A novel alpha-amylase specifically hydrolyzing gelatinized starch and use thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a novel α-amylase that specifically hydrolyzes gelatinized starch and its applications. Background Technology

[0002] The production of dextrins with different DE values ​​is achieved through the hydrolysis of starch. Starch raw materials (such as rice, corn, or wheat) are first gelatinized at high temperatures, i.e., heated and hydrated, causing the starch granules to swell. Then, α-amylase (EC.3.2.1.1) is added, causing the starch molecular chains to break down into shorter sugar chains. Known α-amylases can cleave the α-1,4-glucosidic bonds in both starch and dextrin, and their hydrolysis efficiency for these two substrates is essentially the same. Therefore, known α-amylases cannot effectively distinguish between starch and dextrin. After hydrolyzing gelatinized starch into low-DE dextrin, these α-amylases continue to hydrolyze it into regular dextrin, and then further degrade it into oligosaccharides, and even a large number of monosaccharides. In traditional dextrin production processes, the key is to control the degree of starch hydrolysis by α-amylase; different degrees of hydrolysis will yield dextrins with different DE values.

[0003] Low DE value dextrins refer to a class of maltodextrins with a DE value between 2 and 8. These dextrins can form soft, stretchable, and heat-reversible gels that dissolve instantly in the mouth, producing a fat-like effect. Therefore, they can be used as a fat substitute and have unique application value in food and beverages. In recent years, with increasing health demands, low DE value dextrins have become increasingly popular with consumers. Theoretically, the production of low DE value dextrin requires the use of a special α-amylase that can efficiently hydrolyze starch molecular chains, producing only relatively long sugar chains while producing little or no short sugar chains, or not further hydrolyzing the already produced long sugar chains. However, currently known α-amylases do not meet these requirements. In actual production, ordinary high-temperature α-amylases are still used. However, to achieve the goal of producing more long sugar chains and fewer short sugar chains, current technologies employ strict control of reaction time (short time 5-10 minutes), rapid reduction of reaction temperature (from 130℃ to 30℃), or spray-feeding of starch solution. Therefore, during the production of low DE value dextrins, a relatively large amount of gelatinized starch often remains unhydrolyzed. Meanwhile, due to the poor water solubility of low DE value dextrin, it mixes with residual starch, making conventional filtration or centrifugation techniques ineffective in separating and removing starch. Therefore, the removal of residual starch from low DE value dextrin has always been a challenge.

[0004] To address this, the laboratory purified and expressed the previously screened α-amylase from *Photobacterium gaetbulicola* (Genebank ID: AJR06082) in *E. coli*. The results showed that this α-amylase exhibited unique catalytic properties, effectively hydrolyzing gelatinized starch. However, its hydrolysis efficiency decreased sharply with decreasing sugar molecular weight, reaching only 5.7% of the efficiency of low-DE-value dextrin (DE-value 5) compared to gelatinized starch. In other words, this α-amylase can effectively distinguish between starch and dextrin, effectively hydrolyzing gelatinized starch, but only weakly hydrolyzing low-DE-value dextrin. Therefore, this α-amylase can be used to prepare low-DE-value dextrin. However, in actual research, the specific activity of the above-mentioned α-amylase in hydrolyzing gelatinized wheat starch was only 63.2 ± 1.4 U / mg, indicating a slightly lower preparation efficiency for low-DE-value dextrin.

[0005] Therefore, we can try to optimize the structure of the above-mentioned α-amylase (Genebank number: AJR06082) to obtain a new type of 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 novel α-amylase that can more efficiently and specifically hydrolyze gelatinized starch and its application.

[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0008] A novel α-amylase that specifically hydrolyzes gelatinized starch, the amino acid sequence of which is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0009] As one of the preferred embodiments of the present invention, it is obtained by optimizing and improving the structure of wild-type α-amylase with an amino acid sequence as shown in SEQ ID NO.1.

[0010] As one of the preferred embodiments of the present invention, the structural optimization and improvement are achieved through 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] The encoding gene of the novel α-amylase described above has a nucleotide sequence as shown in SEQ ID NO.4 or SEQ ID NO.5; wherein the sequence of SEQ ID NO.4 is used to encode the α-amylase corresponding to SEQ ID NO.2, and the sequence of SEQ ID NO.5 is used to encode the α-amylase corresponding to SEQ ID NO.3.

[0013] A recombinant expression vector containing the above-mentioned coding gene.

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

[0015] A recombinant engineered bacterium containing the above-mentioned encoding gene.

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

[0017] Application of the above-mentioned novel α-amylase in the preparation of low DE value dextrin.

