Oil-resistant malt amylase and methods and uses of directed evolution thereof
By directing the evolution of malt amylase, mutating its amino acid sequence, and screening for enzymes with better oil resistance, the problem of insufficient oil resistance of malt amylase in flour products was solved, improving enzyme activity and storage stability of flour products, and extending product shelf life.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- XUANCHENG HUADONG LIVESTOCK BREEDING CO LTD
- Filing Date
- 2023-02-23
- Publication Date
- 2026-07-14
AI Technical Summary
Existing malt amylases have poor oil resistance in flour products, which affects enzyme activity and the quality of flour products, leading to aging and quality decline during storage.
The amino acid sequence of malt amylase was mutated using directed evolution. A mutant library was constructed and high-throughput screening was performed to select a superior malt amylase with higher oil resistance and enzyme activity. Specifically, the mutated aspartic acid at position 50 was replaced with threonine, asparagine at position 184 was replaced with aspartic acid, phenylalanine at position 267 was replaced with tyrosine, proline at position 388 was replaced with leucine, and glycine at position 401 was replaced with cysteine. Oil-resistant malt amylase was obtained through error-prone PCR and three rounds of high-throughput screening.
It improves the enzyme activity of malt amylase in a high-oil environment, enhances its heat and acid/alkali resistance, extends the shelf life of flour products, and improves the texture and taste of flour products.
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Figure CN116814591B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of enzyme engineering, and more particularly to an oil-resistant malt amylase, its directed evolution method, and its applications. Background Technology
[0002] As flour products are stored for longer periods, they age, leading to a series of quality problems such as loss of flavor, poor texture, low elasticity, and increased hardness. Maintaining the quality of steamed buns is a major challenge for the industry.
[0003] For example, the main reason for the staling of steamed buns is starch recrystallization. Steamed buns become soft after heating because during baking, the starch changes from a crystalline state (β-starch) to a non-crystalline state (α-starch). However, during storage, hydrogen bonds between adjacent starch molecules reform, and the gelatinized α-starch rearranges into a crystalline state, leading to increased hardness and decreased quality. Maltose amylase is a type of glycoside hydrolase with unique multi-substrate specificity and catalytic multifunctionality. Maltose amylase can hydrolyze amylopectin into maltose and small dextrins, which are too short to crystallize and form crystalline junctions. However, adding maltose amylase to steamed buns can interfere with the entanglement of starch granules and protein macromolecules, reducing starch recrystallization. Therefore, maltose amylase can delay the staling of steamed buns and extend their shelf life, and it is frequently used in steamed bun baking and storage. Steamed buns containing maltose amylase are of higher quality.
[0004] To improve texture and for short-term moisture retention and preservation, flour-based products often incorporate high levels of oil into the dough, which can negatively impact amylase activity. Previously, industry focus on improving the thermal stability of these enzymes has been limited; however, specific oil-resistant amylases remain a gap in research. Summary of the Invention
[0005] The present invention aims to overcome the shortcomings of poor oil resistance of malt amylase in the prior art, and provides an oil-resistant malt amylase, its directed evolution method, and its application.
[0006] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:
[0007] The first objective of this invention is to provide an oil-resistant malt amylase.
[0008] The amino acid sequence of the oil-resistant malt amylase is shown in SEQ ID NO.1.
[0009] Preferably, the oil-resistant malt amylase retains more than 80% activity at a 30% oil content;
[0010] The oil content corresponding to the highest enzyme activity is 15%.
[0011] As a preferred option, the optimal temperature for oil-resistant malt amylase is 55℃-75℃.
[0012] The second objective of this invention is to provide a method for the directed evolution of oil-resistant malt amylase.
[0013] Starting with the malt amylase sequence from NCBI accession number AAA22233.1,
[0014] The aspartic acid at position 50 is mutated to threonine;
[0015] The asparagine at position 184 was mutated to aspartic acid;
[0016] The phenylalanine at position 267 was mutated to tyrosine;
[0017] The proline at position 388 was mutated to leucine;
[0018] The glycine at position 401 is mutated to cysteine.
[0019] As a preferred option, the following steps are included:
[0020] (1) By adding Mn to the PCR reaction system 2+ Genes were randomly mutated to construct a mutation library;
[0021] (2) The dominant preferred strain is obtained from the preferred strain library through three rounds of high-throughput screening, which is the oil-resistant malt amylase.
[0022] Preferably, the Mn in the PCR reaction system 2+ The concentration is 0.1-1.0 mM.
