Heat-stable acid-resistant maltogenic amylase mutant and application thereof

Abstract

The invention discloses a thermostable acid-resistant maltogenic amylase mutant and application thereof. The amino acid sequence of the maltogenic amylase mutant is as shown in SEQ ID NO.1, and the nucleotide sequence of the maltogenic amylase mutant is as shown in SEQ ID NO.2. The optimum temperature is 65 DEG C, the maximum activity is 430.15 U.mg <-1 >, and the action temperature and the hydrolytic activity are improved. The half-life period at the temperature of 60 DEG C and the residual enzyme activity after 60 min are 227 min and 337.43 U.mg <-1 > respectively, and the half-life period and the residual enzyme activity are improved.

Description

Technical Field

[0001] The present invention relates to an acid-tolerant maltogenic amylase derived from Lactobacillus parafoodi, and particularly to a thermostable and acid-tolerant maltogenic amylase mutant, belonging to the technical fields of enzyme engineering and microbial engineering. Background Art

[0002] Maltogenic amylase (EC 3.2.1.133) is a type of α-amylase (EC 3.2.1.1) in the GH13-20 family that acts on starch and related polysaccharides, can hydrolyze α-1,4 and α-1,6-glycosidic bonds, and the main product is maltose. Maltogenic amylase has multi-substrate characteristics and substrate preference selectivity, preferentially hydrolyzing cyclodextrin, followed by amylose, amylopectin, pullulan polysaccharide, etc. When starch is gelatinized by heating, maltogenic amylase can hydrolyze from the end of starch molecules to produce maltose and some small molecular oligosaccharides, interfering with the recrystallization of starch and the entanglement of starch granules and protein macromolecules, thereby reducing the retrogradation rate and recrystallization rate of starch granules, achieving the effect of anti-aging, and can be used to extend the shelf life of baked foods. In addition, compared with traditional chemical reagents such as emulsifiers and gluten improvers, maltogenic amylase is highly effective and meets food safety, without safety hazards. Therefore, maltogenic amylase is regarded as an ideal bread improver.

[0003] Currently, the research on maltogenic amylase mainly focuses on thermophilic microbial sources, such as Bacillus stearothermophilus, Bacillus licheniformis, Bacillus subtilis, and Thermus sp., etc. The maltogenic amylase from the above sources is mainly applied to bread baked at neutral or near-neutral pH. It is sensitive to acidic conditions, so its beneficial effects in the process of making bread using a low-pH formula are very limited. In addition, only two maltogenic amylases from Lactobacillus plantarum and Lactobacillus gasseri have been found to have good acid tolerance, but there are some defects in their thermostability or hydrolysis activity.

[0004] Lactobacillus parafoodi is one of the dominant lactic acid bacteria in sourdough and is a recognized food safety-grade strain. Therefore, Lactobacillus parafoodi is a preferred expression strain for acid-tolerant maltogenic amylase applied to bread improvement. However, at present, the maltogenic amylase derived from Lactobacillus parafoodi has problems of relatively low optimal working temperature and poor thermostability. Therefore, thermostable modification and improvement of the enzyme activity of the maltogenic amylase derived from Lactobacillus parafoodi are of extremely important significance to the baking food industry. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an acid-resistant maltogenic amylase mutant with improved thermal stability and industrial application value.

[0006] Specifically, for the thermostable acid-resistant maltogenic amylase mutant, its amino acid sequence is as shown in SEQ ID NO.1, and its nucleotide sequence is as shown in SEQ ID NO.2.

[0007] The present invention also provides a method for preparing the thermostable acid-resistant maltogenic amylase mutant.

[0008] Specifically, for the method for preparing the thermostable acid-resistant maltogenic amylase mutant, the amino acid sequence of the maltogenic amylase with the NCBI accession number No. WP_025085606.1 is used as the starting sequence, and the alanine at position 375 and the serine at position 257 are respectively mutated into proline.

[0009] The present invention further relates to the application of the thermostable acid-resistant maltogenic amylase mutant.

[0010] Specifically, it relates to the application of the thermostable acid-resistant maltogenic amylase mutant in food preparation. In a specific embodiment, the food is dough or dough products, batter or batter products, or bakery or pastry products.

