Maltogenic amylase mutants, biomaterials, catalysts and applications

Abstract

The present invention discloses a maltogenic amylase mutant, a biomaterial, a catalyst and an application thereof, belonging to the field of protein engineering. The mutant is based on a wild-type maltogenic amylase derived from Geobacillus stearothermophilus and has multiple amino acid residue mutations at positions 244, 274, 214 and 302. The mutant disclosed in the present invention has excellent alkali resistance and, after treatment under pH 8-11 conditions, still has higher enzyme activity than 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 with a buffer solution at pH=10, which can meet the demand for resistance to extreme alkaline environments in industrial applications.

Description

Technical Field

[0001] The present invention relates to the field of protein engineering, and in particular to a maltogenic amylase mutant, a biomaterial, a catalyst and applications. Background Art

[0002] Maltogenic amylase (EC3.2.1.133) is an important glycoside hydrolase in the amylase family. Its core function is to hydrolyze α-1,4-glycosidic bonds in polysaccharides such as starch and dextrin, producing maltose-based products. Its catalytic core domain exhibits a classic (β / α) 8-barrel structure, containing a conserved Asp-Glu-Asp catalytic triad responsible for precise cleavage of glycosidic bonds. The substrate-binding domain recognizes polysaccharide chains through hydrophobic interactions and a hydrogen-bonding network, ensuring reaction specificity. Maltogenic amylase's microbial origin offers excellent industrial potential. It maintains activity at high temperatures (50–70°C) and operates stably over a wide pH range (4.5–8.0). These properties have led to its application in the food industry to delay starch retrogradation and improve the quality of baked goods, in the biofuel sector to promote the efficient conversion of polysaccharides into fermentable sugars, and in the pharmaceutical industry as a green catalyst for the synthesis of carbohydrate compounds.

[0003] Despite its excellent performance in acidic to neutral environments, maltogenic amylase faces significant limitations in industrial applications under strongly alkaline conditions (pH > 8.0). First, the enzyme's active site is highly sensitive to alkaline environments. The glutamic acid residues in the catalytic triad are easily deprotonated under alkaline conditions, resulting in a significant decrease in catalytic efficiency. Second, the enzyme's overall structural stability is easily compromised in alkaline environments. Its auxiliary domains contain numerous negatively charged amino acid residues, which can lead to a loosening of the protein conformation or even irreversible denaturation at high pH due to enhanced electrostatic repulsion. Furthermore, alkaline conditions can trigger side reactions, such as oxidation or isomerization of starch molecules in strongly alkaline solutions, generating modified substrates that are difficult for the enzyme to recognize, further reducing reaction efficiency. These limitations hinder the direct application of maltogenic amylase in typical alkaline industrial applications, such as detergent manufacturing and alkaline textile desizing. Therefore, further optimization of the enzyme's adaptability to alkaline microenvironments is needed to expand its industrial applications. Summary of the Invention

[0004] Objectives of the invention: The purpose of the present invention is to provide a maltogenic amylase mutant that is resistant to alkaline environments; the second purpose is to provide biomaterials and catalysts related to the mutant; and the third purpose is to provide the use of the mutant and the catalyst in catalyzing starch decomposition.

[0005] Technical solution: The maltogenic amylase mutant of the present invention is based on the amino acid sequence of the wild-type maltogenic amylase shown in SEQ ID NO: 1, and has multiple amino acid residue mutations 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 mutates to an arginine residue, the leucine residue at position 274 mutates to a proline residue, the tyrosine residue at position 214 mutates to a phenylalanine residue, and the glutamic acid residue at position 302 mutates 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 nucleotide sequence of the wild-type maltogenic amylase, contains corresponding base mutations, and encodes the amino acid sequence of the aforementioned maltogenic amylase mutant.

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

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

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

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

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

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

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the obtained mutants have excellent alkali resistance and still have higher enzyme activity than the wild-type enzyme after treatment under pH 8-11 conditions. Among them, the mutant T244R / L274P / Y214F has a relative enzyme activity of 2.14 times that of the wild-type enzyme after treatment with a pH=10 buffer, which can meet the requirements of resistance to extreme alkaline environments in industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Geobacillus stearothermophilus Geobacillus stearothermophilus The protein structure of the maltogenic amylase from the source and the amino acid residue positions 244, 274, 214, and 302;

[0017] Figure 2 Glucose-OD obtained in the test example 540 Standard curve graph;

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

[0019] The technical solution of the present invention is further described below.

[0020] The materials used in the examples were obtained from:

[0021] 1. Strains and plasmids

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

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

[0024] 2. Culture medium

[0025] The LB medium used is composed of 5 g / L yeast extract, 10 g / L tryptone, and 5 g / L NaCl. If using solid medium, add 15 g / L agar powder. TB liquid medium is composed of 12 g / L yeast extract, 12 g / L tryptone, 4 mL / L glycerol, 12.5 g / L potassium dihydrogen phosphate, and 2.3 g / L potassium dihydrogen phosphate.

