Mutated pullulanases and uses thereof

Abstract

The application discloses a mechanism of key amino acids in an activity framework of pululanase PulA by a structural analysis-site mutation-function verification approach, and obtains mutant enzymes with high catalytic efficiency, thereby providing a new direction for development of industrial starch processing enzyme resources. The application successfully obtains purified mutant proteins, the N98D increases the enzyme activity of the wild type to 296%, the T175S increases the enzyme activity of the wild type to 157%, and the N379D increases the enzyme activity of the wild type to 170%. The three mutants of N98D, T175S and N379D have important contribution to improvement of the enzyme activity and have wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and specifically relates to a mutated pullulanase. Background Technology

[0002] Starch is a high-molecular-weight carbohydrate composed of α-D-glucopyranose linked by α-1,4 and α-1,6 glycosidic bonds, forming a complex structure of amylose and amylopectin. Amylose typically accounts for 20%-30% of the total starch content and exists in a linear helical structure, while amylopectin accounts for 70%-80%, forming a highly branched dendritic structure through frequent α-1,6 glycosidic bond branching. This unique molecular structure endows starch with unique physicochemical properties, such as the semi-crystalline structure of starch granules, where crystalline and amorphous regions alternate, giving starch properties such as insolubility in cold water and gelatinization.

[0003] However, the physical and chemical properties of natural starch prevent it from being directly applied to industrial processing. Starch gelatinization temperature is high, and high-temperature processing leads to energy waste and damage to heat-sensitive components. Starch has poor shear resistance, making it unsuitable for foods requiring high processing intensity. Natural starch lacks surface activity, antibacterial properties, and other functions, failing to meet the demands of high-end applications. Therefore, it is usually necessary to modify it to obtain better physical and functional properties. Consequently, enzyme modification technology for deep starch processing has developed rapidly in recent years.

[0004] Pullulanase is a starch debranching enzyme that can efficiently hydrolyze α-1,6-glycosidic bonds in starch molecules, degrading complex polysaccharides into linear oligosaccharides (such as maltotriose and glucose), significantly improving saccharification efficiency. It can be used to produce high-concentration maltose syrup and can also be used to process amylopectin to generate linear dextran chains for the production of resistant starch. It has important application value in the starch deep processing industry. However, due to the low enzyme activity of natural pullulanase, it cannot be directly applied to industrial production. Therefore, we need to design and modify it. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a mutated pullulanase (or, referred to as a pullulanase mutant) and its applications.

[0006] On one hand, the present invention provides a mutant pullulanase, wherein the amino acid sequence of the mutant pullulanase is mutated at any one or any few (e.g., two or three) amino acid sites selected from SEQ ID No.1: position 98, position 175, and position 379, compared with SEQ ID No.1.

[0007] The above-mentioned amino acid sites are the amino acid sites starting from the N-terminus of SEQ ID No. 1.

[0008] In one embodiment, the 98th amino acid is mutated to D, the 175th amino acid is mutated to S, and the 379th amino acid is mutated to D.

[0009] In this invention, amino acid residues can be represented by a single letter or by three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), and arginine (Arg, R).

[0010] On the other hand, the present invention also provides the encoding gene of the mutated pullulanase.

[0011] On the other hand, the present invention also provides a recombinant vector containing the encoding gene of the mutated pullulanase; preferably, the recombinant vector is a recombinant expression vector; preferably a pET series vector, such as pET-15b, pET-22b, pET-28a.

[0012] On the other hand, the present invention also provides a recombinant strain containing the above-mentioned recombinant vector, preferably, the recombinant strain is Escherichia coli, such as Escherichia coli BL21.

[0013] On the other hand, the present invention also provides the use of the mutated pullulanase, the encoding gene, the recombinant vector, or the recombinant strain in the degradation of materials containing polysaccharides with α-1,6 glycosidic bonds.

[0014] On the other hand, the present invention also provides a method for hydrolyzing polysaccharide materials containing α-1,6 glycosidic bonds, the method comprising the step of treating the polysaccharide materials containing α-1,6 glycosidic bonds using the above-mentioned mutant pullulanase.

