Amylosucrase mutant with improved enzyme activity and application thereof

Abstract

The method comprises the following steps: by taking an amylosucrase gene derived from a microorganism Calidimertia timidus DSM 17022 as a template, respectively mutating an asparagine residue at the 577th site on an interface between dimers of the amylosucrase into arginine and aspartic acid, so as to obtain mutants N577R and N577D; the aggregation state is changed from a wild type tetramer state to a monomer state, and the total enzyme activity is respectively improved to 2.9 + / -0.2 U / mg and 3.2 + / -0.1 U / mg from 2.7 + / -0.1 U / mg of the wild type, and is respectively improved to 1.07 times and 1.19 times of the wild type.

Description

Technical Field

[0001] This invention belongs to the field of enzyme genetic engineering technology, specifically relating to a starch sucrase mutant with enhanced enzyme activity and its application. Background Technology

[0002] Amylose sucrase (ASase) belongs to the glycoside hydrolase family 13 and is the only polysaccharide-utilizing enzyme in this family with polymerization activity. ASase can catalyze various types of reactions, including polymerization, isomerization, and transglycosylation, using inexpensive sucrose as a substrate. It has broad application potential in the food industry, producing functional sweeteners, dietary fiber, carbohydrate-based encapsulation materials, and bioactive substances. Polymerization reactions start with inexpensive sucrose to synthesize amylose (glucose units linked only by α-1,4 bonds). The self-assembly properties of the amylose product can be used to further synthesize starch-based immunomicroparticles and starch-based nanoparticle encapsulation systems, or coupled with other enzymes (such as cyclodextrin glycosyltransferase, maltodextrin synthase, glycogen branching enzyme, etc.) to produce cyclodextrin, maltodextrin, hyperbranched dextran, etc. Isomerization reactions produce sucrose isomers menobiose and trehalose, both of which are rare functional disaccharides found in nature. When an additional glycosyl acceptor substrate is present in the system besides sucrose, ASase has the ability to transglycosylate. This not only makes it a tool for glycosylation modification of bioactive substances, used in the synthesis of arbutin, flavonoid derivatives, etc., but also used for glycosylation protection and intermediate fragment synthesis in the Shigella antigen polysaccharide synthesis pathway.

[0003] However, currently reported starch sucrase enzymes generally suffer from insufficient catalytic activity, especially at high temperatures, which greatly limits their further development and application. Our research group previously successfully discovered a type of enzyme derived from a thermostable microorganism... Calidithermus timidus DSM 17022's calciferase (CT-ASase) is currently the only reported calciferase existing in solution as a tetramer. Its structure consists of five regions of residues extensively involved in hydrophobic interactions, including amino acid residues Arg368~Leu372, Ala404~His418, Leu573~Val580, Val602~Val609, and Glu630~Val634. Currently, targeted modification of this calciferase to obtain calciferase mutants with enhanced enzyme activity is of great significance for the industrial production and application of calciferase. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects in the prior art and provide a starch sucrase mutant with enhanced enzyme activity and its application. Its aggregation state changes from the tetramer state of the wild type to a monomer, and the enzyme activity is also improved to a certain extent.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A starch sucrase mutant with enhanced enzyme activity, the amino acid sequence of which is shown in SEQ ID NO.4 or SEQ ID NO.6.

[0006] As a further technical solution, the starch sucrase mutant is obtained by site-directed mutation of the asparagine residue at position 577 of the starch sucrase as shown in SEQ ID No. 2 to arginine or aspartic acid.

[0007] As a further technical solution, the amino acid sequence of the starch sucrase is as shown in SEQ ID No. 2, and the nucleotide sequence is as shown in SEQ ID No. 1.

[0008] As a further technical solution, the starch sucrase shown in SEQ ID No. 2 originates from... Warm-blooded timid DSM 17022.

[0009] A gene encoding the starch sucrase mutant.

[0010] As a further technical solution, the nucleotide sequence of the gene encoding the starch sucrase mutant as shown in SEQ ID NO.4 is shown in SEQ ID NO.3.

[0011] As a further technical solution, the nucleotide sequence of the gene encoding the starch sucrase mutant as shown in SEQ ID NO.6 is shown in SEQ ID NO.5.

[0012] A recombinant plasmid carrying the said gene.

[0013] As a further technical solution, the expression vector of the recombinant plasmid is PET-22b(+).