[0023] Preferably, the Mn 2+ Provided by MnCl2.
[0024] Preferably, the PCR reaction system further includes primer F and primer R;
[0025] The sequence of primer F is shown in SEQ ID NO.2;
[0026] The sequence of primer R is shown in SEQ ID NO.3.
[0027] Preferably, the sequence of primer F is:
[0028] TTTAAGAATTCATGCGCAAAGAAGCCATCCATCATC.
[0029] Preferably, the sequence of primer R is:
[0030] TTTAAGGATCCTTACTGGATCGTTTTGCCGGCCG.
[0031] Preferably, the PCR reaction system comprises 0.1–1.0 mM MnCl2, 2.5 mM MgCl2, 0.25 mM per dNTP, 2.5 units of rTaq DNA polymerase, 5 μL 10 × rTaq buffer, 0.5 μL DNA template, and 0.5 μL primers F and R.
[0032] Preferably, Ep-PCR is performed as follows: pre-denaturation at 94°C for 4 minutes, denaturation at 98°C for 30 seconds, annealing at 55°C for 5 seconds, extension at 72°C for 2 minutes, for a total of 30 PCR cycles.
[0033] As a preferred approach, the three-round high-throughput screening process is as follows:
[0034] (2-1) Randomly select the preferred mutants from the mutant library in the step, plate them on LB plates with kanamycin and culture them, then add Lugol solution and select the preferred mutants with obvious transparent circles;
[0035] (2-2) The preferred strains from (2-1) were inoculated into liquid culture medium containing kanamycin. The top 10 colonies with the highest HC values were selected from each plate, and the enzyme activity was determined by the DNS method.
[0036] (2-3) After centrifuging the culture medium of the preferred organisms cultured in (2-2), collect the supernatant, measure the enzyme activity of these positive preferred organisms, and screen out the preferred organisms with an OD value >0.6.
[0037] A third objective of this invention is to provide the application of the oil-resistant malt amylase in flour products.
[0038] The present invention has the following beneficial effects:
[0039] The inventors of this invention constructed a random mutant library of the enzyme using error-prone polymerase chain reaction (EPR) to identify preferred variants with higher activity and oil resistance, and then screened for key point mutations. This directed evolution technique is used to screen malt amylase, identifying oil-resistant malt amylases to overcome the stringent production environment and improve the activity of malt amylase in flour products.
[0040] Furthermore, the cultivation process revealed that the screened enzymes exhibited a certain degree of tolerance to high temperatures and acids / alkalis, indicating broad application prospects and economic value in flour products.
[0041] The results showed that the evolved malt amylase exhibited excellent enzyme activity even at high oil content, while also possessing very good heat and acid / alkali resistance. In experiments, compared to conventional malt amylase, the evolved malt amylase significantly improved the quality of steamed buns and other flour-based products and extended their shelf life. In summary, the above technology effectively improved the oil resistance of malt amylase, and the highly oil-resistant malt amylase helped improve the quality of flour-based products and extend their shelf life. Attached Figure Description
[0042] Figure 1 Enzyme activity diagram of malt amylase produced by different variants obtained after directed evolutionary selection.
[0043] Figure 2 Enzyme activity graphs of wild-type malt amylase and cultivar malt amylase at different oil contents.
[0044] Figure 3 The graph shows the change in hardness of steamed buns containing malt amylase produced by the addition of the preferred agent after different storage days.
[0045] Figure 4 The elasticity changes of steamed buns containing malt amylase produced by the addition of the preferred agent under different storage days.
[0046] Figure 5 Sensory evaluation image of steamed buns after being treated with malt amylase produced by the optimized organism. Detailed Implementation
[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.
[0048] Example 1:
[0049] Construct an optimized somatic library and perform error-prone PCR.
[0050] (1) Using F and R as primers and wild-type genes as templates, mutant libraries were obtained by Ep-PCR.
[0051] in:
[0052] The sequence of primer F is as follows:
[0053] TTTAAGAATTCATGCGCAAAGAAGCCATCCATCATC.
[0054] The sequence of primer R is as follows:
[0055] TTTAAGGATCCTTACTGGATCGTTTTGCCGGCCG.
[0056] (2) The PCR reaction mixture (50 μL) contained 0.1–1.0 mM MnCl2, 2.5 mM MgCl2, 0.25 mM per dNTP, 2.5 units of rTaq DNA polymerase, 5 μL 10 × rTaq buffer, 0.5 μL DNA template, and 0.5 μL primers F and R. Ep-PCR was performed as follows: pre-denaturation at 94 °C for 4 min, denaturation at 98 °C for 30 s, annealing at 55 °C for 5 s, and extension at 72 °C for 2 min. A total of 30 PCR cycles were performed.