[0015] In one embodiment, the processing temperature is 50°C-80°C, preferably 60°C-70°C.

[0016] In one embodiment, the polysaccharide containing α-1,6 glycosidic bonds is starch.

[0017] This invention elucidates the mechanism of action of key amino acids in the active architecture of pullulanase PulA through a "structural analysis-site-directed mutagenesis-functional verification" approach, and obtains mutant enzymes with high catalytic efficiency, providing a new direction for the development of industrial starch processing enzyme resources. This invention successfully obtained purified mutant proteins. The N98D mutation increased the wild-type enzyme activity to 296%, the T175S mutation increased the wild-type activity to 157%, and the N379D mutation increased the wild-type activity to 170%. These three mutants, N98D, T175S, and N379D, make significant contributions to improving enzyme activity. Attached Figure Description

[0018] Figure 1 The active site structure and sequence profile of pullulanase substrate.

[0019] Figure 2 Electrophoresis diagrams of purified pullulanase wild-type and mutant proteins.

[0020] Figure 3 To determine pullulanase activity in wild-type and mutant strains. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0022] 1. Constructing the sequence profile of the active center of the GH13 family

[0023] The target enzyme PDB:3WDJ was selected from the GH13 family as a template for the selection of the active structure center residues, and the substrate was selected in PyMOL software. The active structure of the enzyme was generated by dividing the amino acid residues within the range with the cleavage site (CS) as the boundary, and the sequence profile was plotted using the WebLogo website (http: / / weblogo.berkeley.edu / logo.cgi).

[0024] 2. Site-directed mutagenesis of recombinant plasmids

[0025] Mutations of recombinant plasmids were performed using PCR-based site-directed mutagenesis.

[0026] Using the recombinant plasmid as a template, design mutation primers on the biorun online website (https: / / www.biorun.com / tools / 101_2.html). The primer sequences used for mutation are shown in the table below.

[0027] Table 1. PCR reaction system and reaction procedure

[0028]

[0029] PCR program settings:

[0030]

[0031] Table 2. Mutation primers used in this invention

[0032]

[0033] After confirming the amplification results by performing agarose gel electrophoresis on 3 μL of the PCR product, the PCR product was then mixed with DpnI.

[0034] Incubate at 37°C for 30 minutes. The digestion system is shown in the table below.

[0035] Table 3. Digestive System

[0036]

[0037] The digested PCR products were then purified. The specific steps are as follows:

[0038] Add 500 μl of equilibration buffer BL to adsorption column CB2 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm for 1 min, and discard the waste liquid in the collection tube; add 5 times the volume of PCR reaction solution (binding buffer PB) and mix thoroughly; add the resulting solution to another adsorption column CB2 (place the adsorption column in the collection tube), incubate at room temperature for 2 min, centrifuge at 12,000 rpm for 30-60 sec, discard the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube; add 600 μl of wash buffer PW to adsorption column CB2, centrifuge at 12,000 rpm for 30-60 sec, discard the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube. Repeat the operation twice. Place the adsorption column CB2 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and remove as much wash buffer as possible. The adsorption column CB2 was placed at room temperature for 5 minutes to allow it to dry completely, preventing residual wash solution from affecting subsequent experiments. The column was then placed in a clean centrifuge tube, and 30 μL of dH2O was added dropwise to the center of the adsorption membrane. The tube was incubated at room temperature for 2 minutes. The DNA solution was collected by centrifugation at 12,000 rpm for 2 minutes. The digestion product (10 μL) was then transformed into *E. coli* DH5α. The recombinant plasmid (pET28a) was confirmed by DNA sequencing.

