Acid-resistant and heat-resistant alpha-amylase as well as preparation and application thereof

Abstract

The invention discloses acid-resistant and heat-resistant alpha-amylase as well as preparation and application thereof. The acid-resistant and heat-resistant alpha-amylase is obtained by fusing 37 amino acids at the N end of alpha-amylase Gt-amy with wild type alpha-amylase Ba-amy, deleting 178-site and 179-site amino acids and mutating the 332-site amino acid from valine to aspartic acid, and the specific enzyme activity of the acid-resistant and heat-resistant alpha-amylase is as high as 6734U / mg and is 13 times that of the wild type enzyme; the optimum pH value is 5.5, which is reduced by 0.5 unit compared with that of a wild type enzyme, and 60% of enzyme activity is still retained when the pH value is 4.5; the heat stability is improved, and the half-life period at 90 DEG C is prolonged by 5.8 times compared with that of a wild type enzyme. A pyrodextrin hydrolysis result shows that the method can directly and effectively assist saccharifying enzyme enzymolysis in an acidic environment, avoids repeated adjustment of pH in a reaction process, can improve the yield of resistant dextrin, simplify the process flow and reduce the production cost, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an acid-resistant and heat-resistant alpha-amylase and a preparation and application thereof. Background Art

[0002] Starch is a high molecular carbohydrate whose basic unit is glucose. Starch is widely present in plants and is the main form of energy storage for plants. Starch is also the most important component of people's dietary structure and can be hydrolyzed into glucose by amylase and absorbed by the human body.

[0003] α-Amylase, also known as 1,4-α-D-glucanohydrolase (EC 3.2.1.1), is one of the oldest industrial enzyme preparations. It can cut α-1, 4 glycosidic bonds from the inside of the molecule and hydrolyze starch macromolecules into small molecular dextrins, maltose, glucose and other products. It is a very important starch hydrolyzing enzyme. α-Amylase is one of the most competitive players in the enzyme preparation market. It is widely used in various industrial scenarios based on starch hydrolysis, such as food, washing, agriculture, papermaking, textiles, pharmaceuticals, brewing and fermentation. Not only that, it also has some potential application value, such as the treatment of fermentation wastewater from alcohol plants and the preparation process of resistant dextrins.

[0004] Resistant dextrin is an important low-molecular water-soluble dietary fiber in the modern food industry. It has attracted much attention due to its anti-digestion characteristics. It has broad application prospects in beverages, meat products, pasta products and functional foods. At present, the methods for producing resistant dextrin are mainly divided into two steps: preparing pyrodextrin by acid-heat method and enzymatic separation of pyrodextrin. The pH of the pyrodextrin solution prepared by the acid-heat method is low, and the optimal pH of commercial saccharifying enzymes is also acidic. However, the available acid-resistant α-amylases are few in variety and high in price. Therefore, the existing pyrodextrin enzymatic hydrolysis process often requires first adjusting the pH of pyrodextrin to neutral and using α-amylase for hydrolysis, and then adjusting the pH to acid and using saccharifying enzymes for hydrolysis, which not only increases the production cost, but also increases the ash content in the product, and there is also the risk of polluting the environment. Therefore, there is an urgent need for α-amylases with good acid and heat resistance to be used in the preparation process of resistant dextrin. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide an acid-resistant and heat-resistant α-amylase. The present invention obtains an α-amylase advantage mutant with high catalytic activity and acid-resistant and heat-resistant by molecularly modifying the wild-type α-amylase Ba-amy, with a specific enzyme activity of up to 6734U / mg; the optimum pH is 5.5 and 60% of the enzyme activity is still retained at pH 4.5, the half-life is 85.5min at 90°C, and the thermal stability is 5.8 times higher than that of the wild type. The acid-resistant and heat-resistant α-amylase can be well used in the preparation process of resistant dextrin, which simplifies the process flow and reduces production costs.