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

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

[0028] pET22b-wt was transformed into E.coli DH5α strain, the recombinant bacteriaE.coli DH5α / pET22b-wt was inoculated into a test tube containing 5 mL of LB medium containing ampicillin at a final concentration of 100 μg / mL 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 collected. A high-purity plasmid extraction kit was used to extract the pET22b-wt plasmid as a template for iterative mutagenesis to construct maltogenic amylase mutants.

[0030] At the same time, pET22b-wt was transferred into E.coli BL21 (DE3), construction of recombinant mutant expression strain E.coli BL21 (DE3) / pET22b-wt. The successfully constructed recombinant mutant expression strain was plated onto a plate containing ampicillin at a final concentration of 100 μg / mL and cultured at 37°C for 18 h.

[0031] After the culture was completed, 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 and 200 rpm for 18 h. 2.5 mL of the bacterial liquid 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. After that, filter-sterilized isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the fermentation broth with a final concentration of 0.4 mM and cultured at 22°C and 140 rpm for another 22 h.

[0032] Transfer the recombinant bacterial fermentation broth to a centrifuge tube and collect the cells by centrifugation at 11000 rpm. Resuspend the cells in glycine-sodium hydroxide buffer (pH 10.0) to an OD of 600 = 20, ultrasonically disrupt the cells at 65 W power in an ice bath for 10 min, with 6-s intervals and 3-s rests. Centrifuge again at 12,000 rpm to remove cell debris. The resulting supernatant is the crude enzyme solution containing the wild-type enzyme.

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

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

[0035] Using the maltogenic amylase from Geobacillus stearothermophilus as a starting point, the amino acid sequence shown in SEQ ID NO: 1 was modified by mutating the N-terminal threonine (Thr) at position 244 to arginine (Arg), and the leucine (Leu) at position 274 to proline (Pro). The mutant T244R / L274P was prepared as follows:

[0036] 1. Construction of recombinant plasmid

[0037] The primers for the T244R / L274P mutation site are shown in Table 1 . PCR was performed using the pET22b-wt plasmid carrying the original maltogenic amylase gene as a template. 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 markers in the primers are 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 PCR amplification, the amplified product was detected by 0.9% agarose gel electrophoresis. The results showed that the amplified product was a single band of approximately 7500 bp. The template was digested with the endonuclease DpnI. The template digestion system was prepared as shown in Table 4 and digested at 37°C for 1 h.

[0046] Table 4 Template digestion system

[0047]

[0048] After template digestion, the product was subjected to DNA homologous recombination. A homologous recombination reaction system was prepared as shown in Table 5 and reacted at 37°C for 30 min to construct an expression plasmid for the mutant enzyme.

[0049] Table 5 Homologous recombination system

[0050]

[0051] The recombinant product was transformed into E.coli DH5α competent cells were spread on LB solid culture plates containing 100 μg / mL ampicillin and cultured at 37°C for 12 h.

[0052] After the incubation period, single colonies were picked and cultured in LB liquid containing 100 μg / mL ampicillin. The correctness of the mutation site was verified by sequencing. Once verified, a portion of the culture was stored at -80°C for future use, while another portion was used to extract the recombinant plasmid pET22b-T244R / L274P and stored at -20°C.

[0053] The recombinant expression plasmid pET22b-T244R / L274P that was successfully sequenced was transferred into E.coli BL21 (DE3), construction of recombinant mutant expression strain E.coli BL21(DE3) / pET22b-T244R / L274P.

[0054] 2. Expression of enzyme mutants

[0055] The successful recombinant mutant expression strain E.coli BL21 (DE3) / pET22b-T244R / L274P, spread on LB plates containing 100 μg / mL ampicillin for screening, and culture at 37 °C for 18 h;

[0056] After the incubation period, a single colony was picked and inoculated into a 5 mL test tube containing LB medium at a final concentration of 100 μg / mL ampicillin and cultured at 37°C and 140 rpm for 18 hours. 2.5 mL of the bacterial broth 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 hours. Filter-sterilized isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the fermentation broth at a final concentration of 0.4 mM and cultured at 22°C and 140 rpm for another 22 hours.

[0057] 3. Preparation of crude enzyme solution

[0058] Transfer the recombinant bacterial fermentation broth to a centrifuge tube and collect the cells by centrifugation at 11000 rpm. Resuspend the cells in glycine-sodium hydroxide buffer (pH 10.0) to an OD of 600 = 20, ultrasonically disrupt the cells at 65 W power in an ice bath, with 6-second intervals and 3-second intervals for a total of 10 minutes. The cells were centrifuged again at 12,000 rpm and filtered through a 0.22 μm aqueous filter to remove cell debris. The resulting 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 thermophilic bacteria Geobacillus stearothermophilus ) maltogenic amylase was used as the original enzyme as a template, and the 244th threonine Thr at the N-terminus of its amino acid sequence was mutated to arginine Arg, the 274th leucine Leu was mutated to proline Pro, and the 214th tyrosine Try was mutated to phenylalanine Phe. Based on Example 2, in step 1, in addition to the T244R and L274P mutant primers, a Y214F mutant 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 markers in the primers are mutation sites, “F” represents the upstream primer, and “R” represents the downstream primer.