[0014] A recombinant cell expressing the starch sucrase mutant or the gene is obtained by transforming the recombinant plasmid into a host cell, wherein the host cell comprises E. coli BL21 (DE3).

[0015] A method for preparing the starch sucrase mutant involves inducing and culturing the recombinant cells to produce the starch sucrase mutant.

[0016] As a further technical solution, the induction culture includes: adding the seed culture of the recombinant cells to liquid LB medium containing 50 µg / mL ampicillin, culturing at 37 ℃ and 200 rpm for 2-3 h until the OD value is 0.6~0.8; then adding 1 mM IPTG and inducing in a shaker at 25 ℃ for 6 h to produce the starch sucrase mutant.

[0017] Application of the starch sucrase mutant in the catalytic production of dextran from sucrose.

[0018] As a further technical solution, under the action of a starch sucrase mutant, sucrose is used as a substrate to carry out an enzymatic reaction to produce dextran, wherein the temperature of the enzymatic reaction is 50℃ and the reaction time is 30 min.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses microorganisms Calidithermus timidus Using the DSM 17022 amylose sucrase gene as a template, the asparagine residue at position 577, located at the dimer interface of the enzyme, was mutated to obtain amylose sucrase mutants N577R and N577D. The total enzyme activity of wild-type amylose sucrase catalyzing sucrose at 50 °C was 2.7 ± 0.1 U / mg, while the total enzyme activities of the site-directed mutagenesis mutants N577R and N577D under the same conditions were 2.9 ± 0.2 U / mg and 3.2 ± 0.1 U / mg, respectively, representing increases of 1.07 and 1.19 times compared to the wild type. Furthermore, the aggregation state of the two site-directed mutants, N577R and N577D, in solution changed from the tetrameric state of the wild-type enzyme to a monomeric state. This indicates that mutations based on the protein dimer interface residues can not only alter the aggregation form of ASase protein in solution but also correspondingly change the catalytic activity of the enzyme. Therefore, the enzyme activity modification strategy and positive results provided by this invention have certain guiding value for molecular modification research of other similar enzymes. Attached Figure Description

[0020] Figure 1 This is an SDS-PAGE electrophoresis image of wild-type starch sucrase and its mutants in one embodiment of the present invention; exist Figure 1 In the image, band M: Marker; lane 1: wild type; lane 2: mutant N577R; band 3: mutant N577D.

[0021] Figure 2 This is a graph showing the relative catalytic activity of wild-type starch sucrase and its mutants on sucrose in one embodiment of the present invention. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0024] In this invention, 1. Culture medium (1) Liquid LB medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0025] (2) Expanded culture medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0026] 2. Unless otherwise specified, all raw materials used in this invention are commercially available.

[0027] Example 1: Preparation of recombinant plasmids of starch sucrase mutants 1. Construction of mutant plasmids pET-22b(+)-N577R and pET-22b(+)-N577D (1) Obtaining wild-type plasmids: Wild-type plasmids were obtained from our laboratory and were derived from our laboratory. Warm-blooded timid The starch sucrase (CT-ASase) of DSM 17022, also known as wild-type starch sucrase, has the NCBI database accession number WP_018466847.1; its nucleotide and amino acid sequences are shown in SEQ ID No. 1 and SEQ ID No. 2. (2) Construction of mutant plasmids: Site-directed mutagenesis was performed using the QuikChange™ method. Using the wild-type plasmid as a template, asparagine was mutated to arginine and aspartic acid at the N577 position to construct two single-point mutants (N577R and N577D). The mutation results were verified to be correct by sequencing. The nucleotide and amino acid sequences of mutant N577R are shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleotide and amino acid sequences of mutant N577D are shown in SEQ ID NO.5 and SEQ ID NO.6. Using recombinant wild-type plasmids as templates, forward and reverse primers for mutants N577R and N577D were designed. The mutant primers are shown in Table 1 below, with lowercase letters indicating mutation sites: Table 1 Primers for mutants N577R and N577D

[0028] PCR amplification: The reaction system is shown in Table 2, with a total volume of 50 μL. The reaction program was as follows: 95 ℃, 5 min; 95 ℃, 30 s; 57 ℃, 30 s; 72 ℃, 3 min; 28 cycles; 72 ℃, 5 min; 4 ℃.

