Novel alpha-amylase for specifically hydrolyzing gelatinized starch and application

Abstract

The invention provides a novel alpha-amylase for specifically hydrolyzing gelatinized starch. The amino acid sequence of the novel alpha-amylase is shown as SEQ ID NO.2 or SEQ ID NO.3. The invention also provides a preparation method of the novel alpha-amylase. The invention also provides a coding gene of the alpha-amylase as well as a recombinant expression vector and recombinant engineering bacteria containing the gene. Meanwhile, the invention also provides application of the novel alpha-amylase in preparation of dextrin with a low DE value. According to the invention, RoseTTAFold software is used for modeling wild type alpha-amylase, the structure of the wild type alpha-amylase is analyzed, and finally mutants S263A and I236R / S263A with better starch hydrolysis and gelatinization effects are screened by combining a mutation technology; the two optimized alpha-amylase mutants are used as novel alpha-amylase, not only can specifically hydrolyze gelatinized starch, but also have a better hydrolysis effect compared with a wild type.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a novel alpha-amylase for specifically hydrolyzing gelatinized starch and an application thereof. Background Art

[0002] The production of dextrins with different DE values ​​is achieved through the hydrolysis reaction of starch. The starch raw materials (rice, corn or wheat, etc.) are first subjected to high-temperature gelatinization treatment, that is, heating and adding water to swell the starch granules, and then α-amylase (α-amylase, EC.3.2.1.1) is added to break the starch molecular chains and form short sugar chains. Known α-amylases can cut the α-1,4-glucosidic bonds in starch and dextrin, and the hydrolysis efficiency of these two substrates is basically the same, so the known α-amylases cannot effectively distinguish between starch and dextrin. After these α-amylases hydrolyze gelatinized starch into low-DE dextrins, they will continue to hydrolyze them into ordinary dextrins, and then continue to degrade them into oligosaccharides or even a large amount of monosaccharides. In the traditional process of producing dextrin, the key is to control the degree of hydrolysis of starch by α-amylase. Different degrees of hydrolysis will obtain dextrins with different DE values.

[0003] Low DE dextrin refers to a type of maltodextrin with a DE value between 2 and 8. This type of dextrin can form a soft, stretchable, and thermoreversible gel that dissolves in the mouth and produces a fat-like effect. Therefore, it can be used to replace fat and has unique application value in food and beverages. In recent years, as people's requirements for health have become higher and higher, low DE dextrin has become more and more popular among consumers. When low DE dextrin is produced, in theory, a special α-amylase is required to efficiently hydrolyze the molecular chain of starch and only produce relatively long sugar chains; at the same time, no or less short sugar chains are produced, or the produced long sugar chains will not continue to be hydrolyzed. However, there is no enzyme that meets this requirement among the currently known α-amylases. In actual production, ordinary high-temperature α-amylase is still used. However, in order to achieve the purpose of producing more long sugar chains and less short sugar chains, the current technology is achieved by strictly controlling the reaction time (short time 5 to 10 minutes), rapidly reducing the reaction temperature (130°C to 30°C), or spraying the starch solution. Therefore, when low DE dextrin is produced, more gelatinized starch often remains unhydrolyzed. At the same time, due to the poor water solubility of low DE dextrin, it is mixed with residual starch, and the conventional filtration or centrifugation technology is less effective in separating and removing the starch. Therefore, the removal of residual starch in low DE dextrin has always been a difficult problem.

[0004] In this regard, the laboratory recombined and expressed the α-amylase (Genebank number: AJR06082) from Photobacterium gaetbulicola in Escherichia coli, and found that the α-amylase exhibited unique catalytic properties and could effectively hydrolyze gelatinized starch, but its hydrolysis efficiency would drop sharply with the decrease of the molecular weight of the sugar chain, and the hydrolysis efficiency of low DE dextrin (DE value 5) was only 5.7% of gelatinized starch. In other words, the α-amylase can effectively distinguish between starch and dextrin, effectively hydrolyze gelatinized starch, but only weakly hydrolyze low DE dextrin. Therefore, the α-amylase can be used to prepare low DE dextrin. However, in actual research, it was found that the specific enzyme activity of the above α-amylase in hydrolyzing gelatinized wheat starch was only 63.2±1.4U / mg. If it is used for the actual preparation of low DE dextrin, the preparation efficiency is slightly low.