[0011] The present invention further provides the use of the thermostable and acid-resistant maltogenic amylase mutant as taught herein as a food additive. Meanwhile, a food additive is further provided, which comprises the thermostable and acid-resistant maltogenic amylase mutant described herein in its components. In a specific embodiment, the food additive is a bread improver. In other words, the thermostable and acid-resistant maltogenic amylase mutant as taught herein can be part of a composition, such as a bread improver. Bread improvers (also known as dough conditioners or dough modifiers, modifiers or flour treatment agents) are usually added to dough to improve the texture, volume, flavor and / or freshness of baked products and to enhance the machinability and stability of the dough. Generally, bread improvers consist of, consist essentially of, or consist of: one or more enzymes, such as a combination of one or two of the following types of enzymes, amylases (α-amylase, β-amylase, glucoamylase, raw starch-degrading amylase), xylanases (hemicellulases), cellulases, pectinases, proteases, pectin lyases, oxidases (peroxidase, glucose oxidase, pyranose oxidase, hexose oxidase, L-amino acid oxidase, carbohydrate oxidase, thiol oxidase), lipoxygenase, dehydrogenase, laccase, transglutaminase, acyltransferase, protein disulfide isomerase; one or more oxidants or reductants, such as ascorbic acid, glutathione, cysteine, etc.; one or more emulsifiers, such as diacetyl tartaric acid esters of mono- and diglycerides (DATEM), sodium stearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), glyceryl monostearate (GMS), rhamnolipids, lecithin, sucrose esters, bile salts, etc.; one or more lipid materials, such as margarine, butter, oil, shortening; one or more vitamins, such as pantothenic acid and vitamins, etc.; one or more food gums; (dried) sourdough; and / or one or more fiber sources, such as oat fiber.

[0012] Another aspect of the present invention is to provide a method for preparing food, which involves methods for preparing dough products, flour paste products, and baked products, and the method for baked products is preferred in the present invention. In these methods, it includes the step of mixing the thermostable and acid-resistant maltogenic amylase mutant provided herein with food ingredients.

[0013] In a specific embodiment, if the method is a method for preparing a baked product, the method may include the step of adding the thermostable and acid-resistant maltogenic amylase mutant provided herein to the dough or batter. Preferably, the thermostable and acid-resistant maltogenic amylase mutant provided herein is added to the dough or batter before baking.

[0014] Preferably, in the method provided herein, the tolerance of the dough is improved.

[0015] Consistently, on the other hand, there is provided the use of a thermostable and acid-resistant maltogenic amylase mutant as taught by the present invention in the preparation of a food product having improved dough tolerance, preferably wherein the dough tolerance is at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 10.0%, or at least 15.0% higher than the dough tolerance of a control dough or batter, wherein the control dough or batter is prepared in the absence (i.e., without) of a polypeptide having maltogenic amylase activity as taught herein.

[0016] Preferably, the baked product of the present invention is a bakery or pastry product. Baked or pastry products known in the art include, but are not limited to, those selected from bread, mochi, soft rolls, bagels, doughnuts, Danish pastries, hamburger buns, pizza, pita bread, ciabatta, sponge cakes, butter cakes, pound cakes, muffins, cup cakes, steamed cakes, waffles, brownies, cake doughnuts, yeast-raised doughnuts, baguettes, bread rolls, saltine crackers, sweet crackers, pie crusts, rusks, and / or other baked products. More preferably, the baked product is bread, baguette, and / or bread roll.

[0017] On the other hand, the present invention relates to the use of a thermostable and acid-resistant maltogenic amylase mutant as provided herein in baking and pastry applications. It has been found that the thermostable and acid-resistant maltogenic amylase mutant as provided herein is useful for improving bread volume, texture, and mouthfeel.

[0018] On the other hand, the present invention provides a food product (such as a bread improver), a dough product, a batter product, or a baked product that contains a thermostable and acid-resistant maltogenic amylase mutant as provided herein.

[0019] On the other hand, the present invention provides a food product, a dough product, a batter product, or a baked product obtained by or obtainable by the use or method provided herein.

