Thermostable acid-stable maltogenic amylase mutants and uses thereof

Abstract

The application discloses a thermostable acid-resistant maltogenic amylase mutant and application thereof. The amino acid sequence of the maltogenic amylase mutant is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 2. The optimal temperature is 65 DEG C, the maximum activity is 430.15 U·mg ‑1 , and the half-life at 60 DEG C and the residual enzyme activity after 60 min are 227 min and 337.43 U·mg ‑1 , respectively, so that the half-life and the residual enzyme activity are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to acid-tolerant maltogenic amylase from Lactobacillus sakei, in particular to heat-stable acid-tolerant maltogenic amylase mutants, belonging to the technical field of enzyme engineering and microbial engineering. BACKGROUND

[0002] Maltogenic amylase (EC 3.2.1.133), a kind of GH13-20 family of alpha-amylases (EC 3.2.1.1) that act on starch and related polysaccharides, can hydrolyze alpha-1, 4 and alpha-1, 6-glucosidic bonds, and the main product is maltose. Maltogenic amylase has multi-substrate specificity and substrate preference, and preferentially hydrolyzes cyclic dextrin, followed by amylose, amylopectin and pullulan, etc. After starch is gelatinized by heat, maltogenic amylase can hydrolyze from the end of the starch molecule to produce maltose and some small molecular oligosaccharides, interfere with the recrystallization of starch and the entanglement of starch granules with protein macromolecules, thereby reducing the respiration rate and recrystallization rate of starch granules, achieving the effect of anti-aging, and can be used to prolong the shelf life of baked foods. In addition, compared with traditional emulsifiers and gluten enhancers, maltogenic amylase is efficient and meets food safety requirements, and has no safety hazards. Therefore, maltogenic amylase is considered as an ideal bread improver.

[0003] Currently, the research on maltogenic amylase mainly focuses on heat-tolerant microorganisms, such as Bacillus stearothermophilus, B. licheniformis, B. subtilis and Thermus sp. The maltogenic amylases from the above sources are mainly used in bread baking at neutral or near-neutral pH, and are sensitive to acidic conditions, so their beneficial effects in the process of making bread with low pH formula are very limited. In addition, only two maltogenic amylases from Lactobacillus plantarum and Lactobacillus gasseri are found to be more acid-tolerant, but they have some defects in terms of thermal stability or hydrolysis activity.

[0004] Lactobacillus sakei is one of the dominant lactic acid bacteria in sourdough, and is a recognized food safety level strain. Therefore, Lactobacillus sakei is the preferred expression strain of acid-tolerant maltogenic amylase for bread improvement. However, currently, the maltogenic amylase from Lactobacillus sakei has the problems of low optimal temperature and poor thermal stability. Therefore, it is of great significance for the baking industry to improve the thermal stability and enzyme activity of maltogenic amylase from Lactobacillus sakei. SUMMARY

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

[0006] Specifically, the acid-resistant maltogenic amylase mutant with improved thermal stability has an amino acid sequence as shown in SEQ ID NO. 1 and a nucleotide sequence as shown in SEQ ID NO. 2.

[0007] The present application also provides a preparation method of the acid-resistant maltogenic amylase mutant with improved thermal stability.

[0008] Specifically, the preparation method of the acid-resistant maltogenic amylase mutant with improved thermal stability uses the amino acid sequence of maltogenic amylase with NCBI accession number No. WP_025085606.1 as the starting sequence, and mutates the alanine at position 375 and the serine at position 257 into proline, respectively.

[0009] The present application further relates to the application of the acid-resistant maltogenic amylase mutant with improved thermal stability.

[0010] Specifically, the application relates to the application of the acid-resistant maltogenic amylase mutant with improved thermal stability in food preparation. In specific embodiments, the food is a dough or a dough product, a batter or a batter product, or a bakery or a patisserie product.