[0005] Based on this, an attempt can be made to optimize the structure of the above-mentioned α-amylase (Genebank No.: AJR06082) to obtain a new α-amylase that can more efficiently and specifically hydrolyze gelatinized starch. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a new type of α-amylase and its application which can more efficiently and specifically hydrolyze gelatinized starch.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A novel alpha-amylase for specifically hydrolyzing gelatinized starch, the amino acid sequence of which is shown as SEQ ID NO.2 or SEQ ID NO.3.

[0009] As one of the preferred embodiments of the present invention, the amino acid sequence of the wild-type α-amylase shown in SEQ ID NO.1 is optimized and improved to obtain the amylase.

[0010] As one of the preferred embodiments of the present invention, the structural optimization and improvement is achieved by mutation.

[0011] As one of the preferred embodiments of the present invention, the SEQ ID NO.2 sequence corresponds to the single point mutant S263A: the serine at position 263 of the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine; the SEQ ID NO.3 sequence corresponds to the double point mutant I236R / S263A: the isoleucine at position 236 of the amino acid sequence shown in SEQ ID NO.1 is mutated to arginine, and the serine at position 263 is mutated to alanine.

[0012] A gene encoding the above-mentioned novel α-amylase, whose nucleotide sequence is shown in SEQ ID NO.4 or SEQ ID NO.5; wherein, the SEQ ID NO.4 sequence is used to encode the α-amylase corresponding to SEQ ID NO.2, and the SEQ ID NO.5 sequence is used to encode the α-amylase corresponding to SEQ ID NO.3.

[0013] A recombinant expression vector containing the above encoding gene.

[0014] As one of the preferred embodiments of the present invention, the expression vector is pET-28a.

[0015] A recombinant engineering bacterium containing the above encoding gene.

[0016] As one of the preferred embodiments of the present invention, the host bacteria is Escherichia coli BL21.

[0017] An application of the novel α-amylase in the preparation of low DE value dextrin.

[0018] As one of the preferred methods of the present invention, when using the novel α-amylase to prepare low DE dextrin: using wheat starch with a concentration of 100 g / L as a raw material, adjusting the pH to 8.5, heating with boiling water for 15 minutes, adding α-amylase at a ratio of 100 U / kg dry starch, and hydrolyzing at 25°C for 15 minutes; heating at 60°C for 10 minutes to inactivate the enzyme, and then cooling the sample to room temperature, adjusting the pH to neutral; centrifuging for 20 minutes, taking the supernatant, and freeze-drying, the prepared powder is a low DE dextrin with a DE value of 2 to 5.

[0019] The advantages of the present invention compared to the prior art are:

[0020] (1) In the early stage of the present invention, α-amylase (Genebank: AJR06082) was discovered, and the protein was modeled using RoseTTAFold software, and molecular docking was performed with β-cyclodextrin after modeling; according to the molecular docking results, the key amino acids were mutated, and the molecular docking results were verified by mutation, and α-amylase mutants with better hydrolysis effect on gelatinized starch were screened, among which the specific enzyme activity of mutant S263A on gelatinized wheat starch was 19 times that of its specific enzyme activity on low DE value dextrin, and the specific enzyme activity on gelatinized wheat starch was 2.1 times higher than that of the wild type; the specific enzyme activity of mutant I236R / S263A on gelatinized wheat starch was 20 times that of its specific enzyme activity on low DE value dextrin, and the specific enzyme activity on gelatinized wheat starch was 2.9 times higher than that of the wild type;

