A high-temperature debranching enzyme, a debranching enzyme gene and a preparation method and application thereof

Abstract

The application discloses a high-temperature debranching enzyme, a debranching enzyme gene and a preparation method and application thereof, and belongs to the technical field of enzyme engineering. The optimal temperature interval of the high-temperature debranching enzyme is 81.2-93.8 DEG C, the optimal reaction pH is 6.5-8.0, the residual debranching enzyme activity is 94.3%+ / -0.9% after 100 DEG C heat preservation for 30 min, the industrial gelatinization liquefaction temperature requirement is fully met, one-step gelatinization liquefaction debranching can be realized, the process is simplified, the working hours are shortened, cost reduction and benefit increase are achieved, the debranching pH range of the enzyme is 5.5-8.0, the material pH range is wide, the enzyme can be matched with various amylases, the enzyme also has a wide optimal debranching temperature interval, and the two widen the application scenarios of the high-temperature debranching enzyme, so that the high-temperature debranching enzyme has important application value.

Description

Technical Field

[0001] This invention relates to a high-temperature debranching enzyme, the debranching enzyme gene, its preparation method and application, belonging to the field of enzyme engineering technology. Background Technology

[0002] Starch is an important raw material for food, medicine, and chemicals. Its molecular structure is a dendritic α-1,4-glucan, with shorter α-1,4-glucan branches linked to a longer α-1,6-glycosidic bonds. Starch debranching, which involves hydrolyzing the α-1,6-glycosidic bonds of starch to create easily crystallizable chain molecules, is widely used in the manufacture of health foods (slow-digesting starch for blood sugar and weight control), drug sustained-release agents, and biodegradable bio-based films. Simultaneously, starch debranching is a crucial step in starch sugar production, significantly improving the yield and quality of target sugars. Currently, acid hydrolysis is the main method for preparing debranched starch, but it has disadvantages such as poor hydrolysis selectivity, difficulty in process control, incomplete removal of branch points, and shorter α-glucan chains. Enzymatic debranching, on the other hand, has advantages such as high glycosidic bond selectivity and mild reaction conditions, and is also widely used in starch sugar production.

[0003] Starch debranching enzymes specifically hydrolyze the α-1,6 glycosidic bonds of starch. Currently, the main commercial starch debranching enzymes are isoamylase and pullulanase, both of which are suitable for weakly acidic, mesophilic environments. Pseudomonas amyloderamosa Isoamylase is suitable for pH 3.5 and 40 °C, while Xiasheng pullulanase FDY-2224 is suitable for pH 3.5-6.0 and 40-65 °C. Meanwhile, commercial pullulanases define their enzyme activity by pullulanose hydrolysis activity, while their starch debranching activity is unknown.

[0004] Raw starch is difficult to dissolve at low temperatures, requiring high-temperature gelatinization for starch processing, with industrial gelatinization temperatures often exceeding 90°C. If existing starch debranching enzymes are used for debranching, their poor heat resistance necessitates lowering the starch paste temperature to 60°C and adjusting the pH to around 4.5. Simultaneously, the starch paste needs to be appropriately diluted to reduce viscosity and promote mass transfer. Clearly, improving the heat resistance of debranching enzymes and combining starch gelatinization and debranching into one process can simplify operations, shorten processing time, and thus reduce costs and increase efficiency. Summary of the Invention

[0005] To address the shortcomings of existing technologies and develop high-temperature starch debranching enzymes, this invention predicts enzyme function through protein sequence homology alignment and homology modeling, and screens out enzymes with unknown functions from the NCBI database. Thermuscomposti Through heterologous expression of the GH13 protein gene and functional identification of recombinant proteins, a high-temperature starch debranching enzyme capable of one-step gelatinization, liquefaction, and debranching of starch was obtained, which can be applied to all starch processing processes requiring starch debranching.

[0006] This invention is achieved through the following technical solution: The first objective of this invention is to provide a high-temperature debranching enzyme, wherein the amino acid sequence of the high-temperature debranching enzyme is as follows: (1) The amino acid sequence shown in SEQ ID NO.1; (2) An amino acid sequence that has more than 95% homology with the amino acid sequence shown in SEQ ID NO.1 after substitution, insertion or deletion at one or more positions, and has high-temperature debranching activity.