[0006] Another object of the present invention is to provide the use of the above acid-resistant and heat-resistant α-amylase in the preparation process of resistant dextrin. The hydrolysis results of pyrodextrin solution show that compared with the blank control group, the acid-resistant and heat-resistant α-amylase can hydrolyze more digestible components under pH 4.5, thereby effectively assisting saccharifying enzyme hydrolysis.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] An acid-resistant and heat-resistant alpha-amylase, whose amino acid sequence is shown in SEQ ID NO:3.

[0009] Specifically, the acid-resistant and heat-resistant α-amylase is obtained by fusing the wild-type α-amylase Ba-amy with the amino acid sequence shown in SEQ ID NO: 1, the 37 N-terminal amino acids of the α-amylase Gt-amy with the amino acid sequence shown in SEQ ID NO: 2, deleting the amino acids at positions 178 and 179, and mutating the amino acid at position 332 from valine (Val) to aspartic acid (Asp). The N-terminal fusion fragment can significantly improve the acid resistance of the α-amylase. The deletion of the amino acids at positions 178 and 179 and the mutation of the amino acid at position 332 can significantly improve the thermal stability of the α-amylase.

[0010] An acid-resistant and heat-resistant alpha-amylase gene, the nucleotide sequence of which is shown in SEQ ID NO:5.

[0011] A recombinant expression vector containing the above nucleotide sequence.

[0012] Preferably according to the present invention, the basic vector used by the recombinant expression vector is pET-28a.

[0013] A recombinant expression bacterium comprises the above-mentioned recombinant expression vector.

[0014] Preferably according to the present invention, the basic expression bacteria used in the recombinant expression bacteria are Escherichia coli BL21 (DE3).

[0015] The preparation method of the above-mentioned acid-resistant and heat-resistant α-amylase comprises the following steps: cloning the gene with a nucleotide sequence as shown in SEQ ID NO:5 into the pET-28a expression vector, then transforming it into Escherichia coli BL21 (DE3) for inducing expression, collecting the bacterial precipitate by solid-liquid separation, and obtaining the acid-resistant and heat-resistant α-amylase after purification and dialysis.

[0016] According to the preferred embodiment of the present invention, the specific operation of inducing expression is: inoculating the positive transformant into LB culture medium for cultivation, and when the bacterial solution OD 600 When the pH value reached 0.6-0.8, IPTG was added at a final concentration of 0.01 mmol / L and induced at 18°C ​​for 20 h; the LB culture medium contained 50 μg / mL of kanamycin.

[0017] Preferably, according to the present invention, the specific operation of inoculating the positive transformant into LB culture medium for culturing is:

[0018] (1) culturing the positive transformant in LB culture medium at 37° C. with shaking overnight to obtain a seed solution;

[0019] (2) The seed solution was inoculated into LB medium at a ratio of 1:100 and cultured at 37°C and 200 r / min.

[0020] The purification step is preferably: collecting the supernatant after lysing the cells, passing the supernatant through an affinity chromatography column, and eluting to obtain the purified acid-resistant and heat-resistant α-amylase.

[0021] The method for lysing cells is preferably: lysing cells by ultrasonic disruption in an ice bath.

[0022] In the ultrasonic disruption method, the cells are preferably resuspended in buffer A before the process is performed; the formula of buffer A is 10 mmol / L acetic acid-sodium acetate, 300 mmol / L NaCl, pH=6.0.

[0023] The amount of buffer A is preferably 10 mL of buffer A per gram of bacterial cells.

[0024] The conditions of the ultrasonic disruption method are preferably: power of 150 W, ultrasonication for 3 seconds, interval of 3 seconds, and duration of 20 minutes.

[0025] The operation of collecting the supernatant after lysing the cells is preferably as follows: the lysed bacteria are subjected to freeze centrifugation, the precipitate is discarded, and the cells are filtered through a membrane to obtain the supernatant.

[0026] The conditions of the refrigerated centrifugation are preferably centrifugation at 12000g for 30 min at 4°C.

[0027] The membrane filtration is preferably performed by filtering with a 0.22 μm microporous membrane.