[0018] As one of the preferred embodiments of the present invention, when preparing low DE value dextrin using the novel α-amylase: wheat starch with a concentration of 100 g / L is used as raw material, the pH is adjusted to 8.5, and after heating in boiling water for 15 min, α-amylase is added at a ratio of 100 U / kg dry starch, and hydrolyzed at 25°C for 15 min; the enzyme is inactivated by heating at 60°C for 10 min, and then the sample is cooled to room temperature and the pH is adjusted to neutral; after centrifugation for 20 min, the supernatant is taken and freeze-dried, and the powder prepared is low DE value dextrin with a DE value of 2 to 5.

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

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

[0021] (2) The α-amylase mutant of the present invention can effectively prepare low DE value dextrin (DE value is 2 to 5). The optimal conditions are: 100 g / L of gelatinized wheat starch, 100 U / kg dry starch of enzyme, pH 8.5, reaction temperature 25℃, and reaction time 15 min. Attached Figure Description

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

[0023] Figure 2 This is an SDS-PAGE image of the wild-type α-amylase and the mutants S263A and I236R / S263A purified enzymes from Example 3 (in the image, lane "M" represents the Maker; lane "wild-type" represents the purified wild-type α-amylase; lane "S263A" represents the purified α-amylase S263A; and lane "I236R / S263A" represents the purified α-amylase I236R / S263A).

[0024] Figure 3 This is the optimal temperature curve of wild-type α-amylase and mutant S263A, I236R / S263A pure enzyme in Example 3;

[0025] Figure 4 This is the optimal pH curve of the wild-type α-amylase and the mutant S263A and I236R / S263A pure enzymes in Example 3. Detailed Implementation

[0026] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, 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 this technical field, and will not be described in detail further.

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

[0028] Previously, our laboratory discovered an α-amylase derived from *Photobacterium gaetbulicola* (Genebank ID AJR06082). We commissioned Sangon Biotech (Shanghai, China) to optimize the gene based on *E. coli* codon preferences and synthesize the α-amylase gene (the synthesized α-amylase nucleotide sequence is shown in SEQ ID NO. 6, and the corresponding encoded amino acid sequence is shown in SEQ ID NO. 1). Next, the target gene fragment (SEQ ID NO. 6) was inserted between the BamHI (GGATCC) and XhoI (CTCGAG) sites in the pET-28a vector to construct the recombinant plasmid vector pET-28a-AJR06082 (as shown in SEQ ID NO. 1). Figure 1 (as shown); finally, the recombinant plasmid vector was transformed into Escherichia coli BL21(DE3) to obtain wild-type α-amylase engineered bacteria.

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

[0030] The target amylase-producing strain was inoculated into a test tube containing 5 mL LB and cultured overnight at 37°C and 200 rpm in a shaker. Then, it was transferred to a medium containing 400 mL LB and cultured until OD500 reached. 600 When the α-amylase concentration reached 0.6–0.8, 0.1 mM IPTG was added and the cells were induced overnight at 16°C and 120 rpm. The cells were then centrifuged at 4°C and 4000 rpm for 30 min to collect the cells. After resuspending the cells, the induced cells were disrupted by sonication. The cells were then centrifuged at 4°C and 12000 rpm for 30 min to obtain the supernatant and precipitate. The cell disruption supernatant was purified by nickel column affinity chromatography to obtain the purified α-amylase, which was then analyzed by SDS-PAGE.

[0031] Example 3: Screening and identification of α-amylase mutants:

[0032] (1) Screening of mutation sites

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

[0034] (2) Single point mutation

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

[0036] Site-directed mutagenesis yielded nine single-point mutant transformants at positions D212N, S231A, I236R, E239R, D243C, T262A, S263A, R302A, and D311R (mutation primers are shown in Table 1). Specifically, I236R refers to a single-point mutation of isoleucine at position 236 of the amino acid sequence shown in SEQ ID NO.1 to arginine, S263A refers to a 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] Following the method in Example 2, each single-point mutant was expressed and purified, followed by enzyme activity detection. Gelatinized starch was used as the substrate, and the total reaction volume was 900 μL, including 600 μL of substrate and enzyme. The detection method was as follows: after incubation in pH 8.0 buffer (50 mM Na₂HPO₄ - 50 mM KH₂PO₄) for 10 min, 300 μL of DNS was added, followed by centrifugation, boiling for 10 min, and then cooling. The OD was measured using a UV spectrophotometer. 540 Values. 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. Specific enzyme activities for different single-point mutations are shown in Table 2.

[0041] Table 2. Enzyme activity results for different single-point mutations.

[0042]

[0043]

[0044] Based on the enzyme activity results, the enzyme activity of mutants I236R and S263A was significantly increased, with mutant S263A (amino acid sequence shown in SEQ ID NO.2) being the best.

[0045] (3) Combinatorial double mutation

[0046] Based on the single-point mutation results, the plasmid containing the single-point mutant S263A was further used as a template to perform site-directed mutagenesis at the I236R position through full plasmid amplification (the steps are the same as for single-point mutation), resulting in the combined double mutant transformant I236R / S263A (amino acid sequence as shown in SEQ ID NO.3). The I236R / S263A protein was then expressed and purified according to the method in Example 2.