[0065] The amino acid sequence of the 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 thermophilic bacteria Geobacillus stearothermophilus ) maltogenic amylase was used as the original enzyme as a template, and the 244th threonine Thr at the N-terminus of its amino acid sequence was mutated to arginine Arg, the 274th leucine Leu was mutated to proline Pro, the 214th tyrosine Try was mutated to phenylalanine Phe, and the 302nd glutamic acid Glu was mutated to glutamine Gln. Based on Example 2, in step 1, in addition to the T244R, L274P, and Y214F mutant primers, an E302Q mutant 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 markers in the primers are mutation sites, “F” represents the upstream primer, and “R” represents the downstream primer.

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

[0072] Test Example 1 Determination of alkali resistance of wild-type maltogenic amylase and its mutants

[0073] The activity of the crude maltogenic amylase solution prepared in Examples 1-4 was determined using the DNS method. The DNS method is a commonly used method for determining enzyme activity. It is based on the barbituric acid coloring reaction between reducing sugars produced by the enzyme-catalyzed reaction and dithiobarbituric acid (DNS) under alkaline conditions. The specific method is as follows:

[0074] Prepare 0.1250, 0.2500, 0.5000, 0.7500, 0.1875, and 1.000 mg / mL glucose standard solutions, take 150 μL, react at 65 °C for 5 min, add 150 μL DNS solution, treat the mixed solution in a boiling water bath for 10 min, immediately cool to room temperature in an ice bath, and measure its absorbance at 540 nm to obtain the following: Figure 2 Glucose-540 nm standard curve shown.

[0075] 500 μL of the crude enzyme solutions of the wild-type maltogenic amylase and the maltogenic amylase mutants obtained in Examples 1-4 were treated with 500 μL of 0.1 M glycine-sodium hydroxide buffer at pH 8.0, 9.0, 10.0, and 11.0 at 60°C for 5 min, respectively. 30 μL of each crude enzyme solution was added to 120 μL of 1% starch solution and pipetted evenly. The mixture was reacted at 65°C for 5 min, followed by the addition of 150 μL of DNS solution. The mixed solution was incubated in a boiling water bath for 10 min, immediately cooled to room temperature in an ice bath, and its absorbance was measured at 540 nm.

[0076] One unit (U) of enzyme activity was defined as the amount of enzyme required to catalyze starch hydrolysis to produce 1 nmol of glucose per minute under enzymatic reaction conditions. Specific activity was defined as the catalytic capacity of the enzyme per unit volume of crude enzyme solution (U / mL). The specific activity of the crude enzyme solution was calculated based on the absorbance at 540 nm and a standard curve. Relative enzyme activity was calculated, assuming the specific activity of the wild-type enzyme after treatment with 0.1 M glycine-sodium hydroxide buffer, pH 8.0, was 100%.

[0077] The test results are as follows Figure 3 As shown in Table 8, in a buffer solution with a pH of 10.0, the mutants all had higher enzyme activity than the original enzyme. The mutant enzyme T244R / L274P / Y214F had the best enzyme activity, which was 114% higher than the original enzyme activity, and could meet the requirements for resistance to extreme alkaline environments in industrial applications.

[0078] Table 8 Relative enzyme activity of maltogenic amylase treated under different pH conditions

[0079] .

Claims

1. A maltogenic amylase mutant, characterized in that Based on the amino acid sequence of the wild-type maltogenic amylase shown in SEQ ID NO: 1, the threonine residue at position 244 is mutated to an arginine residue, the leucine residue at position 274 is mutated to a proline residue, or the threonine residue at position 244 is mutated to an arginine residue, the leucine residue at position 274 is mutated to a proline residue, and the tyrosine residue at position 214 is mutated to a phenylalanine residue, and the amino acid sequence of the mutant is shown in SEQ ID NO: 2 or 3.

2. A nucleic acid molecule, characterized in that Based on the nucleotide sequence of the wild-type maltogenic amylase, the amino acid sequence of the maltogenic amylase mutant according to claim 1 is encoded, comprising corresponding base mutations.

3. The nucleic acid molecule according to claim 2, characterized in that The wild-type maltogenic amylase nucleotide sequence is shown in SEQ ID NO: 5, which is a codon-optimized nucleotide sequence of maltogenic amylase derived from Geobacillus stearothermophilus.

4. A recombinant vector, characterized in that The recombinant vector comprises the nucleic acid molecule according to claim 2.

5. A recombinant microorganism, characterized in that The recombinant microorganism comprises the nucleic acid molecule according to claim 2 or the recombinant vector according to claim 4.

6. A catalyst, characterized in that The catalyst comprises the maltogenic amylase mutant according to claim 1.

7. Use of the maltogenic amylase mutant according to claim 1 or the catalyst according to claim 6 in catalyzing starch decomposition.

8. The application according to claim 7, characterized in that: The application is the application in catalyzing starch decomposition at a pH value not higher than 11.0.

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