[0028] The affinity chromatography column is preferably Ni 2+ Tag affinity chromatography column.

[0029] The elution is preferably performed by first balancing with buffer A and then eluting with buffer B; the formula of buffer A is 10mmol / L acetic acid-sodium acetate, 300mmol / L NaCl, pH=6.0; the formula of buffer B is: 500mmol / L imidazole, 10mmol / L acetic acid-sodium acetate and 300mmol / L NaCl, pH=6.0.

[0030] The dialysis step is preferably: dialyzing the purified enzyme solution into a storage buffer to obtain an acid-resistant and heat-resistant α-amylase.

[0031] The storage buffer preferably has a formula of: 10 mmol / L acetic acid-sodium acetate, pH=6.0.

[0032] The dialysis condition is preferably 4° C., a magnetic stirring speed of 100 r / min, and dialysis for 24 hours. Preferably, the storage buffer is replaced once during the process.

[0033] The dialysis is preferably performed in a dialysis bag, and the dialysis bag is preferably a dialysis bag with a molecular weight cutoff of 14 kDa.

[0034] Application of the above acid-resistant and heat-resistant α-amylase in the enzymatic hydrolysis of pyrodextrin.

[0035] Preferably according to the present invention, the enzymatic hydrolysis of pyrodextrin comprises the following steps: adding the acid-resistant and heat-resistant α-amylase into the pyrodextrin solution for enzymatic hydrolysis, and then directly adding saccharifying enzyme for saccharification after enzymatic hydrolysis.

[0036] The conditions for the enzymatic hydrolysis are preferably: temperature of 70±10°C and time of 1±0.5h; more preferably, temperature of 70°C and time of 1h.

[0037] The saccharification conditions are preferably: temperature of 40±5°C, time of 1±0.5h; temperature of 40°C, time of 1h.

[0038] Application of the acid-resistant and heat-resistant α-amylase in the preparation process of resistant dextrin.

[0039] Preferably, according to the present invention, the process for preparing resistant dextrin comprises the following steps:

[0040] S1, subjecting starch to dextrinization reaction by an acid-heat method to obtain pyrodextrin;

[0041] S2. Add the acid-resistant and heat-resistant α-amylase to the pyrodextrin obtained in step S1 for enzymatic hydrolysis, and then directly add saccharifying enzyme for saccharification after enzymatic hydrolysis.

[0042] The pH of the pyrodextrin is preferably 4.5±0.5.

[0043] The conditions of the enzymatic hydrolysis are: temperature 70±10°C, time 1±0.5h;

[0044] The saccharification conditions are preferably: temperature of 40±5°C and time of 1±0.5h.

[0045] Compared with the prior art, the present invention has the following advantages and effects:

[0046] 1. Based on the wild-type α-amylase, the present invention obtains an acid-resistant and heat-resistant α-amylase superior mutant through molecular modification, and its specific enzyme activity is as high as 6734U / mg; the optimum pH is 5.5, and 60% of the enzyme activity is still retained at pH 4.5; the half-life is 85.5min at 90°C, and the thermal stability is improved by 5.8 times compared with the wild type.

[0047] 2. Compared with the existing resistant dextrin preparation process, the acid-resistant and heat-resistant α-amylase provided by the present invention can synergize with saccharifying enzyme under acidic conditions to avoid repeated pH adjustment, which can not only simplify the process flow and reduce production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is the SDS-PAGE picture of AHR-Amy; wherein lane M is the protein marker, and lane 1 is the concentrated enzyme solution after AHR-Amy purification.

[0049] Figure 2 This is the optimal reaction pH diagram of AHR-Amy.

[0050] Figure 3 This is the optimal reaction temperature diagram of AHR-Amy.

[0051] Figure 4 This is the half-life curve of AHR-Amy at 90°C.

[0052] Figure 5 This is the pH stability diagram of AHR-Amy.

[0053] Figure 6 This is a comparison chart of the pH stability of mutant AHR-Amy and wild-type Ba-amy; the red curve is AHR-Amy and the black curve is Ba-amy.

