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

Abstract

The application discloses an acid and heat resistant alpha-amylase as well as preparation and application thereof. The acid and heat resistant alpha-amylase is obtained by fusing 37 amino acids at an N terminal of alpha-amylase Gt-amy to wild-type alpha-amylase Ba-amy, deleting amino acids at positions 178 and 179, and mutating valine at position 332 into aspartic acid, and has a specific enzyme activity of 6734 U / mg, which is 13 times of that of the wild-type enzyme; the optimal pH is 5.5, which is 0.5 units lower than that of the wild-type enzyme, and the enzyme activity still remains 60% at pH 4.5; the thermal stability is improved, and the half-life at 90 DEG C is 5.8 times higher than that of the wild-type enzyme. The pyrodextrin hydrolysis result shows that the acid and heat resistant alpha-amylase can effectively assist the enzymatic hydrolysis of saccharifying enzyme in an acid environment, avoids repeated adjustment of pH in the reaction process, can improve the yield of resistant dextrin, and simplifies the process flow and reduces the production cost, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an acid- and heat-resistant α-amylase and its preparation and application. Background Technology

[0002] Starch is a high-molecular-weight carbohydrate, its basic building block being glucose. Starch is widely found in plants and is their primary form of energy storage. It is also a crucial component of the human diet, as it can be hydrolyzed into glucose by amylases and absorbed and utilized by the body.

[0003] α-Amylase, also known as 1,4-α-D-glucan hydrolase (EC 3.2.1.1), is one of the oldest industrial enzymes. It cleaves the α-1,4 glycosidic bonds within the molecule, hydrolyzing large starch molecules into smaller products such as dextrin, maltose, and glucose. It is a very important starch hydrolase. α-Amylase is one of the most competitive players in the enzyme market, widely used in various industrial applications based on starch hydrolysis, such as food, detergents, agriculture, papermaking, textiles, pharmaceuticals, brewing, and fermentation. Furthermore, it has potential applications, such as the treatment of fermentation wastewater in alcohol plants and the preparation of resistant dextrin.

[0004] Resistant dextrin is an important low-molecular-weight water-soluble dietary fiber in the modern food industry. It has attracted much attention due to its anti-digestive properties and has broad application prospects in beverages, meat products, flour products, and functional foods. Currently, the production methods for resistant dextrin mainly consist of two steps: acid-heat preparation of pyrodextrin and enzymatic hydrolysis of pyrodextrin. The pyrodextrin solution prepared by the acid-heat method has a low pH, and the optimal pH for commercially available saccharifying enzymes is also acidic. However, the types of available acid-resistant α-amylases are limited and expensive. Therefore, existing pyrodextrin enzymatic hydrolysis processes often require first adjusting the pH of the pyrodextrin to neutral for hydrolysis with α-amylase, and then adjusting the pH to acidic for hydrolysis with saccharifying enzyme. This increases production costs, the ash content of the product, and the risk of environmental pollution. Therefore, there is an urgent need for α-amylases with good acid and heat resistance in the preparation process of resistant dextrin. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of existing technologies, the primary objective of this invention is to provide an acid- and heat-resistant α-amylase. This invention, through molecular modification of the wild-type α-amylase Ba-amy, obtains a superior mutant α-amylase with high catalytic activity and excellent acid and heat resistance, exhibiting a specific enzyme activity as high as 6734 U / mg; its optimal pH is 5.5, and it retains 60% of its enzyme activity at pH 4.5; its half-life at 90°C is 85.5 min, and its thermal stability is 5.8 times higher than that of the wild type. This acid- and heat-resistant α-amylase can be effectively used in the preparation process of resistant dextrin, simplifying the process and reducing production costs.

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

[0007] The objective of this invention is achieved through the following technical solution:

[0008] An acid- and heat-resistant α-amylase, the amino acid sequence of which is shown in SEQ ID NO:3.

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

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

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

[0012] According to a preferred embodiment of the present invention, the recombinant expression vector uses pET-28a as the base vector.

[0013] A recombinant expression bacterium containing the aforementioned recombinant expression vector.

[0014] According to a preferred embodiment of the present invention, the recombinant expression bacterium used as the basic expression bacterium is Escherichia coli BL21(DE3).

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

[0016] According to a preferred embodiment of the present invention, the specific operation of the induced expression is as follows: the positive transformant is inoculated into LB culture medium for culture, and when the bacterial culture OD... 600 When the concentration reaches 0.6–0.8, IPTG with a final concentration of 0.01 mmol / L is added, and the mixture is induced at 18°C ​​for 20 h. The LB culture medium contains 50 μg / mL kanamycin.