[0020] In a particular embodiment, the food product is a dough or batter that contains flour and a maltogenic amylase mutant as provided herein, and the addition amount of the maltogenic amylase mutant is 1000 - 100000 U maltogenic amylase units per kilogram of flour, preferably 1000 - 100000 U maltogenic amylase units per kilogram of flour, more preferably 10000 - 100000 U maltogenic amylase units per kilogram of flour.

[0021] Advantages and effects of the present invention:

[0022] (1) The enzymatic properties were determined. The optimal pH of the wild-type maltose amylase derived from Lactobacillus paralimentarius was 5.0, and it had better acid tolerance compared with the maltose amylases derived from the reported Bacillus genus.

[0023] (2) The optimal action temperature was determined. The optimal temperature and maximum activity of the wild-type maltose amylase of the present invention were 50 °C and 274.79 U·mg -1 respectively, and the optimal temperature and maximum activity of its optimal double-point mutant A375P / S257P were 65 °C and 430.15 U·mg -1 respectively, achieving an increase in the action temperature and hydrolysis activity by 15 °C and 156.4%.

[0024] (3) The temperature stability was determined. The half-life and residual enzyme activity after 60 min of the wild-type maltose amylase at 60 °C were 32 min and 85.65 U·mg -1 respectively, and the half-life and residual enzyme activity after 60 min of its optimal double-point mutant A375P / S257P at 60 °C were 227 min and 337.43 U·mg -1 respectively, achieving an increase in the half-life and residual enzyme activity by 7.09 and 3.93 times.

[0025] (4) The kinetic parameters were determined using β-cyclodextrin as the substrate. The catalytic efficiency (k cat / K m ) of the wild-type maltose amylase was 13.36 mM -1 ·s -1 , and the catalytic efficiency (kcat / Km) of its optimal double-point mutant A375P / S257P was 30.42 mM -1 ·s -1 , achieving an increase in the catalytic efficiency (k cat / K m ) by 2.27 times.

[0026] (5) The double-point mutant A375P / S257P obtained in the present invention, while successfully improving the catalytic efficiency (k cat / K m ), also improved the good temperature stability of the wild-type strain, and had more practical industrial application value. Description of the Drawings

[0027] Figure 1 It is for the determination of the optimal action pH of the wild-type maltose amylase.

[0028] Figure 2The optimal temperature and hydrolysis activity of wild type and mutants A375P, S257P and A375P / S257P were determined.

[0029] Figure 3 Temperature stability test of wild type and mutants A375P, S257P and A375P / S257P at 60°C.

[0030] Figure 4 is the half-life of wild type and mutants A375P, S257P and A375P / S257P at 60°C (t 1 / 2 ) fitting.

[0031] Figure 5 The bread storage changes of the maltogenic amylase mutant A375P / S257P, wild-type maltogenic amylase and blank control are shown. DETAILED DESCRIPTION

[0032] The E. coli BL21 (DE3) involved in the following examples was purchased from Beina Biotechnology, the pET-28a (+) plasmid was purchased from Novagen, and the soluble starch and β-cyclodextrin were purchased from Aladdin Biochemical Technology Co., Ltd.

[0033] The culture medium involved in the following examples is as follows:

[0034] LB liquid medium: yeast powder 5.0 g L -1 , Tryptone 10.0 g·L -1 、NaCl 10.0g·L -1 , Kanamycin 50mg·L -1 .

[0035] LB solid medium: yeast powder 5.0 g L -1 , Tryptone 10.0 g·L -1 、NaCl 10.0g·L -1 , agar powder 20g·L -1 , Kanamycin 50mg·L -1 .

[0036] Buffer A: 100 mmol / L Tris, 150 mmol / L NaCl, 20 mmol / L imidazole, pH 7.5.

[0037] Buffer B: 100 mmol / L Tris, 150 mmol / L NaCl, 500 mmol / L imidazole, pH 7.5.

[0038] Example 1 Preparation of maltogenic amylase mutant gene sequence

[0039] The wild-type maltogenic amylase is derived from Lactobacillus paralimentarius.