[0029] Table 2 Composition of PCR reaction system

[0030] (3) Agarose gel electrophoresis verification and template digestion: The size of the PCR product bands was verified by agarose gel electrophoresis according to the marker. After verification, 1 μL of Q.cut was added to the PCR product system. Dpn I and 2 μL of Q.cut Buffer (10×) were used to perform an enzyme digestion reaction in a 37 ℃ water bath for 120 min to remove template DNA from the PCR reaction system.

[0031] (4) Transformation: The digested PCR product was introduced into *E. coli* DH5α competent cells and plated on LB agar plates containing 50 µg / mL ampicillin. The cells were then incubated overnight at 37 °C. Positive clones were subsequently selected for plasmid extraction and DNA sequencing. The successfully sequenced mutant plasmid was then introduced into… E. coli BL21(DE3) competent cells were used to construct a mutant gene recombinant expression strain for the induction expression of mutant enzymes.

[0032] 2. Following the construction methods of the mutant plasmids pET-22b(+)-N577R and pET-22b(+)-N577D described above, other mutant plasmids were constructed. Among them, mutants N577A, N577C, N577E, N577F, N577G, N577H, N577I, N577K, N577L, N577M, N577Q, N577P, N577S, N577T, N577V, N577W, and N577Y were derived from... Calidithermus timidusThe asparagine at position 577 of the starch sucrase in DSM 17022 was obtained by mutating alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, glutamine, proline, serine, histidine, valine, tryptophan, and tyrosine, respectively. The primers involved are shown in Table 3.

[0033] Table 3: Primers for other mutants

[0034]

[0035] Example 2: Expression and purification of mutant enzyme by nickel affinity chromatography 1. Expression and purification of wild-type enzyme, mutant N577R, and mutant N577D. The mutant plasmids pET-22b(+)-N577R and pET-22b(+)-N577D, verified by sequencing in Example 1, and the wild-type recombinant plasmid were introduced into competent cells of the expression host strain *Escherichia coli* BL21(DE3), and plated on LB agar plates containing 50 µg / mL ampicillin and incubated overnight at 37 °C. Positive colonies were picked and incubated overnight at 37 °C and 200 rpm in liquid LB medium containing 50 µg / mL ampicillin. The seed culture was then expanded to 200 mL of liquid LB medium containing 50 µg / mL ampicillin at an inoculation rate of 2 / 1000 and incubated at 37 °C and 200 rpm for 2–3 h until the OD value reached 0.6–0.8. Subsequently, 1 mM IPTG was added to the expansion medium and the culture was incubated at 25 °C in a shaker for 6 h.

[0036] The induced fermentation broth was centrifuged at 8000 rpm for 5 min to collect the bacterial cells. The cells were resuspended in 15 mL of lysis buffer (50 mM Tris, 200 mM NaCl, pH adjusted to 7.0 with HCl). The resuspended cells were placed in an ice-water mixing bath, and the system was then placed in an ultrasonic cell disruptor. The cells were sonicated at 450 kHz for 1 s, paused for 2 s, and then for 20 min to obtain intracellular products. The disrupted broth was centrifuged at 8000 rpm for 5 min in a low-temperature centrifuge, and the supernatant was collected. The supernatant was filtered through a 0.45 μm microporous membrane and stored for later use.

[0037] Protein purification of the crude enzyme solution was performed using nickel column affinity purification. First, the constant flow peristaltic pump and Ni column were connected to the protein purification system. 2+The tubing connecting the affinity chromatography column, 280 nm detector, and other instruments was set to a constant flow rate of 1.5 mL / min and leak checked. Two column volumes of equilibration buffer (50 mM Tris, 500 mM NaCl, pH 7.5) were prepared to equilibrate the nickel ion affinity chromatography column. After equilibration, the crude lysate was pumped into the column at a flow rate of 0.5 mL / min. The column was then continuously washed with equilibration buffer at a flow rate of 1.5 mL / min. Once the UV detector reading stabilized, washing buffer (30 mM imidazole, 50 mM Tris, 500 mM NaCl, pH 7.5) was used to wash away contaminating proteins with weak nickel ion binding affinity. After the UV detector reading stabilized again, elution buffer (500 mM imidazole, 50 mM Tris, 500 mM NaCl, pH 7.5) was pumped in at a flow rate of 1 mL / min, and the washings (the target protein) were collected in a 10 mL centrifuge tube. The target protein was aspirated and transferred to a dialysis bag with a molecular weight cutoff of 14 kDa. The bag was clamped with double dialysis clamps and placed in dialysis solution A (10 mM EDTA·2Na, 50 mM Tris, pH 7.5). Dialysis was performed overnight, followed by dialysis with dialysis solution B (50 mM Tris, pH 7.0) for 6 h. Dialysis with dialysis solution A was performed once, and dialysis with dialysis solution B was repeated twice. The enzyme solution was then collected and stored at 4 °C for later use.

