Maltogenic amylase mutant, biological material, catalyst and application

Abstract

The invention discloses a maltogenic amylase mutant, a biological material, a catalyst and application, and belongs to the field of protein engineering, the mutant is based on wild type maltogenic amylase derived from geobacillus stearothermophilus, and has a plurality of amino acid residue mutations in the 244th site, the 274th site, the 214th site and the 302 site. The mutant disclosed by the invention has excellent alkali resistance and still has higher enzyme activity compared with a wild type enzyme after being treated under the condition that the pH value is 8-11, the relative enzyme activity of the mutant T244R / L274P / Y214F is 2.14 times that of the wild type enzyme after being treated in a buffer solution with the pH value being 10, and the requirement for extreme alkaline environment resistance in industrial application can be met.

Description

Technical Field

[0001] The present invention relates to the field of protein engineering, and particularly to a maltogenic amylase mutant, biological material, catalyst and application thereof. Background Art

[0002] Maltogenic amylase (EC 3.2.1.133) is an important class of glycoside hydrolases belonging to the amylase family. Its core function is to generate maltose - dominated products by hydrolyzing α - 1,4 glycosidic bonds in polysaccharide molecules such as starch and dextrin. Its catalytic core domain presents a classic (β / α)8 - barrel structure, containing a conserved Asp - Glu - Asp catalytic triad responsible for precisely cleaving glycosidic bonds; the substrate - binding domain recognizes polysaccharide chains through hydrophobic interactions and hydrogen - bond networks to ensure reaction specificity. The microbial origin of maltogenic amylase endows it with excellent industrial potential. It can not only maintain activity at high temperatures of 50 - 70 °C but also work stably within a wide pH range of 4.5 - 8.0. These characteristics enable it to be used in the food industry to delay starch retrogradation and improve the quality of baked products, promote the efficient conversion of polysaccharides into fermentable sugars in the biofuel field, and participate in the synthesis of carbohydrate compounds as a green catalyst in the pharmaceutical industry.

[0003] Although maltogenic amylase performs excellently in acidic to neutral environments, its industrial application in strongly alkaline conditions (pH > 8.0) still faces significant limitations. Firstly, the active center of the enzyme is highly sensitive to the alkaline environment. The glutamate residue in the catalytic triad is prone to deprotonation under alkaline conditions, leading to a significant decrease in catalytic efficiency. Secondly, the overall structural stability of the enzyme is easily damaged in the alkaline environment. Its auxiliary domain contains a large number of negatively charged amino acid residues, which may cause the protein conformation to loosen or even irreversibly denature due to enhanced electrostatic repulsion at high pH. In addition, alkaline conditions may trigger side reactions, such as the oxidation or isomerization of starch molecules in strongly alkaline solutions, generating modified substrates that are difficult to be recognized by the enzyme, further reducing the reaction efficiency. These limitations make it difficult for maltogenic amylase to be directly applied to typical alkaline industrial scenarios such as detergent manufacturing and alkaline textile desizing. Therefore, it is still necessary to further optimize the adaptability of the enzyme molecule to the alkaline micro - environment and expand its industrial application boundaries. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide a maltogenic amylase mutant resistant to alkaline environments; the second object is to provide biological materials and catalysts related to the mutant; the third object is to provide the application of the mutant and catalyst in catalyzing starch decomposition.

[0005] Technical solution: The maltogenic amylase mutant of the present invention is based on the wild-type maltogenic amylase amino acid sequence shown in SEQ ID NO: 1, and has mutations in multiple amino acid residues at positions 244, 274, 214, and 302, wherein the reference sequence number of the wild-type maltogenic amylase amino acid sequence is UniProtKB: P19531.2.

[0006] Preferably, in the amino acid mutations, the threonine residue at position 244 is mutated to an arginine residue, the leucine residue at position 274 is mutated to a proline residue, the tyrosine residue at position 214 is mutated to a phenylalanine residue, and the glutamate residue at position 302 is mutated to a glutamine residue.

[0007] Preferably, the maltogenic amylase mutant has an amino acid sequence as shown in SEQ ID NO: 2, or as shown in SEQ ID NO: 3, or as shown in SEQ ID NO: 4.