[0021] (2) The α-amylase mutant of the present invention can effectively prepare low DE value dextrin (DE value is 2-5), and the optimal conditions are: gelatinized wheat starch concentration of 100 g / L, enzyme amount of 100 U / kg dry starch, pH 8.5, reaction temperature of 25°C, and reaction time of 15 min. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a structural diagram of the recombinant plasmid vector pET-28a-AJR06082 in Example 1;

[0023] Figure 2 It is the SDS-PAGE picture of the wild-type α-amylase and the pure enzymes of mutants S263A and I236R / S263A in Example 3 (in the picture, "M" lane: Maker; "wild-type" lane: purified wild-type α-amylase; "S263A" lane: purified α-amylase S263A; "I236R / S263A" lane: purified α-amylase I236R / S263A);

[0024] Figure 3 is the optimum temperature curve of the wild-type α-amylase and the mutants S263A and I236R / S263A pure enzymes in Example 3;

[0025] Figure 4 This is the optimum pH curve of the wild-type α-amylase and the mutants S263A and I236R / S263A pure enzymes in Example 3. DETAILED DESCRIPTION

[0026] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, unless otherwise specified, the carriers, reagents, culture media, methods and equipment used in the present invention are all conventional carriers, reagents, culture media, methods and equipment in the art, and will not be repeated.

[0027] Example 1, Construction of wild-type α-amylase engineering bacteria:

[0028] The laboratory discovered α-amylase (Genebank number AJR06082) from Photobacterium gaetbulicola in the early stage, and commissioned Sangon Biotech (Shanghai, China) to optimize and synthesize the α-amylase gene according to the codon preference of Escherichia coli (the nucleotide sequence of the synthesized α-amylase is shown in SEQ ID NO.6, and the corresponding encoded amino acid sequence is shown in SEQ ID NO.1). Then, the target gene fragment (SEQ ID NO.6) was inserted into the BamHI (GGATCC)-XhoI (CTCGAG) sites of the pET-28a vector to construct the recombinant plasmid vector pET-28a-AJR06082 (as shown in Figure 1 Finally, the recombinant plasmid vector was transformed into Escherichia coli BL21 (DE3) to obtain wild-type α-amylase engineering bacteria.

[0029] Example 2, expression and protein purification of α-amylase:

[0030] The target amylase engineered bacteria strain was inoculated into a test tube containing 5 mL LB, cultured overnight in a shaker at 37°C and 200 rpm, and then transferred to a medium containing 400 mL LB and cultured until OD 600 When the pH reaches 0.6-0.8, 0.1 mM IPTG is added to induce overnight at 16°C and 120 rpm. Then centrifuge at 4°C and 4000 rpm for 30 min to collect the bacteria; after resuspending the bacteria, the induced bacteria are broken by ultrasound. Centrifuge at 4°C and 12000 rpm for 30 min to obtain the supernatant and precipitate after centrifugation respectively; the cell-breaking supernatant is purified by nickel column affinity chromatography to obtain the purified α-amylase, and then tested by SDS-PAGE.

[0031] Example 3, screening and determination of α-amylase mutants:

[0032] (1) Screening of mutation sites

[0033] The wild-type α-amylase protein (amino acid sequence SEQ ID NO.1) was modeled using RoseTTAFold software, and molecular docking was performed with β-cyclodextrin after modeling, and sites with high free energy, i.e., large flexibility (large flexibility is more likely to improve catalytic efficiency) were selected from the results. Corresponding primers were designed according to the selected different sites, and the recombinant plasmid vector pET-28a-AJR06082 prepared in Example 1 was used as a template to construct mutants by whole plasmid PCR to obtain different mutants.

[0034] (2) Single point mutation

[0035] Using wild-type α-amylase as a template, primers for introducing mutation sites were designed and synthesized to perform site-directed mutagenesis on the α-amylase gene. The PCR product was verified by agarose gel electrophoresis, digested with DpnI, introduced into Escherichia coli BL21 (DE3), and plated on LB plates containing kanamycin to obtain a single clone.