[0017] According to a preferred embodiment of the present invention, the specific operation of inoculating the positive transformant into LB culture medium for culture is as follows:

[0018] (1) The positive transformant was cultured in LB medium at 37°C overnight with shaking to obtain seed culture;

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

[0020] The preferred purification step is as follows: after lysing the cells, collect the supernatant, pass the supernatant through an affinity chromatography column, and elute to obtain the purified acid- and heat-resistant α-amylase.

[0021] The preferred method for lysing cells is to use ultrasonic disruption in an ice bath to lyse the cells.

[0022] In the ultrasonic disruption method, it is preferable to resuspend the bacterial cells in buffer A before proceeding; the buffer A is formulated with 10 mmol / L acetate-sodium acetate, 300 mmol / L NaCl, and pH = 6.0.

[0023] The preferred dosage of buffer A is 10 mL of buffer A per gram of bacterial cells.

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

[0025] The preferred method for collecting the supernatant after cell lysis is as follows: after freezing and centrifuging the lysed cells, discard the precipitate, pass the supernatant through a membrane to obtain the supernatant described above.

[0026] The preferred conditions for the refrigerated centrifugation are centrifugation at 12000g for 30 minutes at 4°C.

[0027] The preferred method for filtration is using a 0.22μm microporous membrane.

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

[0029] The preferred elution method is to first equilibrate with buffer A, and then elute with buffer B; the buffer A is formulated as 10 mmol / L acetate-sodium acetate, 300 mmol / L NaCl, pH=6.0; the buffer B is formulated as 500 mmol / L imidazole, 10 mmol / L acetate-sodium acetate and 300 mmol / L NaCl, pH=6.0.

[0030] The preferred step of the dialysis process is to dialyze the purified enzyme solution into a storage buffer to obtain acid- and heat-resistant α-amylase.

[0031] The preferred formulation of the storage buffer solution is: 10 mmol / L acetate-sodium acetate, pH = 6.0.

[0032] The preferred dialysis conditions are 4°C, magnetic stirring speed of 100 r / min, and dialysis for 24 h. Preferably, the storage buffer solution 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] The application of the above-mentioned acid- and heat-resistant α-amylase in the enzymatic hydrolysis of pyrodextrin.

[0035] According to a preferred embodiment of the present invention, the enzymatic hydrolysis of pyrodextrin includes the following steps: adding the acid- and heat-resistant α-amylase to the pyrodextrin solution for enzymatic hydrolysis, and then directly adding a saccharifying enzyme for saccharification after enzymatic hydrolysis.

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

[0037] The preferred saccharification conditions are: temperature 40±5℃, time 1±0.5h; or temperature 40℃, time 1h.

[0038] The application of the above-mentioned acid- and heat-resistant α-amylase in the preparation process of resistant dextrin.

[0039] According to a preferred embodiment of the present invention, the resistant dextrin preparation process includes the following steps:

[0040] S1. Starch is subjected to a dextrinization reaction using an acid-heat method to obtain pyrodextrin;

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

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

[0043] The conditions for enzymatic hydrolysis are: temperature 70±10℃, time 1±0.5h;

[0044] The preferred conditions for saccharification are: a temperature of 40±5℃ and a time of 1±0.5h.

[0045] The present invention has the following advantages and effects compared with the prior art:

[0046] 1. Based on wild-type α-amylase, this invention has obtained an acid- and heat-resistant α-amylase mutant through molecular modification, with a specific enzyme activity as high as 6734 U / mg; the optimal pH is 5.5, and it still retains 60% of the enzyme activity at pH 4.5; the half-life at 90℃ is 85.5 min, and the thermal stability is improved by 5.8 times compared with the wild type.

[0047] 2. Compared with existing resistant dextrin preparation processes, the acid- and heat-resistant α-amylase provided by this invention can work synergistically with saccharifying enzymes under acidic conditions to avoid repeated pH adjustments, which not only simplifies the process but also reduces production costs. Attached Figure Description

[0048] Figure 1 The image shows an SDS-PAGE of AHR-Amy; lane M is the protein marker, and lane 1 is the concentrated enzyme solution after purification of AHR-Amy.

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

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

[0051] Figure 4 The graph shows the half-life of AHR-Amy at 90℃.

[0052] Figure 5 This is a pH stability graph for AHR-Amy.

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

[0054] Figure 7 The graph shows the results of DE value determination for different substrate solutions. Detailed Implementation

[0055] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments are used only for illustration and not for limiting the scope of the invention.

