Isoamylase and gene thereof, engineering bacterium containing gene and application of engineering bacterium

Abstract

The invention discloses isoamylase and a gene thereof, an engineering bacterium containing the gene and application of the engineering bacterium. The invention provides an isoamylase gene, the nucleotide sequence of which is as shown in SEQ ID NO.1, the full length of the gene is 2130 bp, the G + C content is 69.11%, 709 amino acids are encoded, and the amino acid sequence is as shown in SEQ ID NO.2; the isoamylase provided by the invention has wide pH range adaptability, has higher enzyme activity when the reaction is carried out at the pH of 6.0-8.0, and has the highest enzyme activity when the reaction is carried out under the conditions that the temperature is 40 DEG C and the pH is 7.0; the isoamylase provided by the invention has a hydrolysis effect on alpha-1, 4 and alpha-1, 6 glucosidic bonds, the specific activity of hydrolyzed corn starch is 250.46 U / mg, and when the isoamylase is combined with maltogenic amylase BMAL for use, the utilization efficiency of the maltogenic amylase BMAL on a branched chain substrate can be improved.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to an isoamylase and a gene thereof, an engineering bacterium containing the gene and application thereof. Background Art

[0002] Starch debranching enzymes (SDBEs) are a class of hydrolytic enzymes that act on the α-1,6-glycosidic bonds of starch. In the starch processing industry, starch debranching enzymes are used in combination with other amylases that hydrolyze α-1,4-glycosidic bonds to improve the starch hydrolysis rate.

[0003] According to the differences between the catalytic mode and substrate specificity, starch debranching enzymes are divided into pullulanases (EC. 3. 2.1. 41, type I pullulanases), amylopullanases (EC. 3. 2. 1. 41, type II pullulanases) and isoamylases (EC. 3.2.1.68). Pullulanases have typical ( β / α )8 barrel-shaped structure, belonging to the glycoside hydrolase GH13 family. Pullulanase can efficiently hydrolyze pullulan polysaccharide, and the smallest action unit is maltosyl-α-1,6-maltose, and has low hydrolysis activity for large molecular weight amylopectin. In the Carbohydrate Active Enzyme Database (CAZY), starch pullulanase is distributed in the glycoside hydrolase GH13 (( β / α )8 barrel) and glycoside hydrolase GH57 family (( β / α )7 barrels); Amylopectinase is a bifunctional hydrolytic debranching enzyme that can catalyze the hydrolysis of α-1,4-glycosidic bonds and α-1,6-glycosidic bonds at the same time. Isoamylase also belongs to the GH13 family of glycoside hydrolases. It has high hydrolysis activity on amylopectin and glycogen, but low hydrolysis activity on low molecular weight dextrin. The minimum action unit is maltotriosyl-α-1,6-maltotetraose, and it cannot hydrolyze pullulan.

[0004] Since the first Pseudomonas amyloderamosa The gene of the isoamylase from the source was cloned and expressed. After industrial production, the isoamylase was named Promozyme®D2, but the specific enzyme activity of Promozyme®D2 was only 180 U / mg. The specific enzyme activity of the isoamylase IsoM from the myxobacterium EGB was 70600 U / mg, and it had no hydrolysis effect on amylose. The activity of IsoM was measured by iodine staining, and IsoM was expressed in yeast with a very low expression level. The enzyme has not yet been industrialized.

[0005] At present, there are few isoamylases available, and there are problems such as single function, low enzyme activity, and low expression level. In addition, the commercial products on the market are mainly imported enzyme preparations; therefore, it is necessary to develop an isoamylase to improve the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide an isoamylase and its gene, an engineered bacterium containing the gene and its application; the isoamylase provided by the present invention can hydrolyze α-1,4 glycosidic bonds and α-1,6 glycosidic bonds at the same time, and when used in combination with other amylases, the starch hydrolysis rate can be improved, and it can be applied to genetic engineering fields such as starch, food, and feed.

[0007] In a first aspect, the present invention provides an isoamylase gene, the nucleotide sequence of the isoamylase gene is shown as SEQ ID NO.1.

[0008] Furthermore, the full length of the nucleotide sequence of the isoamylase gene is 2130 bp.

[0009] Furthermore, the G+C content of the isoamylase gene is 69.11%.