[0036] Through site-directed mutagenesis, single-point mutation transformants of 9 sites, D212N, S231A, I236R, E239R, D243C, T262A, S263A, R302A, and D311R, were obtained (the mutation primers are shown in Table 1). Among them, I236R refers to the mutation of isoleucine at position 236 of the amino acid sequence shown in SEQ ID NO.1 to arginine, S263A refers to the single-point mutation of serine at position 263 of the amino acid sequence shown in SEQ ID NO.1 to alanine, and so on.

[0037] Table 1 Primers for different single point mutations

[0038]

[0039]

[0040] Referring to the method in Example 2, each single point mutant was expressed and purified, and then the enzyme activity was detected. With gelatinized starch as substrate, the total reaction system was 900 μL, including 600 μL substrate and enzyme. The detection method was: pH 8.0 buffer (50mMNa2HPO4-50MmKH2PO4) was incubated for 10 minutes, 300 μL DNS was added, centrifuged, boiled for 10 minutes, and then cooled and the OD was measured by UV spectrophotometer. 540 The standard curve was determined using glucose as the standard. One standard enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute. The specific enzyme activity results of different single-point mutations are shown in Table 2.

[0041] Table 2 Results of enzyme activity comparison of different single point mutations

[0042]

[0043]

[0044] According to the enzyme activity results, the enzyme activities of mutants I236R and S263A were improved most significantly, and mutant S263A (amino acid sequence as shown in SEQ ID NO.2) was the best.

[0045] (3) Combination of double mutations

[0046] According to the single-point mutation results, the plasmid containing the single-point mutant S263A was further used as a template to perform site-directed mutagenesis of the I236R position by whole-plasmid amplification (the steps were the same as the single-point mutation) to obtain the combined double mutation transformant I236R / S263A (amino acid sequence as shown in SEQ ID NO.3), and the I236R / S263A protein was expressed and purified according to the method in Example 2.

[0047] The enzymatic properties of wild-type α-amylase, single mutant S263A, and double mutant I236R / S263A were tested respectively (SDS-PAGE of purified proteins of wild-type α-amylase, single mutant S263A, and double mutant I236R / S263A is shown in Figure 2). Figure 2 , reaching the purity detected by enzymatic properties).

[0048] ①Optimum temperature and optimum pH

[0049] Referring to the above enzyme activity detection method, samples were taken after incubation at 20-45°C and pH 8.0 (50 mM Na2HPO4-KH2PO4) to determine the activities of wild-type α-amylase and mutants S263A and I236R / S263A. The results are shown in Figure 3 As shown, the optimum temperature of the wild-type α-amylase and mutants S263A and I236R / S263A was 25°C.

[0050] At 25°C, the optimum pH was determined in the range of pH 6.0 to 9.0 according to the above enzyme activity detection method. Figure 4 As shown, the optimum pH of the wild type is 8.0; more than 50% of the activity is retained in the pH range of 6.0-9.0; the optimum pH of mutants S263A and I236R / S263A is 8.5.

[0051] ② Ability to degrade different substrates

[0052] Referring to the aforementioned enzyme activity detection method, gelatinized wheat starch, raw wheat starch, dextrin and low DE value dextrin were used as substrates to determine the hydrolysis ability of wild-type α-amylase and mutants S263A and I236R / S263A. The results are shown in Table 3.

[0053] Table 3 Hydrolysis ability of wild-type α-amylase and mutants S263A and I236R / S263A

[0054]

[0055] As shown in Table 3, the hydrolysis ability of mutants S263A and I236R / S263A on gelatinized wheat starch increased by 2.1 times and 2.9 times respectively; at the same time, the two mutants did not change the hydrolysis preference for gelatinized starch and low DE value dextrin. When the activity of the wild-type enzyme in hydrolyzing gelatinized starch was defined as 100%, its activity in hydrolyzing low DE value dextrin was only 5.7%; the activity of mutant S263A in hydrolyzing low DE value dextrin was 5.2% of its activity in hydrolyzing gelatinized starch; the activity of double mutant I236R / S263A in hydrolyzing low DE value dextrin was 5.0% of its activity in hydrolyzing gelatinized starch.