[0008] The nucleotide sequence of the present invention is based on the wild-type maltogenic amylase nucleotide sequence and contains corresponding base mutations, encoding the amino acid sequence of the aforementioned maltogenic amylase mutant.

[0009] Preferably, the wild-type maltogenic amylase nucleotide sequence is as shown in SEQ ID NO: 5, which is a codon-optimized nucleotide sequence of maltogenic amylase derived from Geobacillus stearothermophilus ( Geobacillus stearothermophilus )

[0010] The recombinant vector of the present invention contains the aforementioned nucleotide sequence.

[0011] The recombinant microorganism of the present invention contains the aforementioned nucleotide sequence or recombinant vector.

[0012] The catalyst of the present invention contains the aforementioned maltogenic amylase mutant.

[0013] Use of the maltogenic amylase mutant or catalyst of the present invention in catalyzing starch decomposition.

[0014] Preferably, the use is in catalyzing starch decomposition at a pH not higher than 11.0.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The obtained mutant has excellent alkali resistance and still has a relatively high enzyme activity after treatment under the conditions of pH 8-11, compared with the wild-type enzyme. Among them, the mutant T244R / L274P / Y214F has a relative enzyme activity of 2.14 times that of the wild-type enzyme after treatment in a buffer solution with pH = 10, which can meet the requirements for an extremely alkaline environment in industrial applications. Description of the drawings

[0016] Figure 1 The protein structure diagram of maltogenic amylase derived from Geobacillus stearothermophilus and the amino acid residue sites at positions 244, 274, 214, and 302; Geobacillus stearothermophilus The glucose-OD obtained in the test example

[0017] Figure 2 Standard curve graph; 540

[0018] Figure 3 The results of the relative enzyme activity assays of the wild-type enzyme and the mutant enzyme after treatment with different pH buffers. Detailed implementation manners

[0019] The technical solutions of the present invention will be further described below.

[0020] Sources of the materials used in the examples:

[0021] 1. Strains and plasmids

[0022] The synthesis of the pET-22b(+) plasmid (pET22b) carrying the wild-type maltogenic amylase gene, the synthesis of the mutant primers and plasmid construction primers, and the sequencing of the gene sequence in the plasmid were completed by Genewiz (Suzhou) Co., Ltd.

[0023] Strains E.coli DH5α was used for plasmid construction, and the strain E.coli BL21(DE3) was used for the expression of foreign proteins, and both were purchased from Shenzhen Kangti Life Technology Co., Ltd.

[0024] 2. Media

[0025] The composition of the LB medium used was: yeast extract 5 g / L, tryptone 10 g / L, NaCl 5 g / L. If a solid medium was used, 15 g / L agar powder was additionally added. TB liquid medium: yeast extract 12 g / L, tryptone 12 g / L, glycerol 4 mL / L, dipotassium hydrogen phosphate 12.5 g / L, potassium dihydrogen phosphate 2.3 g / L.

[0026] Example 1 Construction of the wild-type maltogenic amylase plasmid and preparation of the crude enzyme solution

[0027] The codon-optimized wild-type gene of maltogenic amylase from Geobacillus stearothermophilus ( Geobacillus stearothermophilus ) with the sequence shown in SEQ ID NO: 5 was synthesized by Genewiz (Suzhou) Co., Ltd. and constructed on the pET22b vector to obtain pET22b-wt;

[0028] pET22b-wt was transformed into E.coli the DH5α strain, and the recombinant bacteria​E.coli DH5α / pET22b-wt was inoculated into a test tube containing 5 mL of LB medium with a final concentration of 100 μg / mL ampicillin and cultured with shaking at 37 °C and 220 rpm for 12 h;

[0029] After the culture, the cells were centrifuged at 12,000 rpm for 1 min and the cells were collected. Using a high-purity plasmid miniprep kit, the pET22b-wt plasmid was extracted as a template for iterative mutagenesis for the construction of maltogenic amylase mutants;

[0030] Meanwhile, pET22b-wt was transferred into E.coli BL21(DE3) to construct a recombinant mutant expression strain E.coli BL21(DE3) / pET22b-wt. The successfully constructed recombinant mutant expression strain was spread onto an ampicillin plate containing a final concentration of 100 μg / mL and cultured at 37 °C for 18 h;