[0056] Unless otherwise specified, all reagents and raw materials used in this 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 was obtained by freeze-drying corn starch after undergoing a dextrinization reaction using an acid-heat method.

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

[0059] (1) Based on the amino acid sequence SEQ ID NO:1 of wild-type α-amylase Ba-amy, and compared with the amino acid sequence SEQ ID NO:2 of α-amylase Gt-amy, a 37-amino acid fragment at the N-terminus was obtained. DNA molecules encoding the N-terminal fragment were artificially synthesized using splice PCR. Simultaneously, Guangzhou Aiji Biotechnology Co., Ltd. was commissioned to design the nucleotide sequence SEQ ID NO:4 based on the amino acid sequence of wild-type α-amylase Ba-amy. The vector Ba-amy was constructed using the basic plasmid pET28a, with restriction enzyme sites at the 5' end (BamHI) and the 3' end (XhoI). The vector Ba-amy was then linearized using primers. Finally, the linearized vector Ba-amy and the N-terminal fragment PCR product were purified and recovered separately for homologous recombination.

[0060] The primer sequences for chimeric amplification are as follows:

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

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

[0063] The vector linearization primer sequences are as follows:

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

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

[0066] Using synthetically synthesized DNA molecules encoding a 37-amino acid N-terminus fragment and the Ba-amy vector as templates, PCR amplification was performed with 25 μL of 2×PfuMax HiFi PCR ProMix (Guangzhou Yingzan Biotechnology Co., Ltd., catalog number P217A), 1 μL each of 10 μmol / L forward and reverse primers, and an appropriate amount of sterile water. The amplification conditions were: 98℃ for 30 s; 98℃ for 10 s, 60℃ for 30 s, 72℃ for 30 s, for a total of 30 cycles; 72℃ for 5 min. The amplified products were recovered by agarose gel electrophoresis, and then homologous recombination was performed. The reaction mixture consisted of 5 μL of 2×Hipro DNA Assembly Mix (Guangzhou Yingzan Biotechnology Co., Ltd., K001A), an appropriate amount of recovered product (base count × 0.02 ng), and water added to a final volume of 10 μL. The mixture was incubated at 50℃ for 15 min. The product was transformed into DH 5α competent cells, single colonies were picked for colony PCR identification, and positive monoclonal bacteria were sent to a sequencing company for sequencing verification. Correct competent cells were cultured and plasmids were extracted.

[0067] (2) Using the plasmids that have been verified to be correctly ligated as templates, site-directed mutagenesis was performed. The following four primers were designed:

[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 two amino acids 178 and 179, and FP3 / RP3 was used to mutate V332 to D332). The amplification conditions were: 98℃ for 30s; 98℃ for 10s, 60℃ for 30s, 72℃ for 30s, for a total of 30 cycles; 72℃ 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. Competent cells that were verified to be correct were cultured, and plasmids were extracted. The obtained plasmid is the 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: Induced expression of mutant AHR-Amy

[0075] The recombinant plasmid constructed in Example 1 was transformed into E. coli BL21(DE3) host cells. Single colonies were picked and cultured in 5 mL of LB medium containing 50 μg / mL kanamycin at 37°C overnight with shaking. The seed culture from the overnight culture was then inoculated at a 1:100 ratio into 200 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking until OD (dose elapsed). 600 The concentration of the sample was 0.6-0.8. IPTG was added to a final concentration of 0.01 mmol / L. The sample was induced at 18℃ for 20 h. The cells were collected by centrifugation and weighed. The wet weight of the cells was recorded.

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

[0077] 1. Recombinant bacterial cells were disrupted by ultrasonication.

[0078] Take the induced expression cells frozen at -20℃ and resuspend them in 10 mL of lysis buffer (10 mmol / L acetate-sodium acetate, 300 mmol / L NaCl, pH = 6.0) per gram of cell wet weight, according to the data recorded in Example 3. Lyse the cells using an ultrasonic cell disruptor under the following conditions: 150 W power, 3.0 s sonication, 3.0 s interval, for 20 min. Centrifuge the lysed cells at 12000 g for 30 min at 4℃ for subsequent purification, and filter through a 0.22 μm microporous membrane.

[0079] 2. Nickel ion affinity chromatography purification

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

[0081] HisTrap TM HP 5 mL was connected to the column valve of the rapid protein purification instrument. The system and column were then washed with ultrapure water, and the column was equilibrated with buffer A. The sample to be purified was then loaded into the chromatography column using a sample pump. After loading, the column was washed with buffer A, followed by gradient elution with elution buffer B. The eluted fractions 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 overnight into a storage buffer (10 mmol / L acetate-sodium acetate, pH=6.0) for later use.