[0011] This invention further provides the use of a heat-stable, acid-resistant maltose amylase mutant as taught herein as a food additive. It also further provides a food additive comprising the heat-stable, acid-resistant maltose amylase mutant described herein. In a particular embodiment, the food additive is a bread improver. In other words, the heat-stable, acid-resistant maltose 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 enhancers, improvers, or flour treatment agents) are typically added to dough to improve the texture, volume, flavor, and / or freshness of baked goods and to enhance the machinability and stability of the dough. Typically, bread improvers consist of, or are essentially composed of, one or more enzymes, such as one or a combination of two of the following types of enzymes: amylases (α-amylase, β-amylase, glucosylamylase, native starch-degrading amylase), xylanases (hemicellulases), cellulases, pectinases, proteases, pectin lyases, oxidases (peroxidases, glucose oxidases, pyranose oxidases, hexose oxidases, L-amino acid oxidases, carbohydrate oxidases, thiol oxidases), lipoxygenases, dehydrogenases, laccases, transglutaminases, acyltransferases, and protein disulfides. Bond isomerase; one or more oxidizing or reducing agents, such as ascorbic acid, glutathione, cysteine, etc.; one or more emulsifiers, such as monoglyceride diacetate tartrate (DATEM), sodium stearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), glyceryl monostearate (GMS), rhamnolipid, lecithin, sucrose ester, 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 products, relating to methods for preparing dough products, flour paste products, and baked products, with a preference for methods for preparing baked products. These methods include the step of mixing the heat-stable, acid-resistant maltose amylase mutant provided herein with food ingredients.

[0013] In a particular embodiment, if the method is for preparing a baked product, the method may include the step of adding a heat-stable, acid-resistant maltose amylase mutant, as provided herein, to dough or batter. Preferably, the heat-stable, acid-resistant maltose amylase mutant provided herein is added to the dough or batter prior to baking.

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

[0015] In line with this, in another aspect is provided the use of a thermostable acid-stable maltogenic amylase mutant as taught herein in the manufacture 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 a dough or batter prepared in the absence (i.e. in the absence of) a polypeptide having maltogenic amylase activity as taught herein.

[0016] Preferably, the baked product of the present application is a bakery or patisserie product. Bakery or patisserie products known in the art include, but are not limited to, a product selected from the group consisting of bread, buns, rolls, bagels, donuts, danish, hamburger buns, pizza, pita bread, ciabatta, sponge cake, butter cake, pound cake, muffin, cupcake, steamed cake, waffle, brownie, cake donut, yeast-raised donut, French bread, bun, savoury biscuit, sweet biscuit, pie crust, rusk and / or other baked product. More preferably, the baked product is bread, French bread and / or bun.

[0017] In another aspect, the present application relates to the use of a thermostable acid-stable maltogenic amylase mutant as provided herein in a baking and patisserie application. It has been found that the thermostable acid-stable maltogenic amylase mutants as provided herein are useful in improving bread volume, texture and mouthfeel.

[0018] In another aspect, the present application provides a food product (e.g. a bread improver), a dough product, a batter product or a baked product comprising a thermostable acid-stable maltogenic amylase mutant as provided herein.

[0019] In another aspect, the present application 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 comprising flour and a maltogenic amylase mutant as provided herein, the maltogenic amylase mutant being added in an amount of 1000-100000 U of maltogenic amylase units per kilogram of flour, preferably 1000-100000 U of maltogenic amylase units per kilogram of flour, more preferably 10000-100000 U of maltogenic amylase units per kilogram of flour.

[0021] Advantages and effects of the present application:

[0022] (1) The optimum pH of the wild-type maltogenic amylase from Lactobacillus paralimentarius is 5.0, and the maltogenic amylase has better acid resistance compared with the maltogenic amylases from Bacillus reported.

[0023] (2) The optimum temperature and the maximum activity of the wild-type maltogenic amylase are 50 DEG C and 274.79 U·mg -1 , respectively, and the optimum temperature and the maximum activity of the optimal double-point mutant A375P / S257P are 65 DEG C and 430.15 U·mg -1 , respectively, and the action temperature and the hydrolysis activity are improved by 15 DEG C and 156.4%, respectively.

[0024] (3) The half-life at 60 DEG C and the residual enzyme activity after 60 min of the wild-type maltogenic amylase are 32 min and 85.65 U·mg -1 , respectively, and the half-life at 60 DEG C and the residual enzyme activity after 60 min of the optimal double-point mutant A375P / S257P are 227 min and 337.43 U·mg -1 , respectively, and the half-life and the residual enzyme activity are improved by 7.09 and 3.93 times, respectively.

[0025] (4) The kinetic parameter determination is carried out by taking beta-cyclodextrin as a substrate, and the catalytic efficiency (k cat / K m ) of the wild-type maltogenic amylase is 13.36 mM -1 ·s -1 , and the catalytic efficiency (kcat / Km) of the optimal double-point mutant A375P / S257P is 30.42 mM -1 ·s -1 , and the catalytic efficiency (k cat / K m ) is improved by 2.27 times.