[0054] Figure 7 This is the result diagram of DE value determination of different substrate solutions. DETAILED DESCRIPTION

[0055] The present invention will be further described below in conjunction with specific embodiments, but the embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0056] Unless otherwise specified, all reagents and raw materials used in the present invention are commercially available products or products that can be prepared by known methods.

[0057] The freeze-dried pyrodextrin powder used in the following examples is obtained by freeze-drying corn starch after dextrinization by an acid-heat method.

[0058] Example 1: Construction of a recombinant vector containing the nucleotide sequence of the acid-resistant and heat-resistant α-amylase mutant AHR-Amy, comprising the following steps:

[0059] (1) According to the amino acid sequence SEQ ID NO:1 of the wild-type α-amylase Ba-amy, it was compared with the amino acid sequence SEQ ID NO:2 of the α-amylase Gt-amy to obtain the N-terminal 37 amino acid sequence fragment. The DNA molecule encoding the N-terminal fragment was artificially synthesized by the splicing PCR method. At the same time, Guangzhou Aiji Biotechnology Co., Ltd. was commissioned to design the nucleotide sequence SEQ ID NO:4 according to the amino acid sequence of the wild-type α-amylase Ba-amy, and the basic plasmid pET28a was used to construct the vector Ba-amy, with the restriction sites of BamHI at the 5' end and XhoI at the 3' end. The vector Ba-amy was then linearized with primers. Finally, the linearized vector Ba-amy and the N-terminal fragment PCR product were purified and recovered respectively for homologous recombination.

[0060] The sequences of chimera amplification primers are as follows:

[0061] FP1:5'-ATGGGTCGCGGATCCATGCTGACGTTTCATCGTATTATT-3'

[0062] RP1:5'-CATAGTACCGTTGAAAGGGGCAGCCGCTTTCGCCGGCTGGCCGGTCG-3'

[0063] The sequences of the vector linearization primers are as follows:

[0064] NF:5'-TTCAACGGTACTATGATGCAATATTTCGAATGGTATCTGCCGG-3'

[0065] NR:5'-GGATCCGCGACCCATTTGCTGTCCA-3'.

[0066] The synthetic DNA molecule encoding the 37 amino acid fragment at the N-terminus and the Ba-amy vector were used as templates, and 25 μL 2×PfuMax HiFi PCR ProMix (Guangzhou Yingzan Biotechnology Co., Ltd., catalog number P217A), 1 μL (10 μmol / L) of upstream and downstream primers, and an appropriate amount of sterilized water were added for PCR amplification. The amplification conditions were: 98°C for 30 s; 98°C for 10 s, 60°C for 30 s, and 72°C for 30 s, for a total of 30 cycles; 72°C for 5 min. The amplified products were recovered by agarose gel electrophoresis, and then the recovered products were homologously recombined. The system was 5 μL 2×Hipro DNA Assembly Mix (Guangzhou Yingzan Biotechnology Co., Ltd., K001A), an appropriate amount of recovered products (number of bases × 0.02 ng), water was added to 10 μL, and incubated at 50°C for 15 min. The product was transformed into DH 5α competent cells, single colonies were picked for colony PCR identification, and the positive monoclonal bacteria were sent to a sequencing company for sequencing verification. The correct competent cells were cultured and verified, and the plasmid was extracted.

[0067] (2) Using the plasmids verified to be correctly connected as templates, perform site-directed mutagenesis. Design the following four primers:

[0068] FP2:5'-TTCCGTGGTAAGGCCTGGGACTGGGAA-3'

[0069] RP2:5'-CAGGCCTTACCACGGAACTTGTAGA-3'

[0070] FP3:5'-ATACCGAGCCAGACCAAGCCCTGCAGTCCTGGG-3'

[0071] RP3:5'-TTGGTCTGGCTCGGTATCGTGGTTGTCAACGA-3'.