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

[0085] The mutant AHR-Amy prepared in Example 3 was used to determine enzyme activity according to the following method. A 0.5% (w / v) potato amyl starch substrate solution was prepared using 10 mmol / L acetate-sodium acetate buffer (pH = 6.0). 450 μL of substrate and 50 μL of enzyme solution were added to a 1.5 mL centrifuge tube, and the reaction was carried out at 70 °C for 10 min. Immediately after the reaction, 500 μL of DNS reagent was added. The mixture was then incubated in a boiling water bath for 5 min, and the absorbance was measured at 540 nm after cooling. The buffer solution was used as a blank control. Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of reducing sugar per minute under conditions of pH = 6 and 70 °C. One unit of enzyme activity (U) is defined as this amount of enzyme.

[0086] Enzyme activity was calculated according to Formula 1, and each data point was tested in triplicate.

[0087] Formula 1:

[0088] Where A is the absorbance value of the sample; A0 is the absorbance value of the blank; K is the slope of the standard curve; t is the reaction time; n is the dilution factor; and 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 the enzymatic properties of the mutant AHR-Amy

[0091] 1. Determination of the optimal reaction pH

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

[0093] The effect of pH on AHR-Amy enzyme activity, such as Figure 2 As shown, AHR-Amy exhibits high enzyme activity in the pH range of 4.5-7. The activity initially increases and then decreases with increasing pH, with the best activity observed 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 optimal pH conditions, the enzyme activity of AHR-Amy was measured at 40℃, 50℃, 60℃, 70℃, 80℃ and 90℃ respectively. The group with the highest enzyme activity was taken as 100%, and the relative enzyme activity under other temperature conditions was calculated.

[0096] The effect of temperature on AHR-Amy enzyme activity, such as Figure 3 As shown, the activity of AHR-Amy first increases and then decreases with increasing temperature, exhibiting the best activity at 70℃ and good enzyme activity in the range of 60-80℃.

[0097] 3. Determination of thermal stability

[0098] The enzyme solution was incubated at 90℃. Samples were taken at the following time intervals and placed in an ice box. After all samples were collected, the residual activity relative to the unincubated state was measured at room temperature. The time intervals were 10 min, 20 min, 30 min, 40 min, and 50 min. A linear fit was 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 line is the inactivation rate constant k. The half-life t was calculated using Equation 2. 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℃ is 85.5 min, which is 5.8 times longer than that of wild-type Ba-amy (12.5 min).

[0101] 4. Determination of pH stability

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

[0103] The results are as follows Figure 5 As shown, recombinant AHR-Amy exhibits good pH stability within a pH range of 3-8, with residual activity exceeding 60%, representing a significant improvement in pH stability compared to the wild-type. A comparison of pH stability with wild-type Ba-amy is shown below. Figure 6 As shown.

[0104] Example 6: DE value determination

[0105] Lyophilized pyrodextrin powder and soluble starch were dissolved in water and heated in a microwave oven for 30 seconds to obtain pyrodextrin solution and soluble starch solution. Corn starch, tapioca starch, and potato starch were dissolved in water and gelatinized in a microwave oven for 1 minute to obtain corn starch gelatinized solution, tapioca starch gelatinized solution, and potato starch gelatinized solution, respectively. Pyrodextrin solution, soluble starch solution, corn starch gelatinized solution, tapioca starch gelatinized solution, and potato starch gelatinized solution were prepared as substrates. Appropriately diluted enzyme solution was added, and the reaction was carried out under optimal reaction conditions for 10 minutes. The reducing sugar content in the reaction solution was determined using the DNS method. The DE value was defined as the percentage of reducing sugar (calculated as glucose) in the dry matter of the reaction solution. 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 The dry weight of the sample is (mg).

[0108] The results are as follows Figure 7 As shown, AHR-Amy hydrolyzes tapioca starch more thoroughly and dextrin less effectively.

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

[0110] Lyophilized pyrodextrin powder was dissolved in water and heated in a microwave oven for 30 seconds to obtain a pyrodextrin solution. Corn starch was dissolved in water and gelatinized in a microwave oven for 1 minute to obtain a corn starch gelatinized solution. 2% (w / v) pyrodextrin solution and corn starch gelatinized solution were prepared as substrates, and the pH was adjusted to 4.5 with hydrochloric acid. 500 μL of the acid- and heat-resistant α-amylase AHR-Amy prepared in Example 3 was added to 800 μL of the substrate solution, and the reaction was carried out at 70°C for 1 hour. Then, 100 μL of saccharifying enzyme was added, and the reaction was carried out at 40°C for 1 hour. After the reaction, the reaction solution was placed in a boiling water bath for 10 minutes to inactivate the enzymes. After cooling, the contents of rapidly digestible starch (dextrin), slowly digestible starch (dextrin), and resistant starch (dextrin) in the sample were determined according to the following method.