[0031] After the culture, a single colony was picked and inoculated into a test tube containing 5 mL of LB medium with a final concentration of 100 μg / mL ampicillin and cultured at 37 °C with shaking at 200 rpm for 18 h. 2.5 mL of the bacterial solution was inoculated into 50 mL of TB liquid medium containing 100 μg / mL ampicillin and cultured in a shaker at 37 °C and 140 rpm for 6 h. Then, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.4 mM that had been filter-sterilized was added to the fermentation broth, and the culture was continued at 22 °C and 140 rpm for 22 h;

[0032] The fermentation broth of the recombinant bacteria was transferred to a centrifuge tube and the cells were collected by centrifugation at 11,000 rpm. The cells were resuspended in glycine-sodium hydroxide buffer at pH 10.0 to an OD 600 = 20, and the cells were ultrasonically disrupted at a power of 65 W under ice bath conditions, working for 6 s and then pausing for 3 s, for a total of 10 min to disrupt the cells. The cells were centrifuged again at 12,000 rpm to remove cell debris. The resulting supernatant was the crude enzyme solution containing wild-type enzyme.

[0033] The amino acid sequence of wild-type maltogenic amylase is shown in SEQ ID NO: 1.

[0034] Example 2: Construction of maltogenic amylase mutant T244R / L274P and preparation of the crude enzyme solution.

[0035] Starting from the maltogenic amylase of Geobacillus stearothermophilus, the threonine (Thr) at position 244 at the N-terminus of its amino acid sequence shown in SEQ ID NO: 1 was mutated to arginine (Arg), and the leucine (Leu) at position 274 was mutated to proline (Pro). The preparation method of the mutant T244R / L274P is as follows:

[0036] 1. Construct a recombinant plasmid

[0037] The primers for the T244R / L274P mutation sites are shown in Table 1. Using the pET22b-wt plasmid carrying the original maltogenic amylase gene as a template for PCR, the PCR system is shown in Table 2, and the PCR reaction conditions are shown in Table 3;

[0038] Table 1 T244R / L274P mutation primers

[0039]

[0040] Note: The underlined parts in the primers are the mutation sites. "F" represents the upstream primer, and "R" represents the downstream primer.

[0041] Table 2 PCR reaction system.

[0042]

[0043] Table 3 PCR reaction conditions.

[0044]

[0045] After the PCR amplification is completed, the amplified product is detected by 0.9% agarose gel electrophoresis. The result shows that the amplified product is a single band with a size of about 7500 bp. The template is digested with the restriction enzyme DpnI. Prepare the template digestion system as shown in Table 4 and digest it at 37°C for 1 h.

[0046] Table 4 Template digestion system

[0047]

[0048] After the template digestion, DNA homologous recombination is performed on the product. Prepare the homologous recombination reaction system as shown in Table 5 and react at 37°C for 30 min to construct the expression plasmid of the mutant enzyme.

[0049] Table 5 Homologous recombination system

[0050]

[0051] Transform the recombinant product into E.coli DH5α competent cells, and spread them on an LB solid culture plate containing 100 μg / mL ampicillin and culture at 37°C for 12 h.

[0052] After the cultivation was completed, single colonies were picked and transferred to LB liquid medium containing 100 μg / mL ampicillin. After cultivation, the correctness of the mutation sites was verified by sequencing. After verification, a part of the bacterial liquid was stored at -80 °C for later use, and a part of the bacterial liquid was used to extract the recombinant plasmid pET22b-T244R / L274P, which was stored in a -20 °C refrigerator.

[0053] The successfully sequenced recombinant expression plasmid pET22b-T244R / L274P was transferred into E.coli BL21(DE3) to construct a recombinant mutant expression strain E.coli BL21(DE3) / pET22b-T244R / L274P.