[0007] In one embodiment of the present invention, the high-temperature debranching activity is to debranch starch at a temperature above 80°C.

[0008] A second object of the present invention is to provide a gene encoding the aforementioned high-temperature debranching enzyme.

[0009] In one embodiment of the present invention, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0010] A third objective of this invention is to provide a recombinant cell expressing the aforementioned high-temperature debranching enzyme.

[0011] In one embodiment of the present invention, the host cell of the recombinant cell is a bacterium, fungus, or animal / plant cell. Preferably, the host cell is *Escherichia coli*. More preferably, it is *Escherichia coli*. E. coli BL21.

[0012] In one embodiment of the present invention, the expression plasmid of the recombinant cell includes, but is not limited to, plasmids, granules, bacteriophages, and retroviruses. Preferably, the expression plasmid is a pET series expression vector. Preferably, the expression plasmid is pET28a.

[0013] A fourth objective of this invention is to provide a method for debranching starch under high-temperature conditions. The method involves adding the high-temperature debranching enzyme to a reaction system containing starch and carrying out the debranching reaction under high-temperature conditions, wherein the high-temperature conditions are a temperature not lower than 80°C. Preferably, the high-temperature conditions are 80-105°C, and more preferably, the high-temperature conditions are 90-105°C.

[0014] In one embodiment of the present invention, the pH of the reaction system is 5.0-8.0. Preferably, the pH is 5.5-8.0. More preferably, the pH is 6.5-8.0.

[0015] In one embodiment of the present invention, the starch content in the reaction system is 1%-33% by mass-volume ratio.

[0016] In one embodiment of the present invention, the starch-containing reaction system is obtained by gelatinizing and liquefying starch.

[0017] In one embodiment of the present invention, the starch is amylopectin. Preferably, the starch includes, but is not limited to, corn starch, glutinous rice starch, potato starch, and sweet potato starch.

[0018] The fifth objective of this invention is to provide a method for one-step gelatinization, liquefaction, and debranching of starch. The method involves mixing the crude enzyme solution of the high-temperature debranching enzyme or the high-temperature debranching enzyme obtained from the fermentation of recombinant cells with raw starch in water, and then performing starch gelatinization, liquefaction, and debranching at a temperature of 80-105°C.

[0019] In one embodiment of the present invention, the heat preservation treatment time is 1-10 h.

[0020] In one embodiment of the present invention, the amount of raw starch added to the water is 1%-33% by mass-volume ratio.

[0021] The beneficial effects of this invention are: The optimal debranching temperature range of the high-temperature debranching enzyme provided by this invention is 81.2-93.8 °C, and the optimal reaction pH is 6.5-8.0. After incubation at 100 °C for 30 min, the residual debranching enzyme activity is 94.3%±0.9%, which fully meets the industrial gelatinization and liquefaction temperature requirements. It can realize one-step gelatinization and liquefaction debranching, thereby simplifying the process, shortening the working time, and achieving cost reduction and efficiency improvement. Because the debranching pH range of this enzyme is 5.5-8.0, it is suitable for a wide range of material pH and can be adapted to various amylases. At the same time, it also has a wide optimal debranching temperature range. Both of these factors broaden the application scenarios of this high-temperature debranching enzyme, thus giving it important application value. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 pH-dependent debranching enzymes Tc The effect of P debranching activity; Figure 2 The effect of reaction temperature on debranching enzymes Tc The effect of P debranching activity; Figure 3 The effect of heat treatment temperature on debranching enzymes Tc The effect of residual debranching activity of P; Figure 4 Debranching enzyme Tc Visible absorption spectrum of P debranching product-iodine complex; Figure 5 Debranching enzyme Tc Visible absorption spectrum of starch-iodine complex during P debranching process. Detailed Implementation

[0024] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0025] The terminology involved in this invention: Starch debranching: The term "starch debranching" refers to the hydrolysis of α-1,6 glycosidic bonds in amylopectin, transforming it into easily crystallizing chain-like molecules. Amylopectin is a polysaccharide compound with a dendritic branching structure, its molecules consisting of thousands of glucose residues linked by α-1,4-glycosidic and α-1,6-glycosidic bonds.