[0111] After cooling, 200 μL of a mixed enzyme (porcine pancreatic α-amylase: glucoamylase = 9:1) was added to the reaction solution, and the mixture was reacted at 40 °C for 2 h. The enzyme was inactivated at 0 min, 20 min, and 120 min, and the glucose content was accurately determined using a GOD-POD kit. The glucose content released at these three time points (G0 - free glucose content at 0 min of digestion, G...) was used as the statistical unit. 20 - The amount of glucose released within 20 minutes of digestion, G 120 The content of resistant dextrin was calculated by measuring the amount of glucose released within 120 minutes of digestion. A blank group was used without the addition of AHR-Amy and saccharifying enzyme, and a control group was used without the addition of AHR-Amy. The glucose content released in the reaction solution was calculated using Formula 4. The content of resistant dextrin was calculated using Formulas 5, 6, and 7.

[0112] Formula 4:

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

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

[0115] Formula 6: Slowly digested 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 with added AHR-Amy was increased. This indicates that acid- and heat-resistant α-amylase can effectively assist saccharifying enzymes in hydrolyzing the digestible components in caramelized dextrin and corn starch under acidic conditions, thereby improving the yield of resistant dextrin, effectively simplifying the production process and reducing production costs, and has good economic benefits.

[0118] Table 1. Content of resistant dextrin

[0119]

[0120] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An acid- 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) The acid- and heat-resistant α-amylase gene, the nucleotide sequence of which is shown in SEQ ID NO:5; (2) A recombinant expression vector containing the nucleotide sequence described in (1); (3) Recombinant expression bacteria containing the recombinant expression vector described in (2).

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

4. The method for preparing acid- and heat-resistant α-amylase as described in claim 1, characterized in that: Includes the following steps: The gene with the nucleotide sequence shown in SEQ ID NO:5 was cloned into the pET-28a expression vector, then transformed into Escherichia coli BL21(DE3) for induced expression. The bacterial precipitate was collected by solid-liquid separation, purified and dialyzed to obtain the acid- and heat-resistant α-amylase.

5. The method for preparing acid- and heat-resistant α-amylase according to claim 4, characterized in that: The specific procedure for inducing expression is as follows: positive transformants are inoculated into LB culture medium and cultured until the bacterial culture OD... 600 When the concentration reaches 0.6–0.8, IPTG is added to a final concentration of 0.01 mmol / L, and the mixture is induced at 18 °C for 20 h; the LB culture medium contains 50 μg / mL kanamycin. The specific procedure for inoculating positive transformants into LB medium for culture is as follows: (1) The positive transformant was cultured in LB medium at 37 °C overnight with shaking to obtain seed culture; (2) The seed culture was inoculated into LB medium at a ratio of 1:100 and cultured at 37 °C and 200 r / min. The purification steps are as follows: after lysing the cells, the supernatant is collected, the supernatant is passed through an affinity chromatography column, and after elution, the purified acid- and heat-resistant α-amylase is obtained; The dialysis steps are as follows: the purified enzyme solution is dialyzed into a storage buffer to obtain acid- and heat-resistant α-amylase.

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

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

8. The application according to claim 7, characterized in that: The conditions for the enzymatic hydrolysis are: temperature 70±10℃, time 1±0.5 h; The saccharification conditions are: temperature 40±5℃, time 1±0.5 h.

9. The application according to claim 7, characterized in that: The conditions for the enzymatic hydrolysis are: temperature 70℃ and time 1 h; The conditions for saccharification are: temperature 40℃ and time 1 h.

10. The application of the acid- and heat-resistant α-amylase as described in claim 1 in the preparation process of resistant dextrin, characterized in that: The process for preparing resistant dextrin includes the following steps: S1. Starch is subjected to a dextrinization reaction using an acid-heat method to obtain pyrodextrin; S2. Add the acid- and heat-resistant α-amylase to the caramelized dextrin obtained in step S1 for enzymatic hydrolysis, and then directly add saccharifying enzyme for saccharification. The pH of the pyrodextrin is 4.5 ± 0.5; The conditions for the enzymatic hydrolysis are: temperature 70±10℃, time 1±0.5 h; The saccharification conditions are: temperature 40±5℃, time 1±0.5 h.

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