1 mL of the crude enzyme solution was mixed with 2.5 mL of the enzyme activity reaction system of different pH values ​​for measuring enzyme activity, and the mixture was reacted at 30°C for 30 min. After the reaction, 1.5 mL of DNS reagent was added, boiled for 5 min, and measured at a wavelength of 540 nm using a spectrophotometer. The fructose content after the reaction was calculated by substituting the fructose standard curve to determine the enzyme activity. The results are as follows Figure 2 As shown in A, the pH value at which the highest enzyme activity is measured is 100%. In order 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), and the residual activity was measured at specific time intervals (1, 2, 3, and 4 h). Figure 2 C, the pH value at which the highest enzyme activity was measured was 100%.

[0055] In order 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 min. Figure 2 As shown in B, the temperature value of the highest enzyme activity was taken as 100%. In order 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 the residual activity was measured at specific time intervals (1, 2, 3, 4h). Figure 2 As shown in D, the temperature at which the highest enzyme activity is measured is 100%.

[0056] Example 4 Preparation of Mutansucrase Catalyzed by Mutansucrase MUT-I

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

[0058] The reaction solution after catalysis is filtered, the supernatant is discarded, washed with water, and vacuum dried to obtain the polysaccharide. The scanning electron microscopy results are as follows: Figure 4 shown. Figure 4 AC is an electron microscope image 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. It can be widely used in solvent-based resin systems in the coatings industry, effectively replacing the use of fossil resources in industrial production.

[0059] The GPC spectrum test results of the mutans prepared by using the preferred mutansucrose enzyme MUT-I are shown in Table 2. The molecular weight is about 644,083 Da. The NMR spectrum is shown in Figure 5 As shown, the bond composition of the polysaccharide contains 91% α-1,3 glycosidic bonds and 9% α-1,6 glycosidic bonds. In the present invention, the thermal stability of the polysaccharide product is characterized by thermogravimetric analysis. The Td5% of the product is 287.65°C, which has high thermal stability and lays a good modification foundation for the subsequent preparation of derivatives to replace petrochemical products.

[0060] Table 2 Molecular weight test results of polysaccharides

[0061]

[0062] At present, in the one-step enzymatic synthesis process of the present invention, 200 g / L sucrose substrate is added, and the final fructose concentration detected is 93.15 g / L, the conversion rate is higher than 90%, and the final obtained polysaccharide product is 86.15 g / L, and the final yield is higher than 85%.

[0063] The present invention provides a method and idea of ​​​​mutansucrase and its application. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A mutansucrose enzyme, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. A gene encoding the mutanesucrase according to claim 1, the nucleotide sequence of which is shown in SEQ ID NO.

2.

3. A recombinant expression vector comprising the gene according to claim 2.

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

5. The method for preparing the mutansucrase according to claim 1, characterized in that: The steps include: (1) cloning a gene encoding mutansucrase into an expression vector to obtain a recombinant expression vector, and introducing the vector into a starting bacterium to obtain a genetically engineered strain; (2) inoculating the genetically engineered strain into a culture medium for fermentation to obtain a fermentation broth, crushing and centrifuging to obtain the mutansucrose enzyme.

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

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

8. Use of the mutansucrase according to claim 1, the recombinant expression vector according to claim 3, or the genetically engineered strain according to claim 4 in the preparation of mutans.

9. The use according to claim 8, characterized in that: The weight average molecular weight of the polysaccharide is 600-700 kDa, and contains more than 90% of α-1,3-glycosidic bonds.

10. The use according to claim 8, characterized in that: The method uses mutansucrase to catalyze sucrose to prepare mutansucrase, wherein the amount of mutansucrase 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° C., and the reaction time is 4-6 hours.

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