[0026] Debranching enzymes: The term "debranching enzymes" refers to enzymes that specifically hydrolyze α-1,6 glycosidic bonds in polysaccharides such as starch and glycogen. Based on their substrate specificity, they can be divided into two categories: isoamylases (EC 3.2.1.68) and pullulanases (EC 3.2.1.41). Based on their different amino acid sequences, they belong to the glycoside hydrolase family GH13 and GH57, respectively.

[0027] Expression: The term “expression” refers to any step involving the production of debranching enzymes, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression plasmid: The term "expression plasmid" refers to a linear or circular DNA molecule that contains a polynucleotide encoding a debranching enzyme and is operatively linked to a control sequence that provides for its expression. Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The host cells of the present invention can be selected from bacteria such as *Bacillus* (e.g., *Escherichia coli*), *Bacillus* (e.g., *Bacillus subtilis*), and fungi such as yeasts (e.g., *Pichia pastoris*, *Saccharomyces cerevisiae*). For example, *Escherichia coli* used in the examples. E. coli BL21.

[0028] Recombination: When used to refer to cells, nucleic acids, proteins, or vectors, the term "recombination" means that the cells have been modified from their natural state. Thus, for example, recombinant cells express genes not found in their natural (non-recombinant) form, or express natural genes at different levels or under different conditions compared to those found in nature. The difference between recombinant nucleic acids and their natural sequences lies in the operative linking of one or more nucleotides and / or a heterologous sequence (e.g., a heterologous promoter in an expression vector). The difference between recombinant proteins and their natural sequences may lie in the fusion of one or more amino acids and / or a heterologous sequence. The expression plasmids of this invention can be introduced into host cells for recombination using methods well known in the art, including calcium chloride heat shock, electroporation, PEG-mediated recombination, gene gun methods, etc.

[0029] Mutant: The term "mutant" or "debranching enzyme mutant" refers to a polypeptide with similar debranching enzyme activity that contains alterations (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to the parental debranching enzyme. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

[0030] Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. Furthermore, it should be understood that those skilled in the art can perform nucleotide substitutions using conventional techniques, which will not affect the polypeptide sequence encoded by the polynucleotides used in this invention. Additionally, polynucleotides can be modified using methods known in the art to enhance the activity or survival time of the polynucleotides of this invention in vivo.

[0031] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or commonly used in the industry.

[0032] Example 1: Obtaining the debranching enzyme gene and constructing the recombinant plasmid Enzyme function prediction was performed using protein sequence homology alignment and homology modeling, and enzymes with unknown functions were screened from the NCBI database. Thermus composti The GH13 protein gene sequence (tcp, amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and inserted between the Nco I and Xho I sites of pET28a to construct the expression plasmid pET28a- TCP .

[0033] Example 2: Contains debranching enzyme Tc Construction of recombinant Escherichia coli engineered strains with the P gene by E. coli Using BL21 as the host cell, 0.2 μL of plasmid pET28a- was taken. TCP (0.04 μg / μL) Added to a solution containing 20 μL E. coli BL21 competent cells (prepared by calcium chloride method) were placed in 1.5 mL centrifuge tubes and incubated on ice for 15 min; then subjected to heat shock at 42℃ for 60-90 s; incubated on ice for 2 min; 300 μL of LB medium was added to the centrifuge tubes and cultured at 37℃ with a shaker at 200 r / min for 1 h; the culture was spread onto LB solid plates containing 50 μg / mL kanamycin (5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder) and cultured at 37℃ for 12 h; single colonies were picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin (5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride) and cultured at 37℃ with a shaker at 200 r / min for 8-12 h; after sampling and sequencing verification, the bacterial culture was preserved with 12% glycerol at -80℃.

[0034] Example 3: Debranching enzyme Tc Preparation of P enzyme solution (1) Debranching enzyme Tc P-induced expression First, the frozen recombinant bacterial strain was streaked onto an LB agar plate containing 50 μg / mL kanamycin and incubated overnight at 37 ℃. A single colony was then picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin and incubated for 12 h. Finally, 500 μL of the bacterial culture was inoculated into 50 mL of LB liquid medium containing 50 μg / mL kanamycin and incubated at 37 ℃ until OD (dose elapsed). 600 =0.6; Add IPTG to a final concentration of 0.1 mmol / L, incubate at 25 ℃ for 18 h; collect bacteria by centrifugation at 7000 r / min for 5 min. Store the remaining bacterial cells at -80 ℃.