[0072] The template was amplified by PCR using FP2 / RP2 and FP3 / RP3 respectively (FP2 / RP2 was used to delete the two amino acids 178 / 179, and FP3 / RP3 was used to mutate V332 to D332). The amplification conditions were: 98°C for 30s; 98°C for 10s, 60°C for 30s, and 72°C for 30s, for a total of 30 cycles; 72°C for 5min.

[0073] The amplified product was transformed into DH5α competent cells, single colonies were picked for colony PCR identification, and positive single clones were sent to a sequencing company for sequencing verification to confirm the correctness of the DNA molecule encoding the mutant AHR-Amy. The competent cells that were verified to be correct were cultured and the plasmid was extracted. The obtained plasmid was a recombinant vector containing the DNA molecule encoding the mutant AHR-Amy. The amino acid sequence of the mutant AHR-Amy is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 5.

[0074] Example 2: Inducible expression of mutant AHR-Amy

[0075] The recombinant plasmid constructed in Example 1 was transformed into the host cell E. coli BL21 (DE3), and a single colony was picked and placed in 5 mL LB medium containing 50 μg / mL kanamycin, and cultured overnight in a 37°C shaker; the seed solution of the overnight culture was inoculated into 200 mL LB medium containing 50 μg / mL kanamycin at a ratio of 1:100, and the culture was continued in a 37°C shaker until the OD 600 The pH value was 0.6-0.8, IPTG was added to a final concentration of 0.01 mmol / L, and the cells were induced at 18°C ​​for 20 h. The induced cells were collected by centrifugation and weighed, and the wet weight of the cells was recorded.

[0076] Example 3: Isolation and purification of mutant AHR-Amy

[0077] 1. Ultrasonic disruption of recombinant bacteria

[0078] Take the induced expression bacteria frozen at -20°C, and according to the wet weight of the bacteria recorded in Example 3, add 10mL lysis buffer (10mmol / L acetic acid-sodium acetate, 300mmol / L NaCl, pH=6.0) per gram of bacteria to resuspend the bacteria, and use an ultrasonic cell disruptor to lyse the bacteria. The ultrasonic conditions are: power 150W, ultrasound 3.0s, interval 3.0s, and last 20min. The lysed bacteria are placed in a high-speed refrigerated centrifuge and centrifuged at 12000g for 30min at 4°C for subsequent purification, and filtered with a 0.22μm microporous filter membrane.

[0079] 2. Nickel ion affinity chromatography purification

[0080] The chromatography column used is HisTrap TMHP 5mL (purchased from GE Healthcare), binding buffer buffer A is: 10mmol / L acetic acid-sodium acetate and 300mmol / L NaCl, pH 6.0; elution buffer buffer B is: 500mmol / L imidazole, 10mmol / L acetic acid-sodium acetate and 300mmol / L NaCl, pH 6.0, filtered through 0.22μm filter membrane for later use.

[0081] HisTrap TM HP 5mL was connected to the column valve of the rapid protein purifier, and then the system and column were cleaned with ultrapure water, and then the column was balanced with buffer A. Then the sample to be purified was loaded into the chromatography column with a sample pump. After loading, the column was first cleaned with buffer A, and then gradient eluted with elution buffer buffer B. The eluted components were collected and analyzed by SDS-PAGE. The results showed that a relatively pure target enzyme solution was successfully eluted ( Figure 1 ).

[0082] 3. Dialysis storage

[0083] The purified enzyme solution was dialyzed into storage buffer (10 mmol / L acetic acid-sodium acetate, pH=6.0) overnight for later use.

[0084] Example 4: Activity determination of mutant AHR-Amy

[0085] Take the mutant AHR-Amy prepared in Example 3, and determine the enzyme activity according to the following method. Prepare a 0.5% (w / v) potato amylose substrate solution with 10mmol / L acetic acid-sodium acetate buffer (pH=6.0), add 450μL substrate and 50μL enzyme solution to a 1.5mL centrifuge tube, react at 70°C for 10min, and immediately add 500μL DNS reagent after the reaction. Color develops in a boiling water bath for 5min, and after cooling, measure the absorbance at 540nm. The buffer solution is used as a blank control. Enzyme activity definition: Under the conditions of pH=6 and 70°C, the amount of enzyme required to catalyze the production of 1μmol of reducing sugar per minute is one enzyme activity unit (U).