[0047] The enzymatic properties of wild-type α-amylase and its single mutant S263A and double mutant I236R / S263A were tested (SDS-PAGE of purified proteins of wild-type α-amylase, single mutant S263A, and double mutant I236R / S263A are shown in [reference needed]). Figure 2 (to achieve the purity required for enzyme property testing).

[0048] ①Optimal temperature and optimal pH

[0049] Following the aforementioned enzyme activity detection method, samples were incubated at 20–45°C and pH 8.0 (50 mM Na2HPO4-KH2PO4) before measurement of the activities of wild-type α-amylase and mutants S263A and I236R / S263A. The results are as follows: Figure 3 As shown, the optimal temperature for wild-type α-amylase and mutants S263A and I236R / S263A is 25℃.

[0050] The optimal pH was determined at 25°C within the pH range of 6.0–9.0, following the aforementioned enzyme activity assay method. The results are as follows: Figure 4 As shown, the optimal pH for the wild type is 8.0; more than 50% of the activity is retained in the pH range of 6.0 to 9.0; the optimal pH for mutants S263A and I236R / S263A is 8.5.

[0051] ② Ability to degrade different substrates

[0052] Referring to the aforementioned enzyme activity detection method, using gelatinized wheat starch, raw wheat starch, dextrin, and low DE value dextrin as substrates, the hydrolytic capacity of wild-type α-amylase and mutants S263A and I236R / S263A was determined, and the results are shown in Table 3.

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

[0054]

[0055] Table 3 shows that the mutants S263A and I236R / S263A exhibited 2.1-fold and 2.9-fold increased hydrolytic capacity for gelatinized wheat starch, respectively. Furthermore, neither mutant altered their preference for hydrolyzing 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; and the activity of the double mutant I236R / S263A in hydrolyzing low-DE-value dextrin was 5.0% of its activity in hydrolyzing gelatinized starch.

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

[0057] Example 4: Preparation of low DE value dextrin using 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 allow the raw starch granules to absorb water, swell, and gelatinize, thus obtaining a starch slurry. Add the α-amylase of this invention (using the double mutant I236A / S263R as an example) to the obtained starch slurry at a dosage of 100U / kg dry starch. Hydrolyze at 25℃ for 15min, then heat (60℃, 10min) to inactivate the enzyme. Cool the hydrolyzed sample to room temperature and adjust the pH to neutral. Then centrifuge the hydrolyzed sample at 6000×g for 20min, and freeze-dry the supernatant in a freeze dryer until the sample becomes powder.

[0060] 2. DE value determination

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

[0062] Calculation method:

[0063]

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

[0065] The dextrin finally prepared in this embodiment has a DE value of 2 to 5, and is confirmed to be a low DE value dextrin.

[0066] The above description is merely 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 within the protection scope of the present invention.

Claims

1. A novel α-amylase which specifically hydrolyzes gelatinized starch, characterized in that, The amino acid sequence is shown as SEQ ID NO. 2 or SEQ ID NO.

3.

2. The novel α-amylase specific for hydrolysis of gelatinized starch according to claim 1, characterized in that, The structure optimization improvement is obtained by optimizing and improving the wild-type alpha-amylase structure with the amino acid sequence shown as SEQ ID NO.

1.

3. The novel α-amylase hydrolyzing specifically gelatinized starch according to claim 2, characterized in that, The structure optimization improvement is achieved by mutation.

4. The novel α-amylase specific for hydrolysis of gelatinized starch according to claim 3, characterized in that, The sequence of SEQ ID NO. 2 corresponds to a single-point mutant S263A, i.e. the 263th serine in the amino acid sequence shown as SEQ ID NO. 1 is mutated to alanine; the sequence of SEQ ID NO. 3 corresponds to a double-point mutant I236R / S263A, i.e. the 236th isoleucine in the amino acid sequence shown as SEQ ID NO. 1 is mutated to arginine, and the 263th serine is mutated to alanine.

5. A novel α-amylase-encoding gene according to any one of claims 1 to 4, characterized in that, The nucleotide sequence is shown as SEQ ID NO. 4 or SEQ ID NO. 5; wherein the sequence of SEQ ID NO. 4 is used for encoding the alpha-amylase corresponding to SEQ ID NO. 2, and the sequence of SEQ ID NO. 5 is used for encoding the alpha-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 of claim 6, wherein, The expression vector is pET-28a.

8. A recombinant engineering bacterium containing the coding gene according to claim 5.

9. The recombineering bacteria of claim 8, wherein, The host bacterium is Escherichia coli BL21.

10. Use of a novel α-amylase according to any one of claims 1 to 4 for the production of a low DE dextrin, characterized in that, The low DE value is 2-5.

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