[0057] (3) The PCR products were separated by 1% agarose gel electrophoresis and purified using the Pure Link PCR Purification Kit.
[0058] By using wild-type genes as templates, error-prone PCR was performed to construct a mutant library from which preferred mutants could be screened.
[0059] The results showed that, through DNA sequencing, five amino acid substitutions were found in the preferred mutant compared to the wild type: aspartic acid at position 50 was mutated to threonine; asparagine at position 184 was mutated to aspartic acid; phenylalanine at position 267 was mutated to tyrosine; proline at position 388 was mutated to leucine; and glycine at position 401 was mutated to cysteine. Furthermore, the double-mutant preferred mutant exhibited significantly enhanced activity compared to the wild type.
[0060] Example 2:
[0061] Through three rounds of high-throughput screening, explicit preferred variants were obtained from the preferred variant library.
[0062] (1) The preferred body was prepared using 50 μg·mL −1 Kanamycin was spread on LB plates and incubated for 12 h. Then, a Lugol solution of 0.3% I2 and 0.6% KI was added to the surface of the plates, and the preferred specimens with obvious transparent circles were selected.
[0063] (2) The second round of inoculation was performed in 96-well plates, inoculated into 100 μL of premium broth (TB) liquid medium (0.5% glycerol, 2.4% yeast extract, 1.25% K2HPO4·3H2O, 0.23% KH2PO4, 1.2% trypsin, 50 μg·mL). −1 Incubate with kanamycin at 33°C for 48 hours.
[0064] (3) On a new 96-well plate, the OD values of these preferred specimens were determined by the DNS method, and the preferred specimens with OD values above 0.6 were screened for a third round of screening.
[0065] (4) After culturing in 25 mL TB liquid medium at 33℃ for 48 h, the fermentation broth was centrifuged at 8000×g for 15 min, and the supernatant was collected for enzyme activity determination. Positive isolates with extremely high activity were selected for further experiments.
[0066] To achieve targeted evolutionary selection for oil resistance, the applicant invented a three-round high-throughput screening technique. The malt amylase produced from the optimized strains obtained through this three-round high-throughput screening exhibits better and higher activity in environments with high oil content. The results show that the malt amylase produced from the optimized strains obtained through this three-round high-throughput screening has superior oil resistance compared to ordinary malt amylase, demonstrating the effectiveness of this technique.
[0067] Table 1. Comparison of enzyme activity and optimal environmental oil content between wild-type and preferred enzymes.
[0068] .
[0069] The numerical differences after different lowercase letters in the same line were significant (p<0.05).
[0070] Example 3: Activity of malt amylase produced by the preferred body
[0071] The applicant used the DNS method to determine enzyme activity.
[0072] (1) Put 50 mM phosphate buffer (pH 6.5), 500 μL, and 1% soluble starch solution into an Erlenmeyer flask and preheat at 60°C for 10 minutes.
[0073] (2) Add enzyme solution (100 μL) to the preheated Erlenmeyer flask and incubate at 60°C for another 10 minutes.
[0074] (3) Add DNS (600 μL) to the mixture to stop the reaction.
[0075] (4) Add the reaction mixture (200 μL) to a 96-well plate and measure its OD value at 540 nm using a microplate reader.
[0076] One unit of malt amylase is defined as the amount of enzyme that releases the equivalent of 1 μmol of glucose per minute of reducing sugar under the conditions described above.
[0077] The results are as follows Figure 1 As shown, highly active malt amylases exist in large quantities among different variants. Furthermore, the malt amylase produced through directed evolution exhibits an overall activity increase of approximately 48.5% compared to wild-type malt amylase, demonstrating superior catalytic activity. This indicates that highly active malt amylases can be obtained through directed selection.
[0078] Example 4: Oil tolerance of malt amylase produced by the preferred organism
[0079] (1) Take eight Erlenmeyer flasks and fill them with 50 mM phosphate buffer (pH 6.5) and 500 μL 1% soluble starch solution, and preheat them at 60°C for 10 minutes.
[0080] (2) Prepare solutions from the eight Erlenmeyer flasks with oil concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 35%, and 40%, respectively.
[0081] (2) Add enzyme solution (100 μL) to eight different Erlenmeyer flasks and incubate at 60°C for 10 minutes.