[0035] (2) Preparation of crude enzyme solution Take 1 mL of water to resuspend the bacterial cells in 5 mL of bacterial solution in a 5 mL centrifuge tube; place in an ice-water bath and sonicate to disrupt the cells. Use a 6 mm probe, 70% power, sonicate for 3 s, pause for 2 s, and disrupt for 3 min; centrifuge the clear disruption solution at 12000 r / min for 10 min, and take the supernatant to obtain the crude enzyme.

[0036] Example 4: Determination of debranching enzymes by iodine staining method Tc P debranching activity Substrate solution composition: 16 μL of 10% (w / v) corn amylopectin, 16 μL of 200 mM citrate-disodium hydrogen phosphate pH buffer, and 28 μL of H2O.

[0037] Iodine staining agent: Take 4.0 μL of 1% I2-KI (1.0 g I2 + 8.0 g KI + 100 mL H2O) and add 1 mL of 100 mM HCl and mix well.

[0038] Add 60 μL of substrate solution to a PCR tube (200 μL) and place it in the PCR instrument's temperature control module. Cool to 4 °C, then add 20 μL of crude enzyme solution and mix well. Inflate to the set temperature and maintain for a certain time. Cool to 4 °C and add 80 μL of 1 M NaOH and mix well. Add 5.7 μL of alkalization reaction solution to a 96-well microplate containing 195 μL of iodine stain, mix well, and measure OD using a microplate reader. 620 .

[0039] Enzyme activity unit definition, under experimental conditions, OD 620 The amount of enzyme required for a change of 0.01 per hour is 1 U.

[0040] Example 5: Debranching enzyme Tc Enzymatic properties of P and characteristics of debranching products (1) Debranching enzyme Tc The optimal pH for debranching of P Debranching enzymes by iodine staining method Tc P was reacted at 77 ℃ for 30 min to hydrolyze 2% (w / v, e.g., 20 g starch added to 1 L of water) corn amylopectin at pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 to enhance its debranching activity. Three replicates were set up for each treatment. Results are as follows: Figure 1 As shown, debranching enzyme Tc The debranching pH range of P is 5.5-8.0, with the optimum pH being 6.5-8.0.

[0041] (2) Debranching enzyme Tc Optimal temperature for P debranching Debranching enzymes by iodine staining method Tc P was used to hydrolyze 2% (w / v) corn amylopectin at pH 5.5 for 30 min at 71.8, 73.5, 77.0, 81.2, 85.4, 89.6, 93.8, 98.0, 101.5, 103.3, and 105.0 ℃ to enhance its debranching activity. Three replicates were set up for each treatment. Results are as follows... Figure 2 As shown, debranching enzyme Tc The optimal temperature for P debranching is 81-94 ℃.

[0042] (3) Debranching enzymes Tc Thermal stability of P Debranching enzymes Tc P was incubated at 71.5, 73.0, 76.0, 79.6, 83.2, 86.8, 90.4, 94.0, 97.0, 98.5, and 100.0 ℃ for 30 min, and then rapidly cooled to 4 ℃. Each treatment was repeated in triplicate, with 20 μL of enzyme solution per sample. The debranched enzyme content after incubation was determined by iodine staining. Tc To determine the residual enzyme activity of P, 60 μL of a 2% (w / v) corn amylopectin solution (pH 5.5) was added to the enzyme solution, mixed well, and incubated at 77 ℃ for 30 min. The results are as follows: Figure 3 As shown, debranching enzyme Tc After incubation at 100℃ for 30 min, the residual debranching enzyme activity was 94.3% ± 0.9%.

[0043] (4) Debranching enzymes Tc Characteristics of debranching products of P The debranched product-iodine complex obtained in the debranching enzyme activity assay was subjected to visible absorption spectrum measurement using an ELISA reader, with a wavelength scanning range of 400-700 nm. The results are as follows: Figure 4 As shown, corn amylopectin is processed by debranching enzymes. Tc After debranching, the maximum absorption wavelength of the starch-iodine complex shifted from 536 nm to 560 nm, indicating that amylopectin was converted into amylose.