[0026] (5) The double-point mutant A375P / S257P obtained in the application has the improved catalytic efficiency (k cat / K m ) and the good temperature stability of the wild-type strain, and has the practical industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the optimum pH determination of the wild-type maltogenic amylase.

[0028] Figure 2Determination of optimum temperature and hydrolytic activity of wild type and mutant A375P, S257P and A375P / S257P.

[0029] Figure 3 Determination of temperature stability of wild type and mutant A375P, S257P and A375P / S257P at 60℃.

[0030] Figure 4 Determination of half-life (t 1 / 2 ) of wild type and mutant A375P, S257P and A375P / S257P at 60℃.

[0031] Figure 5 Bread storage profile of maltogenic amylase mutant A375P / S257P, wild type maltogenic amylase and blank control. DETAILED DESCRIPTION

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

[0033] Culture medium used in the following examples was as follows:

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

[0035] LB solid medium: yeast powder 5.0 g·L -1 , tryptone 10.0 g·L -1 , NaCl 10.0 g·L -1 , agar powder 20 g·L -1 , kanamycin 50 mg·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 the maltogenic amylase was chemically synthesized on a vector pET28a and transformed into E. coli BL21 (DE3) to form the wild-type pET28a-LpaMA / E. coli BL21 of the maltogenic amylase, and the NCBI accession number of the gene is No. WP_025085606.1. The site-directed mutation was performed by using the obtained recombinant plasmid pET28a-LpaMA as a template by using the whole-plasmid PCR technology to obtain the 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] Wherein, the primer used for mutation A375P is as follows:

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

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

[0078] The primer used for mutation S257P (A375P / S257P mutant) is as follows:

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

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

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

[0082] The PCR amplification product was detected by 1% agarose gel electrophoresis. After detection, 0.5 μL of methylation template digestion enzyme (Dpn I) was added to 10 μL of the amplification product, which was mixed by blowing and sucking with a syringe head. The mixture was reacted at 37°C for 1.5 h. The amplification product treated by Dpn I was transformed into E. coli BL21 (DE3). The transformation product was spread on LB solid medium and cultured at 37°C for 8-10 h. Twenty transformants were picked from the LB solid medium and inoculated into LB liquid medium. After being cultured at 37°C for 10 h, the plasmid was extracted. The plasmid was sequenced, and the recombinant E. coli containing the gene encoding the mutant A375P, S257P, and A375P / S257P was obtained.

[0083] Example 2 Induced culture and protein purification of maltogenic amylase mutants

[0084] The maltogenic amylase with the amino acid sequence shown in SEQ ID No. 1 was used as the wild type. The obtained recombinant E. coli pET28a-LpaMA / E. coli BL21 and the recombinant E. coli containing the gene encoding the mutant A375P, S257P, and A375P / S257P were spread on LB solid medium and cultured at 37°C for 8-10 h to obtain single colonies. The single colonies were picked and inoculated into LB liquid medium and 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), and then cultured at 37°C and 200 rpm for 2-3 h. Then, IPTG was added to the fermentation liquid at a final concentration of 0.1 mmol / L, and the fermentation was continued at 17°C and 200 rpm for 12-17 h to obtain fermentation liquid. The fermentation liquid was centrifuged at 4°C and 8000 rpm for 5 min, and the supernatant was discarded. The pellet was washed with 9% physiological saline twice to obtain wet bacterial cells of the wild type, the mutant A375P, S257P, and A375P / S257P.

[0085] The wet bacteria were resuspended in buffer A and ultrasonically broken, centrifuged at 10000 rpm, 4°C for 30 min to obtain a crude enzyme solution. After filtration with a 0.22 um water filter, the sample was slowly added to a Ni-NAT affinity chromatography column, washed with buffer A first and then eluted with buffer B gradient, and the elution peak corresponding to 300 mmol / L imidazole was collected. Then, the desalting column was used to remove imidazole from the pure enzyme, and the pure enzymes of wild type, mutant A375P, S257P and A375P / S257P were obtained.