[0054] 2. Express the enzyme mutant

[0055] The successfully constructed recombinant mutant expression strain E.coli BL21(DE3) / pET22b-T244R / L274P was spread on an LB plate containing 100 μg / mL ampicillin for screening and cultured at 37 °C for 18 h;

[0056] After the cultivation was completed, single colonies were picked and inoculated into a test tube containing 5 mL of LB medium with a final concentration of 100 μg / mL ampicillin and cultured at 37 °C at 140 rpm for 18 h. 2.5 mL of the bacterial liquid was taken and inoculated into 50 mL of TB liquid medium containing 100 μg / mL ampicillin, and cultured in a shaker at 37 °C and 140 rpm for 6 h. Isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.4 mM that had been filter-sterilized was added to the fermentation broth, and the culture was continued at 22 °C and 140 rpm for 22 h;

[0057] 3. Prepare the crude enzyme solution

[0058] The recombinant bacterial fermentation broth was transferred to a centrifuge tube, and the cells were collected by centrifugation at 11,000 rpm. The cells were resuspended in glycine-sodium hydroxide buffer at pH 10.0 to an OD 600 = 20, and the cells were ultrasonically disrupted at a power of 65 W under ice bath conditions, with a 3 s interval for every 6 s of work, and the cells were disrupted for a total of 10 min. Then, it was centrifuged again at 12,000 rpm, and the cell debris was removed by filtration through a 0.22 μm aqueous filter head. The obtained supernatant was the crude enzyme solution containing the mutant T244R / L274P;

[0059] The amino acid sequence of the maltogenic amylase mutant T244R / L274P is shown in SEQ ID NO: 2.

[0060] Example 3: Construction of Maltogenic Amylase Mutant T244R / L274P / Y214F and Preparation of Crude Enzyme Solution

[0061] In this example, the maltogenic amylase of Geobacillus stearothermophilus ( Geobacillus stearothermophilus ) was used as the original enzyme template. The threonine (Thr) at the 244th position at the N-terminus of its amino acid sequence was mutated to arginine (Arg), the leucine (Leu) at the 274th position was mutated to proline (Pro), and the tyrosine (Try) at the 214th position was mutated to phenylalanine (Phe). On the basis of Example 2, in step 1, in addition to the T244R and L274P mutation primers, the Y214F mutation primer was added, and the other conditions remained unchanged. The primers are shown in Table 6:

[0062] Table 6 Y214F Mutation Primers

[0063]

[0064] Note: The underlined positions in the primers are the mutation sites. "F" represents the upstream primer, and "R" represents the downstream primer.

[0065] The amino acid sequence of maltogenic amylase mutant T244R / L274P / Y214F is shown in SEQ ID NO: 3.

[0066] Example 4: Construction of Maltogenic Amylase Mutant T244R / L274P / Y214F / E302Q and Preparation of Crude Enzyme Solution

[0067] In this example, the maltogenic amylase of Geobacillus stearothermophilus ( Geobacillus stearothermophilus ) was used as the original enzyme template. The threonine (Thr) at the 244th position at the N-terminus of its amino acid sequence was mutated to arginine (Arg), the leucine (Leu) at the 274th position was mutated to proline (Pro), the tyrosine (Try) at the 214th position was mutated to phenylalanine (Phe), and the glutamate (Glu) at the 302nd position was mutated to glutamine (Gln). On the basis of Example 2, in step 1, in addition to the T244R, L274P, and Y214F mutation primers, the E302Q mutation primer was added, and the other conditions remained unchanged. The primers are shown in Table 7:

[0068] Table 7 E302Q Mutation Primers

[0069]

[0070] Note: The underlined positions in the primers are the mutation sites. "F" represents the upstream primer, and "R" represents the downstream primer.

[0071] The amino acid sequence of maltogenic amylase mutant T244R / L274P / Y214F / E302Q is shown in SEQ ID NO: 4.

[0072] Test Example 1 Determination of the Alkali Resistance of Wild-Type Maltogenic Amylase and Mutants

[0073] The DNS method was used to determine the enzyme activity of the crude maltogenic amylase solution prepared in Examples 1-4. The DNS method is one of the commonly used methods for determining enzyme activity, which is based on the coloring reaction of the reducing sugar produced by the enzyme-catalyzed reaction with dithiosalicylic acid (DNS) under alkaline conditions. The specific method is as follows:

[0074] Glucose standard solutions with concentrations of 0.1250, 0.2500, 0.5000, 0.7500, 0.1875, and 1.000 mg / mL were prepared respectively. Take 150 μL, react at 65 °C for 5 min, then add 150 μL DNS solution. The mixed solution was treated in a boiling water bath for 10 min, immediately cooled in an ice bath to room temperature, and its absorbance was measured at 540 nm to obtain the glucose-540 nm standard curve as Figure 2 shown.