[0010] Furthermore, the isoamylase gene encodes a total of 709 amino acids.

[0011] In a second aspect, the present invention provides an isoamylase protein encoded by the isoamylase gene, and the amino acid sequence of the isoamylase protein is shown in SEQ ID NO.2.

[0012] In a third aspect, the present invention provides a recombinant vector, which includes the isoamylase gene and an empty vector.

[0013] Furthermore, the empty vector includes any one of pET-29a, pET-28a, pET-42a, pUC18, and pUC19.

[0014] Preferably, the empty vector is pET-29a.

[0015] In a fourth aspect, the present invention provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium comprises the isoamylase gene, Escherichia coli BL21DE3.

[0016] In a fifth aspect, the present invention provides an isoamylase encoded by the isoamylase gene, wherein the isoamylase is capable of simultaneously hydrolyzing α-1,4 glycosidic bonds and α-1,6 glycosidic bonds.

[0017] Furthermore, the optimum temperature of the isoamylase is 40°C and the optimum pH is 7.0.

[0018] In a sixth aspect, the present invention provides an application of an isoamylase in improving the hydrolysis rate of starch, wherein the application comprises using the isoamylase in combination with other isoamylases to improve the hydrolysis rate of starch.

[0019] Furthermore, the other isoamylases include α-1,6 endoamylase and maltogenic amylase.

[0020] In a seventh aspect, the present invention provides an application of an isoamylase gene in starch processing, wherein the application includes starch hydrolysis, food, and feed.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a novel isoamylase gene, the nucleotide sequence of which is shown in SEQ ID NO.1, the full length of which is 2130 bp, the G+C content is 69.11%, and it encodes a total of 709 amino acids, the amino acid sequence of which is shown in SEQ ID NO.2; (2) The present invention provides a novel isoamylase, which is encoded by an isoamylase gene and has bifunctional properties. It can hydrolyze α-1,4 glycosidic bonds and α-1,6 glycosidic bonds at the same time, and its enzyme activity is highest at 40°C and pH 7.0. (3) The isoamylase provided by the present invention can improve the starch hydrolysis rate, and the specific activity of hydrolyzing corn starch is 250.46 U / mg, which is 1.39 times the specific enzyme activity of the commercial isoamylase Promozyme® D2; and the isoamylase provided by the present invention, after being placed at 35-40°C for 12 hours, still retains more than 70-75% of its activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the electrophoresis diagram of PCR amplification of isoamylase gene; Figure 2 is the SDS-PAGE protein electrophoresis diagram of the isoamylase prepared in Example 1; Figure 3 The optimum temperature determination result of the isoamylase prepared in Example 1; Figure 4 The optimum pH measurement result of the isoamylase prepared in Example 1; Figure 5 The results of the thermal stability test of the isoamylase prepared in Example 1 are as follows; Figure 6 The pH stability test results of the isoamylase prepared in Example 1; Figure 7 The results of thin layer chromatography analysis of the hydrolysis products of different substrates by the isoamylase prepared in Example 1; Figure 8 Analysis of the degree of polymerization of the hydrolysis product of amylopectin by the isoamylase prepared in Example 1; Fig. 9The results show that the utilization efficiency of amylopectin was improved by using the isoamylase prepared in Example 1 in combination with the maltogenic amylase BMAL. DETAILED DESCRIPTION

[0023] The present invention will be further described by the accompanying drawings and the following examples.

[0024] In a first aspect, an isoamylase gene is provided in an embodiment of the present invention, and the nucleotide sequence of the isoamylase gene is shown in SEQ ID NO.1.

[0025] In some embodiments, the full length of the nucleotide sequence of the isoamylase gene is 2130 bp.

[0026] In some embodiments, the G+C content of the isoamylase gene is 69.11%.

[0027] In some embodiments, the isoamylase gene encodes a total of 709 amino acids.

[0028] In a second aspect, an embodiment of the present invention provides an isoamylase protein encoded by the isoamylase gene, and the amino acid sequence of the isoamylase protein is shown in SEQ ID NO.2.

[0029] In a third aspect, an embodiment of the present invention provides a recombinant vector, which includes the isoamylase gene and an empty vector.