[0086] Example 3 Hydrolysis activity of different maltogenic amylase mutants on starch

[0087] The reaction system was obtained by adding soluble starch (10 mg / mL) in 50 mol / L citric acid buffer (pH 4.0-6.0), phosphate buffer (pH 6.0-7.0) and Tris-HCl buffer (pH 7.0-9.0), respectively. After incubation at 40-70°C for 3 min, 100 ul of the pure enzyme 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 the other components were the same. The reaction was carried out at 40-70°C for 10 min, 10 ul of the product was diluted 20 times and an equal volume of 200 ul DNS (3,5-dinitrosalicylic acid) was added to terminate the reaction, and then it was placed in boiling water at 100°C for 5 min and cooled on ice to room temperature. The change in absorbance at 540 nm was recorded to obtain the hydrolysis activity of wild type, mutant A375P, S257P and A375P / S257P on starch.

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

[0089] Wherein, Ew is the absorbance change value at 540 nm within 10 min; k is the reducing sugar standard curve coefficient, with the unit of umol / mL; V is the volume of enzyme activity determination 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 liquid dilution multiple; L is the volume of added enzyme liquid, with the unit of mL, which is 0.1 here; the protein concentration of enzyme liquid is determined by Bradford protein kit (Bradford protein kit for determining protein concentration can 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 Interactions 1 [J]. Cancer Research, 1993, 53(21): 5121-5126.), with the unit of mg / mL.

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

[0091] Example 4 Temperature stability of different maltogenic amylase mutants

[0092] The enzyme liquids of the wild type, mutant A375P, S257P and A375P / S257P obtained in Example 2 are warmed in a water bath at 60℃ for 60 min, and samples are taken every certain time interval, and the catalytic activities of the wild type, mutant A375P, S257P and A375P / S257P of the maltogenic amylase on starch after the warming are determined at 50℃, and the activity before the warming is taken as 100%, and the relative activity is calculated by comparing the residual activity after the warming, so as to investigate the temperature stability and half-life of the wild type, mutant A375P, S257P and A375P / S257P of the maltogenic amylase. The residual activity and the corresponding warming time are linearly fitted by using Origin software to obtain the half-life (t 1 / 2 ) of the enzyme.

[0093] The residual relative activity of the mutants A375P, S257P and A375P / S257P was 70.63%, 47.59% and 95.27%, respectively, and the half-life was 109 min, 56 min and 227 min, respectively; the residual relative activity and the 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 improved the thermal stability on the basis of the single-point mutants A375P and S257P.

[0094] Example 5 Catalytic activity of wild-type maltogenic amylase and double-point mutant

[0095] In a phosphate buffer (50 mmol / L, pH 5.0) reaction system containing 0-30 mg / mL β-cyclodextrin, 100 ul of the wild type and double-point mutant A375P / S257P pure enzyme obtained in Example 2 was added to start the reaction, and the control group did not contain enzyme liquid, and the other components were the same; the reaction was carried out at the optimum temperature for 10 min to obtain the hydrolysis activity of the wild type and double-point mutant A375P / S257P to β-cyclodextrin; the calculated hydrolysis activity and the corresponding substrate concentration were non-linearly fitted using Origin software to obtain the catalytic efficiency (k cat / K m ) of the enzyme.

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

[0097] Example 6 Preparation of bread using maltogenic amylase mutant A375P / S257P

[0098] (1) 300 grams of high-gluten flour, 4.5 g of yeast powder, 18 g of white sugar, 3 grams of salt and activated maltogenic amylase mutant A375P / S257P (HLpaMAA375P / S257P) were stirred in a stirring cylinder until the ingredients were uniform. The addition amount of the maltogenic amylase mutant was 50,000 U of maltogenic amylase unit per 300 grams of flour. Add 180 g of water, and the temperature of the water is 18℃. Stir at low speed for 3 min and at high speed for 5 min to complete the dough mixing.

[0099] (2) After taking out the dough and moderately manually mixing, cover it with plastic wrap for 5 min;

[0100] (3) The relaxed dough is roughly cut into about 90 g per piece, and after the initial shaping and bubble venting operation, it is covered with plastic wrap again and allowed to rise for the first time at room temperature for 5 min;

[0101] (4) Each small dough is rolled out in turn, ensuring that the bubbles are exhausted, and then rolled up and placed in a baking mold, and allowed to rise for the second time at 37°C and 85% relative humidity for 90 min;

[0102] (5) The bread that has finished rising is baked at a temperature of 170°C top and 210°C bottom for 21 min;

[0103] (6) The freshly baked bread is cooled at room temperature for 1-2 h and then stored 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 white sugar, 3 g of salt, and activated wild-type maltogenic amylase (LpaMA) are stirred in a stirring cylinder until the ingredients are evenly distributed. The addition amount of wild-type maltogenic amylase is 50,000 U of maltogenic amylase units per 300 g of flour. Add 180 g of water, and the temperature of the water is 18°C. Stir at low speed for 3 min and at high speed for 5 min to complete the dough mixing.