[0086] The enzyme activity was calculated according to Formula 1, and three parallel tests were performed for each data.

[0087] Formula 1:

[0088] Among them, A is the sample absorbance value; A0 is the blank absorbance value; K is the slope of the standard curve; t is the reaction time; n is the dilution multiple; 342.3 is the relative molecular weight of maltose.

[0089] The results showed that the specific enzyme activity of AHR-Amy was 6734±386.38U / mg, which was 13 times higher than that of wild-type Ba-amy (477.43±5.87U / mg).

[0090] Example 5: Determination of enzymatic properties of mutant AHR-Amy

[0091] 1. Determination of the optimal reaction pH

[0092] The enzyme activity of AHR-Amy was measured at 70°C under different pH conditions (4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0), and the highest activity was taken as 100%, and the relative enzyme activity under other pH conditions was calculated. The pH 4.0-6.0 was 50 mM acetic acid-sodium acetate buffer, and the pH 6.0-8.0 was 50 mM PBS buffer.

[0093] Effect of pH on the activity of AHR-Amy enzyme Figure 2 As shown, AHR-Amy has high enzyme activity in the range of pH = 4.5-7. As the pH increases, the activity first increases and then decreases, and the best activity is shown at pH 5.5, which is 0.5 units lower than that of wild-type Ba-amy (pH 6.0).

[0094] 2. Determination of the optimal reaction temperature

[0095] Under the optimal pH conditions, the enzyme activity of AHR-Amy was measured at 40°C, 50°C, 60°C, 70°C, 80°C and 90°C, respectively. The group with the highest enzyme activity was taken as 100%, and the relative enzyme activity under other temperature conditions was calculated.

[0096] Effect of temperature on AHR-Amy enzyme activity Figure 3 As shown, with the increase of temperature, the activity of AHR-Amy showed a trend of first increasing and then decreasing, showing the best activity at a temperature of 70°C and having good enzyme activity within 60-80°C.

[0097] 3. Determination of thermal stability

[0098] Incubate the enzyme solution at 90°C, take samples at the following intervals and place them in an ice box. After all samples are taken, measure their residual activity relative to that before incubation at room temperature. The intervals are: 10min, 20min, 30min, 40min, and 50min. A linear fit is performed with Ln (relative residual activity) as the ordinate and time as the abscissa (min) to obtain the half-life curve. The slope of the straight line is the inactivation rate constant k. Using formula 2, the half-life t is calculated. 1 / 2 (90℃)

[0099] Formula 2:

[0100] The results are as follows Figure 4 As shown, the half-life of recombinant AHR-Amy at 90°C was 85.5 min, which was 5.8 times higher than that of wild-type Ba-amy (12.5 min).

[0101] 4. Determination of pH stability

[0102] The enzyme solution was incubated for 2 h under different pH conditions. After the incubation was completed, the solution was adjusted to the optimal pH and the relative residual activity of AHR-Amy was measured at the optimal pH and temperature, and the enzyme activity of the unincubated enzyme was defined as 100%.

[0103] The results are as follows Figure 5 As shown in Figure 2, the recombinant AHR-Amy has good pH stability in the pH range of 3-8, and the residual activity is greater than 60%. Its pH stability is greatly improved compared with the wild type. Figure 6 shown.