[0039] 3. Heterologous expression of recombinant plasmids

[0040] Heat shock transformation of recombinant plasmid: Add 50 μL of competent cells to a centrifuge tube, add 5 μL of recombinant plasmid, mix gently, place on ice for 30 min, then place in a 42°C water bath for 90 s, and immediately remove and place on ice for 5 min. Add 1 mL of LB medium to the centrifuge tube in a clean bench, incubate at 37°C for 1 h, centrifuge at 8000 rpm for 5 min; in a clean bench, discard the supernatant, gently mix by pipetting, transfer to a plate containing kanamycin (100 μg / mg) for inoculation, and incubate the inoculated plate upside down in a 37°C incubator overnight. Transfer 4 mL of bacterial culture to 400 mL of LB liquid medium containing kanamycin. Co-express each protein in 1600 mL of medium. Incubate at 37°C and 200 rpm for about 3 h until the OD600 of the bacterial culture is 0.3-0.4 as measured by a microplate reader. Then add 200 μL of 1 M IPTG and incubate at 16°C and 200 rpm for 20 h.

[0041] 4. Purification of the target protein

[0042] Cells were collected by centrifugation at 8000 rpm for 5 min and resuspended in Tris-HCl buffer (50 mM Tris, 100 mM NaCl, pH 8.0). Cells were lysed by sonication (35% power, 2 s sonication, 2 s rest, 45 min). The target protein containing the 6×His tag was purified by nickel column affinity chromatography, eluting the protein with imidazole-Tris-HCl buffer at concentrations of 5 mM, 10 mM, 15 mM, 250 mM, and 300 mM, and collected in 10 mL EP tubes.

[0043] 5. SDS-PAGE and protein concentration determination

[0044] Mix the sample and SDS buffer at a 4:1 ratio. Take 20 μL of sample and 5 μL of buffer, and incubate at 105℃ for 10 min. For sample loading, load 10 μL of sample and 7 μL of marker. Perform electrophoresis at 80V. When the protein sample forms a straight line (approximately 30 min), adjust the voltage to 180V (approximately 1 h). After electrophoresis, stain with Coomassie Brilliant Blue for at least 30 min and destain with destaining solution for approximately 2 h. Observe the gel using a scanner. Perform ultrafiltration at 4℃ using 50 mM Na2HPO4–citrate buffer (pH 6.0) to replace the buffer until the protein buffer pH = 6.0. Transfer the ultrafiltered enzyme solution to a new centrifuge tube using a 0.22 μm sterile filter tip and store at -20℃.

[0045] The Bradford method was used, with bovine serum albumin as the standard, to determine protein concentration. 200 μL of Coomassie brilliant blue staining solution and 20 μL of protein solution were added to each tube, and the mixture was shaken well. After standing for 5 min, OD595 was measured. Each group was divided into three replicates, with a total of nine replicates. The protein amount and concentration were calculated based on the standard curve.

[0046] 6. Enzyme activity assay

[0047] Add 200 μL of enzyme solution and 200 μL of 2% pullulan solution to a reaction tube. Incubate the reaction mixture at 60 °C for 30 minutes. Terminate the reaction by adding 800 μL of DNS solution. Heat the reaction mixture in a boiling water bath for 10 minutes, cool to room temperature, and measure the absorbance at 540 nm. Construct a standard curve using standard glucose solution to calculate enzyme activity. Perform triplet assays for each group, for a total of nine replicates.

[0048] By analyzing the sequence and structure of pullulanase PulA derived from Bacillus, a triplet of amino acids (Asp435, Glu464, Asp554) and three key sites (N300, T377, N581) in the catalytic center of pullulanase PulA were identified. It is speculated that these sites influence substrate binding and catalytic efficiency through the distance and number of hydrogen bonds. Figure 1 As shown.

[0049] In this embodiment, the catalytic domain of PulA was obtained, the amino acid sequence of which is shown in SEQ ID No. 1, and the DNA sequence encoded by it is shown in SEQ ID No. 2.