[0040] The gene encoding maltogenic amylase was chemically synthesized on the vector pET28a and transformed into Escherichia coli BL21(DE3), forming the wild-type pET28a-LpaMA / E. coli BL21 of this maltogenic amylase. The NCBI accession number of the gene is No. WP_025085606.1. Using the whole plasmid PCR technique, site-directed mutagenesis was carried out with the obtained recombinant plasmid pET28a-LpaMA as the template to obtain mutants A375P, S257P, and A375P / S257P.

[0041] Amino acid sequence:

[0042] MGNTPAIYHRPDSEFAYLYKDNLMHIRLRTARGDIKEVGLLHGDPYTLDTDHWQDQPTPMKRYLTTDLYDF

[0043] WTIEVTEPFKRISYAFKVTGNDGITIFYGDHGVFSFEKSVYDSPDNYFRLPYFHEVDRFKAPEWVKKTVWYQIF

[0044] PERFANGDKSNDPENTLPWGSKTPSATDFFGGDLQGVIDHLDHLTDLGVNGIYFCPIFKAKSNHKYDTIDYMEI

[0045] DPAFGDKATFKKLVQECHKRGIKVMLDAVFNHMGDSSPQWLDVVKNGKNSKYADWFHINKFPVSYEDDGF

[0046] DQAKNISYDTFAFTPHMPKLNTANPEVKEYLLKIAKYWIEEFDIDAWRLDVANEVDHEFWRDFRRACDSVKK

[0047] DFYILGEVWHSSQPWLQGDQFSAVMNYAYTDSISKYLIKKQIPIEKMVSDINDQLMLYRDQTDQIQFNVLDSH

[0048] DTARLLTETNDDKDLMKQVLAFTYLQPGVPCIYYGDEIGMDGGNDPECRKCMVWDKSQQDLNLYDFFKHLI

[0049] AFRKDNQKVLSEGEMVWEKVCDDGLLILSRKLNGTEVKVILNAGDTPKQVTTNQSVVLSNLVTENNHELTVDPKGFALVK.

[0050] Nucleotide sequence:

[0051] ATGGGCAATACACCAGCAATTTATCATCGCCCAGACAGCGAATTTGCATATCTATACAAAGATAATTTAATGC

[0052] ATATTAGATTAAGAACCGCTCGCGGAGACATTAAAGAGGTAGGTTTACTTCACGGTGATCCGTATACGCTAG

[0053] ATACAGACCACTGGCAAGATCAACCTACGCCGATGAAACGTTATTTAACGACTGATTTGTATGATTTTTGGA

[0054] CTATTGAAGTTACAGAGCCGTTCAAACGTATTTCCTATGCTTTCAAGGTTACTGGTAACGATGGCATTACCAT

[0055] ATTCTATGGTGATCATGGTGTGTTCTCATTTGAAAAGAGCGTCTACGATAGTCCTGATAATTACTTTAGATTG

[0056] CCATATTTCCACGAAGTAGATCGTTTCAAGGCACCAGAGTGGGTCAAAAAGACTGTCTGGTATCAAATTTTC

[0057] CCCGAAAGATTTGCTAATGGGGATAAATCTAATGATCCCGAAAATACTTTGCCATGGGGTTCTAAAACTCCA

[0058] AGTGCTACGGATTTCTTTGGTGGTGATCTACAAGGTGTTATTGACCATCTAGATCATTTAACTGATTTGGGTG

[0059] TCAATGGAATCTATTTCTGTCCTATCTTCAAAGCTAAGTCTAATCACAAATACGACACAATTGATTATATGGA

[0060] AATAGATCCAGCCTTTGGCGACAAAGCTACTTTTAAAAAATTAGTTCAAGAATGTCACAAACGTGGCATTA

[0061] AAGTCATGCTAGATGCGGTTTTCAATCATATGGGTGACAGTTCACCGCAATGGTTGGATGTCGTTAAAAATG

[0062] GTAAGAATTCTAAATACGCCGATTGGTTCCATATCAATAAATTCCCAGTTTCATATGAAGATGATGGCTTTGAT

[0063] CAAGCTAAGAATATTTCCTATGATACTTTTGCCTTCACACCACATATGCCAAAATTGAATACTGCTAATCCAG

[0064] AAGTTAAGGAGTATTTGTTGAAGATTGCTAAGTATTGGATTGAAGAATTCGATATTGATGCTTGGAGATTAGA

[0065] TGTGGCAAATGAAGTTGATCATGAATTCTGGCGTGACTTTAGAAGAGCATGTGACAGTGTTAAAAAGGATT

[0066] TCTATATTCTAGGTGAAGTTTGGCACTCCTCACAACCGTGGCTCCAAGGAGATCAGTTTAGTGCTGTAATGA

[0067] ATTATGCGTATACTGATTCAATTTCCAAATACTTAATTAAAAAACAAATTCCAATTGAAAAAATGGTTTCAGA

[0068] TATCAATGATCAATTGATGCTCTATCGTGATCAAACAGATCAAATTCAATTCAATGTTTTGGATTCACATGATA

[0069] CAGCTCGATTGTTGACTGAAACAAATGATGATAAAGATTTAATGAAACAAGTACTAGCTTTCACATATCTAC

[0070] AACCAGGTGTTCCATGTATATATTATGGTGATGAAATCGGTATGGATGGCGGTAATGATCCTGAATGTCGTAA

[0071] GTGTATGGTTTGGGATAAATCTCAACAAGATTTAAATCTATATGATTTCTTCAAACACTTGATTGCTTTTAGA

[0072] AAAGATAATCAAAAAGTGCTTTCTGAAGGAGAGATGGTTTGGGAGAAGGTCTGTGATGATGGATTATTGAT

[0073] CTTGAGTCGTAAGTTGAATGGTACTGAAGTAAAGGTTATTTTGAACGCAGGCGATACTCCTAAACAAGTAA

[0074] CAACTAATCAAAGCGTAGTTTTAAGTAACTTAGTTACAGAAAATAATCATGAGTTAACGGTTGATCCTAAAGGATTTGCATTAGTTAAA.

[0075] Among them, the primers used for the mutation A375P are as follows:

[0076] A375P-For: TCACAACCGTGGCTCCAAGGAGATCAGTTTAG (SEQ ID No.3);

[0077] A375P-Rev: TGGAGCCACGGTTGTGAGGAGTGCCAAACTTCA (SEQ ID No.4).

[0078] The primers used for the mutation S257P (A375P / S257P mutant) are as follows:

[0079] S257P-For: CAGTTCACCGCAATGGTTGGATGTCGTTAAAAAT (SEQ ID No.5);

[0080] S257P - Rev: ACCATTGCGGTGAACTGTCACCCATATGATTGAAA (SEQ ID No.6).

[0081] The PCR reaction was carried out in a 50 μL system. The reaction conditions were pre - denaturation at 94 °C for 4 min; then entered 30 cycles: denaturation at 98 °C for 10 s, annealing at 55 °C for 5 s, extension at 72 °C for 5 min; finally, extension at 72 °C for 10 min and incubation at 4 °C.

[0082] The PCR amplification products were detected by 1% agarose gel electrophoresis. After the detection, 0.5 μL of methylation template digestion enzyme (Dpn I) was added to 10 μL of the amplification products, and they were mixed by pipetting. The reaction was carried out at 37 °C for 1.5 h. The amplified products treated with Dpn I were transformed into Escherichia coli E. coli BL21(DE3). The transformed products were spread on LB solid medium and cultured at 37 °C for 8 - 10 h. 20 transformants were picked from the LB solid medium, inoculated into LB liquid medium and cultured at 37 °C for 10 h, then the plasmids were extracted. When the sequencing was correct, recombinant Escherichia coli containing the genes encoding mutants A375P, S257P and A375P / S257P was obtained.

[0083] Example 2 Inductive Cultivation and Protein Purification of Maltogenic Amylase Mutants

[0084] Using the maltogenic amylase with the amino acid sequence shown in SEQ ID NO.1 as the wild type, the obtained recombinant Escherichia coli pET28a - LpaMA / E. coli BL21 and the recombinant Escherichia coli containing the genes encoding mutants A375P, S257P and A375P / S257P were spread on LB solid medium and cultured at 37 °C for 8 - 10 h to obtain single colonies. Single colonies were picked and inoculated into LB liquid medium, cultured at 37 °C and 200 rpm for 6 - 8 h to obtain seed liquid. The seed liquid was inoculated into LB liquid medium at an inoculation amount of 2% (v / v), cultured at 37 °C and 200 rpm for 2 - 3 h, then IPTG with a final concentration of 0.1 mmol / L was added to the fermentation broth, and the induction culture was continued at 17 °C and 200 rpm for 12 - 17 h to obtain the fermentation broth. The fermentation broth was centrifuged at 4 °C and 8000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed twice with 9% normal saline to obtain the wet cells of the wild type, mutants A375P, S257P and A375P / S257P.