[0104] Example 6: DE value determination

[0105] Take freeze-dried pyrodextrin powder and soluble starch, dissolve them in water, and heat them in a microwave oven for 30 seconds to obtain pyrodextrin solution and soluble starch solution; take corn starch, cassava starch, and potato starch and dissolve them in water, gelatinize them in a microwave oven for 1 minute, and obtain corn starch gelatinized liquid, cassava starch gelatinized liquid, and potato starch gelatinized liquid; prepare pyrodextrin solution, soluble starch solution, corn starch gelatinized liquid, cassava starch gelatinized liquid, and potato starch gelatinized liquid as substrates, add appropriately diluted enzyme solution and react for 10 minutes under optimal reaction conditions, and determine the reducing sugar in the reaction solution by DNS method, and the percentage of reducing sugar (in terms of glucose) in the reaction solution to dry matter is the DE value. The DE values ​​of several substrates were calculated using formula 3.

[0106] Formula 3:

[0107] Where A is the absorbance of the sample; A0 is the absorbance of the blank sample; k is the slope of the standard glucose solution curve; D is the dilution factor; w t is the sample dry weight (mg)

[0108] The results are as follows Figure 7 As shown, AHR-Amy hydrolyzes cassava starch more thoroughly and hydrolyzes pyrodextrin the worst.

[0109] Example 7: Application of mutant AHR-Amy in the preparation process of resistant dextrin

[0110] Take freeze-dried pyrodextrin powder, dissolve it in water, and heat it in a microwave oven for 30s to obtain a pyrodextrin solution; dissolve corn starch in water, gelatinize it in a microwave oven for 1min, and obtain corn starch gelatinization liquid; prepare 2% (w / v) pyrodextrin solution and corn starch gelatinization liquid as substrates, and adjust the pH to 4.5 with hydrochloric acid. Add 500μL of the acid-resistant and heat-resistant α-amylase AHR-Amy prepared in Example 3 to 800μL of substrate solution, and react at 70℃ for 1h. Add 100μL of saccharifying enzyme and react at 40℃ for 1h. After the reaction is completed, place the reaction solution in a boiling water bath to inactivate the enzyme for 10min. After cooling, determine the content of rapidly digestible starch (dextrin), slowly digestible starch (dextrin) and resistant starch (dextrin) in the sample according to the following method.

[0111] The reaction solution was cooled and 200 μL of mixed enzyme (porcine pancreatic α-amylase: saccharifying enzyme = 9:1) was added. The mixture was reacted at 40°C for 2 h. The reaction solution was taken at 0 min, 20 min, and 120 min to inactivate the enzyme and its glucose content was accurately determined using the GOD-POD kit. The glucose content released in the three time periods (G0-0 min digestion, G0-10 min digestion, G0-20 min digestion, G0-30 min digestion, G0-40 min digestion, G0-50 min digestion, G0-60 min digestion, G0-70 min digestion, G0-80 min digestion, G0-90 min digestion, G0-100 min digestion, G0-110 min digestion, G0-120 min digestion, G0-120 min digestion, G0-110 min digestion, G0- 20 -The amount of glucose released within 20 minutes of digestion, G 120 -The amount of glucose released within 120 min of digestion) was used to calculate the content of resistant dextrin. The blank group was not added with AHR-Amy and saccharifying enzyme, and the control group was not added with AHR-Amy. The content of glucose released in the reaction solution was calculated using Formula 4. The content of resistant dextrin was calculated using Formula 5, Formula 6, and Formula 7.

[0112] Formula 4:

[0113] Among them, A t is the absorbance of the test solution; V t is the total volume of the test solution (mL); c is the concentration of standard glucose (mg / mL); D is the dilution factor; A s is the absorbance value of standard glucose; w t is the weight of the sample (mg).

[0114] Formula 5: Rapidly digestible starch (dextrin) RDS (%) = (G 20 -G0)*0.9

[0115] Formula 6: Slowly digestible starch (dextrin) SDS (%) = (G 120 -G 20 )*0.9

[0116] Formula 7: Resistant starch (dextrin) RS (%) = 100% - RDS (%) - SDS (%).

[0117] The results are shown in Table 1. Compared with the blank group and the control group, the final content of resistant starch (dextrin) in the experimental group added with AHR-Amy was increased, indicating that the acid-resistant and heat-resistant α-amylase can effectively assist the saccharifying enzyme to hydrolyze the digestible components in pyrodextrin and corn starch under acidic conditions, thereby helping to increase the yield of resistant dextrin, effectively simplifying the production process and reducing production costs, with good economic benefits.