[0082] (3) Add DNS (600 μL) to the mixture to stop the reaction.
[0083] (4) Add the reaction mixture (200 μL) to a 96-well plate and measure its OD value at 540 nm using a microplate reader.
[0084] One unit of malt amylase was defined as the amount of enzyme that releases the equivalent of 1 μmol of reducing sugar per minute of glucose under the conditions described above. The enzyme's oil tolerance was determined by measuring the residual enzyme activity after incubation in phosphate buffer (pH 6.5) at different oil concentrations (2%–16%) for 30 minutes. The enzyme activity of wild-type malt amylase at a 5% oil concentration was defined as 100%.
[0085] The results are as follows Figure 2 As shown, the malt amylase produced by the preferred strain exhibits better overall enzyme activity compared to the wild-type malt amylase. Furthermore, while the wild-type malt amylase reaches its peak activity at an oil content of 5%, the malt amylase produced by the preferred strain reaches its peak activity at an oil content of 15%, achieving an activity exceeding 120%, which is more than 20% higher than the highest enzyme activity of the wild-type malt amylase. This demonstrates that directed evolutionary selection can yield malt amylases with better oil tolerance.
[0086] Example 5: The effect of malt amylase produced by the preferred body on the quality of steamed buns.
[0087] The test temperature conditions are as follows:
[0088] (1) Maintain a temperature of 30℃ for 8 minutes.
[0089] (2) At 4 ℃·min −1 Increase the temperature from 30°C to 90°C and then maintain it for 7 minutes.
[0090] (3) At 4 ℃·min −1 Lower the temperature from 90°C to 50°C and then maintain this temperature for 5 minutes.
[0091] Steamed buns were prepared using the following materials: 100% flour, 1% yeast, 7% sugar, 1% salt, 65% water, and 45 ppm of preferred maltose amylase or 60 ppm of wild-type maltose amylase. Using 60 ppm (mg of maltose amylase per kilogram of flour) of wild-type maltose amylase as a control, the effect of the enzyme on dough properties was investigated by adding the preferred maltose amylase to the dough.
[0092] The ingredients were first mixed thoroughly and then fermented at 30°C and 85% relative humidity for 40 minutes. After resting for 5 minutes, the dough was proofed for another 30 minutes at 30°C and 85% relative humidity. The dough was then heated in an oven for 32 minutes. The final product was stored at 25°C and 33% relative humidity. The effects of enzymes on the quality of steamed buns were studied by measuring their specific volume, hardness, elasticity, and sensory evaluation characteristics.
[0093] The results are as follows Figure 3 As shown in Figure 4, the steamed buns with added malt amylase and wild-type malt amylase exhibited better elasticity and firmness than the blank control group without malt amylase. Furthermore, the steamed buns with added malt amylase showed better quality than those with added wild-type malt amylase under different storage conditions over various days. This indicates that adding malt amylase produced by a selectively evolved strain can improve the quality of steamed buns during storage.
[0094] Example 6: Sensory evaluation of steamed buns containing malt amylase produced by the preferred formulation
[0095] Sensory evaluation is also an important indicator of the quality of steamed buns. Steamed buns stored for 7 days were evaluated using a nine-point scale (moderately like, very like, like, like, like, neither like nor dislike, dislike, moderately dislike, very dislike, extremely dislike) for appearance, color, smoothness, texture, flavor, taste, air pockets, and overall acceptability, with scores ranging from 1 to 9.
[0096] The results are as follows Figure 5 As shown, steamed buns with added malt amylase produced by the preferred strain had the highest sensory evaluation score, steamed buns without added malt amylase had the lowest sensory evaluation score, and steamed buns with added wild-type malt amylase had a moderate sensory evaluation score. This indicates that adding malt amylase produced by the preferred strain after directed evolutionary selection can improve the quality of steamed buns during storage.
Claims
1. An oil-resistant malt amylase, characterized in that, The amino acid sequence of the oil-resistant malt amylase is shown in SEQ ID NO.
1.
2. A method for preparing the oil-resistant malt amylase of claim 1, Its features are, This includes: site-directed mutagenesis of the amino acid sequence of malt amylase with NCBI accession number AAA22233.1, wherein the site-directed mutagenesis includes: The aspartic acid at position 50 is mutated to threonine; The asparagine at position 184 was mutated to aspartic acid; The phenylalanine at position 267 was mutated to tyrosine; The proline at position 388 was mutated to leucine; The glycine at position 401 is mutated to cysteine.
3. The application of the oil-resistant malt amylase of claim 1 in flour products.