[0085] The wet bacterial cells were resuspended in Buffer A and sonicated, and then centrifuged at 10,000 rpm and 4 °C for 30 min to obtain the crude enzyme solution. After filtration with a 0.22-μm aqueous filter membrane, the sample was slowly loaded onto a Ni-NAT affinity chromatography column. After loading, it was first washed with Buffer A and then eluted with a gradient of Buffer B. The elution peak corresponding to 300 mmol / L imidazole was collected. Subsequently, the imidazole in the pure enzyme was removed using a desalting column to obtain the pure enzymes of wild type, mutant A375P, S257P, and A375P / S257P.

[0086] Example 3 Hydrolysis Activity of Different Maltogenic Amylase Mutants on Starch

[0087] Soluble starch (10 mg / mL) was added to 50 mmol / L citrate buffer (pH 4.0 - 6.0), phosphate buffer (pH 6.0 - 7.0), and Tris-HCl buffer (pH 7.0 - 9.0) respectively to obtain reaction systems. 500 μL of each reaction system was incubated at 40 - 70 °C for 3 min, and then 100 μL of the pure enzymes of wild type, mutant A375P, S257P, and A375P / S257P obtained in Example 2 was added to start the reaction. The control group did not contain enzyme solution, and other components were the same. The reaction was carried out at 40 - 70 °C for 10 min. 10 μL of the product was diluted 20 times and an equal volume of 200 μL DNS (3,5-dinitrosalicylic acid) was added to end the reaction. After a 5-min boiling water bath at 100 °C, it was cooled on ice to room temperature, and the absorbance change at 540 nm was recorded to obtain the hydrolysis activities of wild type, mutant A375P, S257P, and A375P / S257P on starch.

[0088] Hydrolysis activity (U / mg) = Ew × k × V / t / N × L × protein concentration of pure enzyme solution

[0089] Among them, Ew is the absorbance change value at 540 nm within 10 min; k is the reducing sugar calibration curve coefficient, with the unit of μmol / mL; V is the volume of the enzyme activity assay reaction system, with the unit of mL, which is 0.6 here; t is the reaction time, with the unit of min, which is 10 here; N is the enzyme solution dilution factor; L is the volume of the added enzyme solution, with the unit of mL, which is 0.1 here; the protein concentration of the enzyme solution is measured using a Bradford protein assay kit (For the method of measuring protein concentration using a Bradford protein assay kit, please refer to the literature: Zhou-Pan X R, ESérée, Zhou X J, et al. Involvement of Human Liver Cytochrome P450 3A in Vinblastine Metabolism: Drug Interactions1[J]. Cancer Research, 1993, 53(21): 5121 - 5126.), with the unit of mg / mL.

[0090] The results were as follows: The optimal working temperature and pH of the wild type were 50 °C and 5.0 respectively, and the maximum hydrolysis activity was 274.79 U·mg -1 ; the optimal working temperatures of mutants A375P, S257P, and A375P / S257P were 60, 60, and 65 °C respectively; the maximum hydrolysis activities were 425.89, 325.82, and 430.15 U·mg -1 .

[0091] Example 4 Temperature Stability of Different Maltogenic Amylase Mutants

[0092] The wild type, mutant A375P, S257P, and A375P / S257P enzyme solutions obtained in Example 2 were incubated in a water bath at 60 °C for 60 min. Samples were taken at regular intervals, and the catalytic activities of the wild type, mutant A375P, S257P, and A375P / S257P of maltogenic amylase on starch after incubation were measured at 50 °C. Taking the activity before incubation as 100%, the relative activity was calculated by comparing with the remaining activity after incubation to investigate the temperature stability and half-life of the wild type, mutant A375P, S257P, and A375P / S257P of maltogenic amylase. The Origin software was used to perform a linear fit on the calculated residual activity and the corresponding incubation time to obtain the half-life (t 1 / 2 ).