[0118] Table 1 Resistant dextrin content

[0119]

[0120] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An acid-resistant and heat-resistant α-amylase, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

3.

2. A biomaterial, characterized in that: The biomaterial is any one or more of the following biomaterials: (1) an acid-resistant and heat-resistant α-amylase gene, the nucleotide sequence of which is shown in SEQ ID NO: 5; (2) a recombinant expression vector comprising the nucleotide sequence described in (1); (3) A recombinant expression bacterium containing the recombinant expression vector described in (2).

3. The biomaterial according to claim 2, characterized in that: The basic vector used in the recombinant expression vector is pET-28a; The basic expression bacteria used in the recombinant expression bacteria are Escherichia coli BL21 (DE3).

4. The method for preparing the acid-resistant and heat-resistant α-amylase according to claim 1, characterized in that: The steps include: The gene with the nucleotide sequence shown in SEQ ID NO:5 is cloned into the pET-28a expression vector, and then transformed into Escherichia coli BL21 (DE3) for induced expression, the bacterial precipitate is collected by solid-liquid separation, and the acid-resistant and heat-resistant α-amylase is obtained after purification and dialysis.

5. The method for preparing the acid-resistant and heat-resistant α-amylase according to claim 4, characterized in that: The specific operation of inducing expression is as follows: the positive transformant is inoculated into LB culture medium for cultivation. 600 When the pH value reaches 0.6-0.8, add IPTG with a final concentration of 0.01 mmol / L and induce at 18°C ​​for 20 hours; the LB culture medium contains 50 μg / mL of kanamycin; The specific operation of inoculating the positive transformant into LB culture medium for cultivation is as follows: (1) culturing the positive transformant in LB culture medium at 37° C. with shaking overnight to obtain a seed solution; (2) The seed solution was inoculated into LB medium at a ratio of 1:100 and cultured at 37°C and 200 rpm; The purification step is: collecting the supernatant after lysing the cells, passing the supernatant through an affinity chromatography column, and eluting to obtain the purified acid-resistant and heat-resistant α-amylase; The dialysis step is: dialyzing the purified enzyme solution into a storage buffer to obtain acid-resistant and heat-resistant α-amylase.

6. Use of the acid-resistant and heat-resistant α-amylase described in claim 1 in the enzymatic hydrolysis of pyrodextrin.

7. The use according to claim 6, characterized in that: The enzymatic hydrolysis of pyrodextrin comprises the following steps: adding the acid-resistant and heat-resistant α-amylase into the pyrodextrin solution for enzymatic hydrolysis, and then directly adding saccharifying enzyme for saccharification after enzymatic hydrolysis.

8. The use according to claim 7, characterized in that: The conditions of the enzymatic hydrolysis are: temperature 70±10°C, time 1±0.5h; The saccharification conditions are: temperature of 40±5°C and time of 1±0.5h.

9. The use according to claim 7, characterized in that: The conditions of the enzymatic hydrolysis are: temperature 70°C, time 1h; The saccharification conditions are: temperature of 40° C. and time of 1 h.

10. Use of the acid-resistant and heat-resistant α-amylase according to claim 1 in the process for preparing resistant dextrin, characterized in that: The resistant dextrin preparation process comprises the following steps: S1, subjecting starch to dextrinization reaction by an acid-heat method to obtain pyrodextrin; S2, adding the acid-resistant and heat-resistant α-amylase to the pyrodextrin obtained in step S1 for enzymolysis, and then directly adding saccharifying enzyme for saccharification after enzymolysis; The pH of the pyrodextrin is 4.5±0.5; The conditions of the enzymatic hydrolysis are: temperature 70±10°C, time 1±0.5h; The saccharification conditions are preferably: temperature of 40±5°C and time of 1±0.5h.

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