[0050] LAKTNIPKPSMSLFISMTDAIIYEMHIRDFTIHHESGVRQKGKYVGLTERGTTGPNGTLTGLSYIKQLGVTHVQLMPVQDFEGVDELQPLKMYNWGYNTVHYNA PEGSYATDPDDPYARIIELKRAIRAFQQEGIRVILDVVYNHVYVRETSSFEHLVPGYYFRYERNGYPSNGTGVGNDLASERKMVKKFIIDSVTYWLKEYGVDGFR FLMGILDIDTMNDVRRAIDEIDPTVIILGEGWDLATPLPSEKKTTIANAKHTPRIAYFNDRFRDYVKGSTFDIHERGFALGDCSYKEAVIGAIRGSIHLFFSPR QSVNYVECHDNHTLWDKMAVANAHESEYIRRKRQKLATAIVLLSQGIPFLHSGQEFYRTKKGVENSYNSPDEVNQVDWNEKSRWEEDVREIMKLIELRKKH(SEQ ID No.1);

[0051]

[0052] Based on the predicted key amino acid sites of PulA (N300, T377, N581), the corresponding sites of the PulA catalytic domain (SEQ ID No. 1) are N98, T175, and N379, respectively.

[0053] In this embodiment, amino acids at positions 98, 175, and 379 of SEQ ID No. 1 were mutated to N98D (N at position 98 of SEQ ID No. 1 was mutated to D), T175S (T at position 175 of SEQ ID No. 1 was mutated to S), and N379D (N at position 379 of SEQ ID No. 1 was mutated to D), respectively. These proteins were then heterologously expressed and purified to obtain N98D, T175S, and N379D proteins, as well as the wild-type protein (WT, shown in SEQ ID No. 1). Electrophoresis images are shown below. Figure 2 As shown.

[0054] In this embodiment, N98D can also be referred to as N300D, T175S as T377S, and N379D as N581D based on the amino acid site correspondence of the PulA catalytic domain according to the full-length PulA sequence.

[0055] Pullulan enzyme activity was measured using the above-described enzyme activity assay method for wild-type PulA protein (SEQ ID No. 1) and three site-directed mutants (N300D, T377S, and N581D). Figure 3 As shown, the results indicate that the enzyme activities of the three mutant proteins were increased to 296%, 157%, and 170% of the wild-type enzyme activities, respectively, with the N300D mutant protein exhibiting the highest enzyme activity.

[0056] This invention elucidates the mechanism of action of key amino acids in the active architecture of pullulanase PulA through a "structural analysis-site-directed mutagenesis-functional verification" approach, and obtains mutant enzymes with high catalytic efficiency, providing a new direction for the development of industrial starch processing enzyme resources. This invention successfully obtained purified mutant proteins. The N300D mutation increased the wild-type enzyme activity to 296%, the T377S mutation increased the wild-type activity to 157%, and the N581D mutation increased the wild-type activity to 170%. The mutation directions of the N300D, T377S, and N581D mutants significantly contribute to improving enzyme activity.

[0057] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A mutant pullulanase, wherein the amino acid sequence of the mutant pullulanase, compared with SEQ ID No. 1, contains a mutation at any of the following amino acid sites selected from SEQ ID No. 1: position 98, position 175, and position 379; wherein the amino acid at position 98 is mutated to D, the amino acid at position 175 is mutated to S, and the amino acid at position 379 is mutated to D.

2. The gene encoding the mutant pullulanase of claim 1.

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

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector is a recombinant expression vector.

5. A recombinant strain comprising the recombinant vector of claim 3 or 4.

6. The use of the mutant pullulanase of claim 1, or the gene of claim 2, or the recombinant vector of claim 3 or 4, or the recombinant strain of claim 5 in the degradation of polysaccharides containing α-1,6 glycosidic bonds.

7. The application according to claim 6, characterized in that, The polysaccharide containing α-1,6 glycosidic bonds is starch.

8. A method for hydrolyzing a polysaccharide containing an α-1,6 glycosidic bond, the method comprising the step of treating the polysaccharide containing an α-1,6 glycosidic bond using the mutant pullulanase of claim 1.

9. The method according to claim 8, characterized in that, The processing temperature is 50℃-80℃.

10. The method according to claim 8 or 9, characterized in that, The polysaccharide containing α-1,6 glycosidic bonds is starch.

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