[0044] Example 6: Debranching enzyme Tc The one-step gelatinization and debranching process of P Take four 200 μL PCR tubes and add 20 μL to each tube. Tc The crude enzyme solution and 80 μL of 4% (w / v) raw corn amylopectin were mixed and aliquoted into the temperature control modules of four PCR instruments. The mixtures were incubated at 95 ℃ for 15 min, 30 min, 60 min, and 120 min, respectively. After incubation, 20 μL of the reaction solution was added to 20 μL of 1 M NaOH and mixed. 5.7 μL of the alkalization reaction solution was added to a 96-well microplate containing 195 μL of iodine stain, mixed, and the visible absorption spectrum was scanned using a microplate reader. The results are as follows: Figure 5 As shown, the maximum absorption wavelength of the debranching product-iodine complex red-shifted to 562 nm with increasing incubation time, indicating that the debranching enzyme under the experimental conditions... Tc P can complete the gelatinization and debranching of raw amylopectin at 95℃ for 120 min.

[0045] The sequences used in this invention: Debranching enzymes Tc The amino acid sequence of P is SEQ ID NO:1: MAWYEGAFFYQIFPDRFFRAGPPGKPAPAGPFEPWEAPPTLRGFKGGTLWGVAEKLPYLLDLGVEALYLNPVFASTANHRYHTTDYFQVDPILGGNEALRHLLEVAHAHGVRVILDGVFNHTGRGFFAFQHLLENGEQSPYRDWYHVKGFPLKAYTAHPNYEAWWGNPELPKLKVETPAVREYLLSVAEHWIRFGADGWRLDVPNEIQDPEFWRAFRRRVKGVNPEAYIVGEIWEEADFWLQGDMFDAVMNYPLSRAILGFVGGEALDRELAGRSGLGSIEPLQALAFSHRLEALFGRYRPEVVRAQMNLLTSHDTPRLLTLMRGGVERARLALALLFLLPGNPTVYYGEEVGMEGGHDPENRGGMVWEEARWRKELRETVRRMARLRKEHPELRTAPYWRVYAADGHLAFTRGPYLVVVNATPEPFLQDFPLHGAFPRGGRLLDLLSGAVCTPQGGRLCGPALPPFSVAVWKEA Debranching enzyme Tc Nucleotide sequence of the P gene SEQ ID NO:2: The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A high-temperature debranching enzyme, characterized in that, The amino acid sequence of the high-temperature debranching enzyme is as follows: (1) The amino acid sequence shown in SEQ ID NO.1; (2) An amino acid sequence that has more than 95% homology with the amino acid sequence shown in SEQ ID NO.1 after substitution, insertion or deletion at one or more positions, and has high-temperature debranching activity.

2. The high-temperature debranching enzyme according to claim 1, characterized in that, The high-temperature debranching activity involves debranching starch at temperatures above 80°C.

3. A gene encoding the high-temperature debranching enzyme of claim 1 or 2.

4. A recombinant cell expressing the high-temperature debranching enzyme of claim 1 or 2.

5. A method for debranching starch under high temperature conditions, characterized in that, The method involves adding the high-temperature debranching enzyme described in claim 1 or 2 to a reaction system containing starch, and carrying out the debranching reaction under high-temperature conditions, wherein the high-temperature conditions are a temperature not lower than 80°C.

6. The method according to claim 5, characterized in that, The pH of the reaction system is 5.0-8.

0.

7. The method according to claim 5, characterized in that, The starch-containing reaction system is obtained by gelatinizing and liquefying starch, and the starch content is 1%-33% by mass-volume ratio.

8. A method for one-step gelatinization, liquefaction, and debranching of starch, characterized in that, The method involves mixing the crude enzyme solution of the high-temperature debranching enzyme or the high-temperature debranching enzyme obtained from the fermentation of the recombinant cells with raw starch in water, and then treating the starch gelatinization, liquefaction and debranching at 80-105℃.

9. The method according to claim 8, characterized in that, The heat preservation treatment time is 1-10 h.

10. The method according to claim 8, characterized in that, The amount of raw starch added to water, by mass-volume ratio, is 1%-33%.

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