[0038] The purified target recombinant wild-type enzyme protein (also known as wild-type amylose sucrase protein), mutant N577R protein (amylose sucrase mutant N577R protein), and mutant N577D protein (amylose sucrase mutant N577D protein) prepared above were analyzed by protein electrophoresis. Specific experiments are as follows: (1) Sodium dodecyl sulfate polyacrylamide gel was prepared using a pre-formed gel kit, with a top stacking gel concentration of 4% and a bottom separating gel concentration of 12.5%; (2) Take 20 μL of the target protein solution, add 5 μL of protein loading buffer, mix well, heat in a boiling water bath for 5 min, and then centrifuge at 10000 r / min for 5 min to remove insoluble precipitate. (3) Take 10 μL of the supernatant obtained in (2) and add it to the polyacrylamide gel. Run the gel at 120 V until the bromophenol blue indicator in the loading buffer appears at the end of the gel. (4) Remove the gel, stain it with staining solution for 18-20 min, then destain it with destaining solution until the background color of the electrophoresis gel becomes colorless, and take pictures for observation using a gel imaging system.

[0039] Protein electrophoresis analysis showed that the target recombinant wild-type enzyme protein, mutant N577R protein, and mutant N577D protein exhibited single bands, indicating electrophoretic purity, and were ready for further processing. Simultaneously, the electrophoresis results showed that the molecular weight of a single subunit of the wild-type enzyme protein, mutant N577R protein, and mutant N577D protein was approximately 66 kDa.

[0040] 2. Following the above expression and protein purification methods, express and purify other mutant enzymes and proteins to obtain purified mutant N577A, mutant N577C, mutant N577E, mutant N577F, mutant N577G, mutant N577H, mutant N577I, mutant N577K, mutant N577L, mutant N577M, mutant N577Q, mutant N577P, mutant N577S, mutant N577T, mutant N577V, mutant N577W, and mutant N577Y proteins.

[0041] Example 3: Determination of the catalytic ability of starch sucrase mutants to sucrose The purpose of this embodiment is to compare the changes in the catalytic ability of the mutant and wild-type enzymes on the substrate sucrose before and after mutation. The specific method is as follows: Using 0.1 mol / L sucrose as the substrate, the reaction was carried out at 50 °C for 30 min in 50 mmol / L sodium phosphate buffer (pH 7.0), and finally terminated by heating at 95 °C for 15 min. The amount of fructose released in the reaction system represents the total enzyme activity, and the amount of polymerase released minus the amount of glucose released represents the polymerase activity. Unless otherwise specified, the enzyme activity of CT-AS is assumed to be the total enzyme activity, defined as the amount of enzyme required to catalyze the release of 1 μmol of fructose per minute. When it is necessary to determine the glucose and fructose released in the reaction solution, a high-performance liquid chromatography (HPLC) system connected to a differential refractive index detector and a cation exchange column (Sugar-Pak™) was used for analysis. The mobile phase was ultrapure water containing 50 mg / L of calcium disodium EDTA, the column temperature was 85 °C, the flow rate was 0.4 mL / min, and the run time for each sample was 20 min. Standard curves were prepared using glucose and fructose standard samples to determine the actual content of products in the reaction solution.

[0042] The pure enzyme solution obtained in Example 2 was subjected to an enzymatic reaction under optimal conditions, and the results were determined by high-performance liquid chromatography to compare the degradation ability of the mutant enzyme of the present invention with that of the wild-type enzyme. The total enzyme activity of the wild-type enzyme catalyzing sucrose at pH 7.0 and 50 °C was 2.7 ± 0.1 U / mg. The total enzyme activities of the starch sucrase mutants N577R and N577D catalyzing sucrose under the same conditions (pH 7.0 and 50 °C) were 2.9 ± 0.2 U / mg and 3.2 ± 0.1 U / mg, respectively, which were 1.07 and 1.19 times that of the wild-type starch sucrase. By observing the position of N577 in the crystal structure, it was found that N577 is close to the adjacent subunit G412, and a 3.29 Å hydrogen bond is formed between ND2 of N577 and O of the main chain of G412. After mutating it to charged residues arginine and aspartic acid, the interaction between residues was further enhanced, resulting in a certain increase in the total enzyme activity of sucrose catalysis compared with wild-type starch sucrase.