[0093] The results were as follows: the residual relative activities of mutants A375P, S257P, and A375P / S257P were 70.63%, 47.59%, and 95.27%, respectively, and their half-lives were 109, 56, and 227 min, respectively; the residual relative activity and half-life of the wild type under the same conditions were 30.73% and 32 min, respectively. It can be seen that the double-point mutant A375P / S257P further achieved excellent thermal stability on the basis of the improved thermal stability of single-point mutants A375P and S257P.

[0094] Example 5 Catalytic Activities of Wild-Type Maltogenic Amylase and Double-Point Mutant

[0095] In a reaction system of phosphate buffer (50 mmol / L, pH 5.0) containing 0 - 30 mg / mL β-cyclodextrin, 100 μL of the pure enzymes of the wild type and double-point mutant A375P / S257P obtained in Example 2 were added to start the reaction. The control group did not contain the enzyme solution, and other components were the same; the reaction was carried out at the optimal temperature for 10 min to obtain the hydrolysis activities of the wild type and double-point mutant A375P / S257P towards β-cyclodextrin; Origin software was used to perform non-linear fitting on the calculated hydrolysis activities and the corresponding substrate concentrations to obtain the catalytic efficiency (k cat / K m ) of the enzyme.

[0096] The results were as follows: the catalytic efficiency (k cat / K m ) of the wild type towards β-cyclodextrin was 13.36 mM -1 ·s -1 ; the catalytic efficiency (k cat / K m ) of the double-point mutant A375P / S257P towards β-cyclodextrin was 30.42 mM -1 ·s -1 .

[0097] Example 6 Preparation of Bread Using Maltogenic Amylase Mutant A375P / S257P

[0098] (1) 300 g of high-gluten flour, 4.5 g of yeast powder, 18 g of white granulated sugar, 3 g of salt, and the activated maltogenic amylase mutant A375P / S257P (HLpaMAA375P / S257P) were stirred in a mixing bowl until the ingredients were evenly mixed. The addition amount of the maltogenic amylase mutant was 50,000 U of maltogenic amylase units per 300 g of flour. 180 g of water was added, and the temperature of the water was 18 °C. Stir at low speed for 3 min and at high speed for 5 min to complete the kneading of the dough.

[0099] (2) Take out the dough, knead it manually moderately, and then cover it with plastic wrap for 5 min;

[0100] (3) Roughly cut the relaxed dough into pieces of about 90 g each. After preliminary shaping and degassing, cover it with plastic wrap again and let it proof for the first time at room temperature for 5 min.

[0101] (4) Roll out each small dough piece in turn. After ensuring that the air bubbles are exhausted, roll it up and put it into a baking mold, and let it proof for the second time at 37 °C and a relative humidity of 85% for 90 min.

[0102] (5) Bake the proofed bread at 170 °C for the upper heat and 210 °C for the lower heat for 21 min.

[0103] (6) Cool the freshly baked bread at room temperature for 1 - 2 h and then store it at 4 °C.

[0104] Control group 1: Wild - type maltogenic amylase (LpaMA)

[0105] (1) 300 g of high - gluten flour, 4.5 g of yeast powder, 18 g of granulated sugar, 3 g of salt, and the activated wild - type maltogenic amylase (LpaMA) are stirred in a mixing bowl until the ingredients are evenly mixed. The addition amount of wild - type maltogenic amylase is 50,000 U of maltogenic amylase units / 300 g of flour. Add 180 g of water, and the water temperature is 18 °C. Stir at low speed for 3 min and at high speed for 5 min to complete the kneading of the dough.

[0106] (2) Take out the dough and knead it manually moderately, then cover it with plastic wrap for 5 min.

[0107] (3) Roughly cut the relaxed dough into pieces of about 90 g each. After preliminary shaping and degassing, cover it with plastic wrap again and let it proof for the first time at room temperature for 5 min.

[0108] (4) Roll out each small dough piece in turn. After ensuring that the air bubbles are exhausted, roll it up and put it into a baking mold, and let it proof for the second time at 37 °C and a relative humidity of 85% for 90 min.