[0075] Take 500 μL of the wild-type maltogenic amylase and the mutant crude maltogenic amylase solution obtained in Examples 1-4, and treat them with 500 μL of 0.1M glycine-sodium hydroxide buffer solution with pH values of 8.0, 9.0, 10.0, and 11.0 at 60 °C for 5 min respectively. Then take 30 μL of the crude enzyme solution and add it to 120 μL of 1% starch solution, and mix well by pipetting. After reacting at 65 °C for 5 min, add 150 μL DNS solution. The mixed solution was treated in a boiling water bath for 10 min, immediately cooled in an ice bath to room temperature, and its absorbance was measured at 540 nm.

[0076] Under the conditions of the enzymatic reaction, the amount of enzyme required to catalyze the hydrolysis of starch to produce 1 nmol of glucose per 1 min is defined as one enzyme activity unit (U). The specific enzyme activity is the catalytic ability of the enzyme per unit volume of the crude enzyme solution (U / mL); according to the absorbance at 540 nm and the standard curve, the specific enzyme activity of the crude enzyme solution was calculated. Taking the specific enzyme activity of the wild type treated with 0.1M glycine-sodium hydroxide buffer solution at pH 8.0 as 100%, the relative enzyme activity was calculated.

[0077] The test results are as Figure 3 shown in Table 8. In the buffer solution with pH 10.0, compared with the original enzyme, the mutants all had higher enzyme activities. The mutant enzyme T244R / L274P / Y214F had the best enzyme activity, and its relative enzyme activity increased by 114% compared with the original enzyme, which could meet the requirements for resistance to extreme alkaline environments in industrial applications.

[0078] Table 8 Relative Enzyme Activities of Maltogenic Amylase Treated under Different pH Conditions

[0079] .

Claims

1. A maltogenic amylase mutant, characterized in that, Based on the wild-type maltogenic amylase amino acid sequence shown in SEQ ID NO: 1, there are mutations in multiple amino acid residues at positions 244, 274, 214, and 302.

2. The maltogenic amylase mutant according to claim 1, wherein Among the amino acid mutations, the threonine residue at position 244 is mutated to an arginine residue, the leucine residue at position 274 is mutated to a proline residue, the tyrosine residue at position 214 is mutated to a phenylalanine residue, and the glutamate residue at position 302 is mutated to a glutamine residue.

3. The maltogenic amylase mutant according to claim 1, wherein The maltogenic amylase mutant has an amino acid sequence as shown in SEQ ID NO: 2, or as shown in SEQ ID NO: 3, or as shown in SEQ ID NO:

4.

4. A nucleotide sequence, characterized in that, Based on the wild-type maltogenic amylase nucleotide sequence, it contains corresponding base mutations and encodes the amino acid sequence of the maltogenic amylase mutant according to any one of claims 1-3.

5. The nucleotide sequence according to claim 4, wherein The wild-type maltogenic amylase nucleotide sequence is as shown in SEQ ID NO: 5, which is a codon-optimized nucleotide sequence of maltogenic amylase from Geobacillus stearothermophilus.

6. A recombinant vector, characterized in that, The recombinant vector contains the nucleotide sequence according to claim 4.

7. A recombinant microorganism, characterized in that, The recombinant microorganism contains the nucleotide sequence according to claim 4 or the recombinant vector according to claim 6.

8. A catalyst, characterized in that, The catalyst contains the maltogenic amylase mutant according to any one of claims 1-3.

9. Use of the maltogenic amylase mutant according to any one of claims 1-3 or the catalyst according to claim 8 in catalyzing starch decomposition.

10. The application according to claim 9, characterized in that The use is in catalyzing starch decomposition at a pH not higher than 11.0.

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