[0043] Assuming the total enzyme activity of wild-type starch sucrase is 100%, calculate the relative enzyme activity of each mutant, and the results are as follows: Figure 2 As shown.

[0044] from Figure 2 It can be seen that among the mutants obtained by site-directed mutagenesis of amino acid 577 in wild-type starch sucrase, the relative enzyme activity of the starch sucrase mutants N577R and N577D is increased to a certain extent compared with the wild type, while the other mutants are decreased to varying degrees compared with the wild type.

[0045] Example 4: Determination and comparison of the total molecular weight of wild-type starch sucrase and mutant enzymes The oligomeric state of recombinant proteins in solution was determined using gel size exclusion chromatography, as follows: The oligomeric state of recombinant proteins (wild-type amylose WT, amylose sucrase mutant N577R, and amylose sucrase mutant N577D) in solution was analyzed using a Superdex 200 gel size exclusion column connected to a UV detector. The mobile phase was 20 mmol / L Tris-hydrochloric acid buffer (pH 7.0) containing 150 mmol / L sodium chloride, and the flow rate was set to 1 mL / min. A gel size exclusion protein standard kit was used as the molecular weight standard, comprising 13.7 kDa bovine pancreatic ribonuclease, 44.3 kDa chicken ovalbumin, 150 kDa alcohol dehydrogenase, and 670 kDa bovine thyroglobulin. The molecular weights of the standard proteins (…) M w ) and its retention time in gel size exclusion chromatography Rt The interval satisfies the equation Log M w =7.96-0.179× R t Similarly, based on the retention time of the recombinant protein in the test sample on gel size exclusion chromatography, the total molecular weight of the recombinant protein in the test sample can be calculated, and the results are shown in Table 4.

[0046] Table 4. Comparison of total molecular weight between wild-type and mutant amylases

[0047] As shown in Table 4, the molecular weights of the starch sucrase mutants N577R and N577D are both around 70 kDa, suggesting they exist in a single-subunit form and are monomers. In contrast, the total molecular weight of wild-type starch sucrase (CT-ASase wild-type enzyme) is around 240 kDa, exhibiting a tetramer structure. The structure shows that residue N577 is located in region 3 of the dimer interface, which is also spatially close to the inner interface of the dimer, indicating that mutations at this site also affect the overall oligomeric state of the protein.

[0048] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A starch sucrase mutant with enhanced enzyme activity, characterized in that, The amino acid sequence of the starch sucrase mutant is shown in SEQ ID NO.4 or SEQ ID NO.

6.

2. The starch sucrose enzyme mutant with enhanced enzyme activity according to claim 1, characterized in that, The starch sucrase mutant was obtained by site-directed mutation of the asparagine residue at position 577 of the starch sucrase as shown in SEQ ID No. 2 to arginine or aspartic acid.

3. The starch sucrose enzyme mutant with enhanced enzyme activity according to claim 2, characterized in that, The amylose sucrase shown in SEQ ID No. 2 originates from... Calidithermus timidus DSM 17022.

4. A gene encoding the starch sucrase mutant of claim 1.

5. A recombinant plasmid carrying the gene of claim 4.

6. The recombinant plasmid according to claim 5, characterized in that, The expression vector for the recombinant plasmid is PET-22b(+).

7. A recombinant cell expressing the starch sucrase mutant of claim 1 or the gene of claim 4, characterized in that, The recombinant plasmid of claim 5 is obtained by transforming it into a host cell, wherein the host cell comprises... E. coli BL21 (DE3).

8. A method for preparing the starch sucrase mutant as described in claim 1, characterized in that, The recombinant cells of claim 7 were induced and cultured to produce a starch sucrase mutant.

9. The application of the starch sucrase mutant as described in claim 1 in the catalytic production of dextran from sucrose.

10. The application according to claim 9, characterized in that, Dextran was produced by enzymatic reaction using sucrose as a substrate under the action of a starch sucrase mutant. The temperature of the enzymatic reaction was 50℃ and the reaction time was 30 min.

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