[0109] (5) Bake the proofed bread at 170 °C for the upper heat and 210 °C for the lower heat for 21 min.

[0110] (6) Cool the freshly baked bread at room temperature for 1 - 2 h and then store it at 4 °C.

[0111] Control group 2: Blank control (Control)

[0112] (1) 300 g of high-gluten flour, 4.5 g of yeast powder, 18 g of granulated sugar, and 3 g of salt are stirred in a mixing bowl until the ingredients are evenly mixed. Then add 180 g of water at a temperature of 18 °C. Stir at low speed for 3 min and at high speed for 5 min to complete the kneading of the dough.

[0113] (2) Take out the dough, knead it manually moderately, and then cover it with plastic wrap for 5 min;

[0114] (3) Divide the relaxed dough into pieces of about 90 g each. After preliminary shaping and degassing, cover it with plastic wrap again and let it proof for the first time at room temperature for 5 min;

[0115] (4) Roll out each small dough in turn. After ensuring that the air bubbles are exhausted, roll it up and put it into a baking mold. Let it proof for the second time at 37 °C and a relative humidity of 85% for 90 min;

[0116] (5) Bake the proofed bread at 170 °C for the upper fire and 210 °C for the lower fire for 21 min;

[0117] (6) Cool the freshly baked bread at room temperature for 1 - 2 h and then store it at 4 °C.

[0118] As shown in Table 1 and Figure 5 as shown, the volume of the bread obtained by applying the maltogenic amylase mutant A375P / S257P of the present invention is larger than that of the control group, the bread crumb is denser, and the softness is better.

[0119] Table 1: Hardness change of bread stored for 7 days (unit: g)

[0120]

[0121] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A heat-stable and acid-resistant maltogenic amylase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.

2.

2. The preparation method of the thermally stable acid-resistant maltogenic amylase mutant according to claim 1, characterized in that, Using the amino acid sequence of maltogenic amylase with the NCBI accession number No.WP_025085606.1 as the starting sequence, alanine at position 375 and serine at position 257 were respectively mutated to proline by whole plasmid PCR.

3. The preparation method of the thermally stable and acid-resistant maltogenic amylase mutant according to claim 1, characterized in that, The primers for mutating alanine at position 375 are as follows: A375P-For: TCACAACCGTGGCTCCAAGGAGATCAGTTTAG; A375P-Rev: TGGAGCCACGGTTGTGAGGAGTGCCAAACTTCA; The primers for mutating serine at position 257 are as follows: S257P-For: CAGTTCACCGCAATGGTTGGATGTCGTTAAAAAT; S257P-Rev: ACCATTGCGGTGAACTGTCACCCATATGATTGAAA.

4. Use of the thermostable and acid-resistant maltogenic amylase mutant according to claim 1 in food preparation.

5. Use of the thermostable acid-resistant maltogenic amylase mutant according to claim 4 in food preparation, characterized in that, The food is dough or dough products, or batter or batter products, or bakery or pastry products.

6. Use of the thermostable and acid-resistant maltogenic amylase mutant according to claim 1 as a food additive.

7. The use according to any one of claims 4 - 6 for improving the shelf life and / or texture of bakery or pastry products.

8. Use of the thermostable and acid-resistant maltogenic amylase mutant according to claim 1 as a bread improver.

9. Use of the thermostable and acid-resistant maltogenic amylase mutant according to claim 1 in improving dough tolerance.

10. Food additive, characterized in that, Comprising the thermostable and acid-resistant maltogenic amylase mutant according to claim 1.

11. The food additive according to claim 10, characterized in that, The food additive is a bread improver.

12. A method for preparing food, characterized in that, Including the step of mixing the thermostable and acid-resistant maltogenic amylase mutant according to claim 1 with food ingredients.

13. The method for preparing food according to claim 12, characterized in that, The food is a bakery or pastry product, and the thermostable and acid-resistant maltogenic amylase mutant according to claim 1 is added to the dough or batter and mixed.

14. A food comprising the thermostable and acid-resistant maltogenic amylase mutant of claim 1, prepared by the method according to claim 11 or claim 12.

15. The food according to claim 14, characterized in that, The food is a dough product, a batter product or a baked product.

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