[0056] In summary, both α-amylase mutants S263A and I236R / S263A can be excellent enzymes for producing low DE dextrins, but the double mutant I236R / S263A is the best.

[0057] Example 4: Preparation of low DE value dextrin by the α-amylase mutant of the present invention:

[0058] 1. Preparation of low DE value dextrin

[0059] Add pure water to 10g of raw wheat starch to adjust the dry matter concentration to 100g / L, adjust the pH to 8.5, and heat in boiling water for 15min to make the raw starch particles absorb water, swell and gelatinize to obtain starch slurry. Add the α-amylase of the present invention (taking the double mutant I236A / S263R as an example) to the prepared starch slurry, the enzyme addition amount is 100U / kg dry starch, and heat (60℃, 10min) after hydrolysis at 25℃ for 15min to inactivate the enzyme. After cooling the hydrolyzed sample to room temperature, adjust the pH to neutral, and then centrifuge the hydrolyzed sample at 6000×g for 20min, take the supernatant and put it into a freeze dryer for freeze drying until the sample becomes powder.

[0060] 2. DE value determination

[0061] Determination of DE of hydrolyzed sample: weigh 1.0g of powder prepared from hydrolyzate into a conical flask, add 24mL ultrapure water, and record the actual weight of the hydrolyzed sample. Add 10mL Fehling's reagent A solution and 10mL Fehling's reagent B solution into the conical flask. The subsequent titration steps are the same as above. After the titration is completed, record the volume of sodium thiosulfate consumed, which is the titration value of the sample.

[0062] Calculation method:

[0063]

[0064] T wb : The volume of sodium thiosulfate consumed by the water blank, in mL; T s : The volume of sodium thiosulfate consumed by the sample, in mL; T dex: Volume of sodium thiosulfate consumed by 1% glucose, unit: m; W: weight of sample, unit: g; %DS: dry matter content of sample, unit: %.

[0065] The DE value of the dextrin finally prepared in this example is 2-5, which is confirmed to be a low DE value dextrin.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A novel α-amylase for specifically hydrolyzing gelatinized starch, characterized in that: Its amino acid sequence is shown in SEQ ID NO.2 or SEQ ID NO.

3.

2. The novel α-amylase for specifically hydrolyzing gelatinized starch according to claim 1, characterized in that: The amino acid sequence is obtained by optimizing and improving the wild-type α-amylase structure as shown in SEQ ID NO.

1.

3. The novel α-amylase for specifically hydrolyzing gelatinized starch according to claim 2, characterized in that: The structural optimization and improvement is achieved through mutation.

4. The novel α-amylase for specifically hydrolyzing gelatinized starch according to claim 3, characterized in that: The SEQ ID NO.2 sequence corresponds to the single point mutant S263A: the serine at position 263 of the amino acid sequence shown in SEQ ID NO.1 is mutated to alanine; the SEQ ID NO.3 sequence corresponds to the double point mutant I236R / S263A: the isoleucine at position 236 of the amino acid sequence shown in SEQ ID NO.1 is mutated to arginine, and the serine at position 263 is mutated to alanine.

5. A gene encoding a novel α-amylase according to any one of claims 1 to 4, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.4 or SEQ ID NO.5; wherein, the SEQ ID NO.4 sequence is used to encode the α-amylase corresponding to SEQ ID NO.2, and the SEQ ID NO.5 sequence is used to encode the α-amylase corresponding to SEQ ID NO.

3.

6. A recombinant expression vector containing the coding gene according to claim 5.

7. The recombinant expression vector according to claim 6, characterized in that The expression vector is pET-28a.

8. A recombinant engineered bacterium containing the encoding gene according to claim 5.

9. The recombinant engineered bacterium according to claim 8, characterized in that The host bacteria was Escherichia coli BL21.

10. Use of the novel α-amylase according to any one of claims 1 to 4 in the preparation of low DE value dextrin.

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