[0106] (2) After taking out the dough and moderately manually mixing, cover it with plastic wrap for 5 min;

[0107] (3) The relaxed dough is roughly cut into about 90 g per piece, and after the initial shaping and bubble venting operation, it is covered with plastic wrap again and allowed to rise for the first time at room temperature for 5 min;

[0108] (4) Each small dough is rolled out in turn, ensuring that the bubbles are exhausted, and then rolled up and placed in a baking mold, and allowed to rise for the second time at 37°C and 85% relative humidity for 90 min;

[0109] (5) The bread that has finished rising is baked at a temperature of 170°C top and 210°C bottom for 21 min;

[0110] (6) The freshly baked bread is cooled at room temperature for 1-2 h and then stored 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 white sugar and 3 g of salt are stirred in a stirring cylinder until the ingredients are uniform, 180 g of water is added, and the temperature of the water is 18°C. The dough is mixed by stirring at low speed for 3 min and at high speed for 5 min.

[0113] (2) After the dough is moderately manually mixed, it is covered with plastic wrap for 5 min;

[0114] (3) The relaxed dough is roughly cut into pieces of about 90 g each, and after preliminary shaping and bubble venting, it is again covered with plastic wrap and allowed to rise at room temperature for the first time for 5 min;

[0115] (4) Each small piece of dough is rolled out in turn, ensuring that the bubbles are vented, and then rolled up and placed in a baking mold, and allowed to rise for the second time for 90 min at 37°C and 85% relative humidity;

[0116] (5) The finished bread is baked at an upper temperature of 170°C and a lower temperature of 210°C for 21 min;

[0117] (6) The freshly baked bread is cooled at room temperature for 1-2 h and then stored at 4°C.

[0118] As shown in Table 1 and Figure 5 , the bread obtained using the maltogenic amylase mutant A375P / S257P of the present application has a larger volume and a more fluffy crumb and better softness than the control group.

[0119] Table 1: Change in hardness of bread stored for 7 days (unit: g)

[0120]

[0121] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the scope of protection of the present application should be defined by the claims.

Claims

1. A thermostable acid-stable maltogenic amylase mutant, characterized in that, The amino acid sequence is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO.

2.

2. The process for the production of a thermostable acid-stable maltogenic amylase mutant according to claim 1, characterized in that, The amino acid sequence of maltogenic amylase with NCBI accession No. WP_025085606.1 is used as the starting sequence, and the alanine at position 375 and the serine at position 257 are mutated to proline by whole plasmid PCR.

3. The process for preparing a thermostable acid-stable maltogenic amylase mutant according to claim 2, characterized in that, The primer for mutating alanine at position 375 is as follows: A375P-For: TCACAACCGTGGCTCCAAGGAGATCAGTTTAG; A375P-Rev: TGGAGCCACGGTTGTGAGGAGTGCCAAACTTCA; The primer for mutating serine at position 257 is as follows: S257P-For: CAGTTCACCGCAATGGTTGGATGTCGTTAAAAAT; S257P-Rev: ACCATTGCGGTGAACTGTCACCCATATGATTGAAA.

4. Use of the heat-stable acid-tolerant maltogenic amylase mutant of claim 1 in food preparation.

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

6. Use of the heat-stable acid-tolerant maltogenic amylase mutant of claim 1 as a food additive.

7. Use according to any one of claims 4-6, for improving the shelf life and / or texture of a bakery or patisserie product.

8. Use of the heat-stable acid-tolerant maltogenic amylase mutant of claim 1 as a bread improver.

9. Use of the heat-stable acid-tolerant maltogenic amylase mutant of claim 1 for improving dough tolerance.

10. A food additive characterized by, The heat-stable acid-tolerant maltogenic amylase mutant of claim 1.

11. The food additive of claim 10, wherein the food additive is a food additive for use in the preparation of a food product. The food additive is a bread improver.

12. A method of preparing a food product, characterized by, The step of mixing the heat-stable acid-tolerant maltogenic amylase mutant of claim 1 with food ingredients.

13. The method of preparing a food product according to claim 12, wherein, The food is a bakery or patisserie product, and the heat-stable acid-tolerant maltogenic amylase mutant of claim 1 is added to the dough or batter for mixing.

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