[0030] In some embodiments, the empty vector comprises any one of pET-29a, pET-28a, pET-42a, pUC18, and pUC19.

[0031] Specifically, the empty vector is pET-29a.

[0032] In a fourth aspect, an embodiment of the present invention provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium comprises the isoamylase gene, Escherichia coli BL21DE3.

[0033] In a fifth aspect, an embodiment of the present invention provides an isoamylase encoded by the isoamylase gene, wherein the isoamylase can hydrolyze α-1,4 glycosidic bonds and α-1,6 glycosidic bonds at the same time.

[0034] In some embodiments, the optimal reaction temperature of the isoamylase is 40° C., and the optimal reaction pH is 7.0.

[0035] In a sixth aspect, an embodiment of the present invention provides an application of an isoamylase in improving the hydrolysis rate of starch, wherein the application comprises using the isoamylase in combination with other amylases to improve the hydrolysis rate of starch.

[0036] In some embodiments, the other amylase comprises a maltogenic amylase.

[0037] In a seventh aspect, an embodiment of the present invention provides an application of an isoamylase gene in the starch processing industry, wherein the application includes starch hydrolysis, food, and feed processing industries.

[0038] In some embodiments, the composition of LB medium is: 5.0 g / L yeast powder, 10.0 g / L tryptone, and 10.0 g / L NaCl.

[0039] The materials and reagents used in the examples of the present invention are all commercially available; the vector pET-29a is from Invitrogen; the Escherichia coli Escherichia coli BL21 (DE3) was from Invitrogen; Cosmosil Sugar-D chromatographic column was from Nacalai Tesuque, Kyoto, Japan; restriction endonucleases were from Takara; seamless cloning kit was from Nanjing Novozymes; and oligosaccharide standard kit was from Sigma.

[0040] Example 1

[0041] Example 1 of the present invention provides a method for cloning an isoamylase gene and constructing an expression vector, comprising the following steps: S1. PCR amplification of isoamylase gene S11. Reference Myxobacteria Myxococcus sp. V11 genome sequence was performed orf Prediction: Based on the amino acid sequence of isoamylase (named MDBE), primers were designed according to the restriction sites of the nucleic acid sequence. The upstream primer F sequence is shown in SEQ ID NO.3, and the downstream primer R sequence is shown in SEQ ID NO.4; S12, according to the primers designed in S11, the isoamylase gene ( mdbe ) Full-length PCR amplification; The PCR reaction system is as follows: Myxococcus sp.V11 total DNA 1.0 μL, primers F and R 1.0 μL each, PrimeSTAR® Max DNA Polymerase 25.0 μL, add ddH2O to make the total volume of the system 50.0 μL; PCR amplification conditions were: Pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 55 °C for 22 s, extension at 72 °C for 2 min, a total of 30 cycles, and a final extension at 72 °C for 10 min; mdbe The full-length sequence of Figure 1 As shown, Figure 1 Lane M in the figure is the DL5000 nucleic acid marker, and lane 1 is the PCR amplification product of the isoamylase gene; S2, recombinant vector pET-29a- mdbe Construction and verification of S21, performing double enzyme digestion on pET-29a to obtain double enzyme digestion products; The double enzyme digestion system is: Bm I 2.0 μL, Hind III2.0 μL, 10×H Buffer 5.0 μL, pET-29a20.0 μL, add ddH2O to make the total volume of the system 50.0 μL; S22, the double enzyme digestion product was recovered by DNA agarose gel recovery kit and compared with the purified mdbe The PCR amplification products were subjected to enzyme ligation; Enzyme-linked system: 4.0 μL purification mdbe PCR amplification product, 2.0 μL double enzyme digestion product, 2.0 μL 5ⅹCE Buffer, 1.0 μL ExnaseⅡ, add ddH2O to make the total volume of the system 10.0 μL; connect the enzyme system at 37 ℃ for 1 h to obtain the enzyme-linked product; S23, take 10 μL of enzyme-linked product and add it to 100 μL E. coli In DH5α competent cells, place on ice for 30 min; heat shock in a 42°C water bath for 90 s, then quickly transfer to an ice bath and cool for 5 min; S24, add 800 μL of liquid LB medium to the cooled competent system, and incubate at 37 °C with a shaker at 110 rpm for 45 min; S25. After incubation, centrifuge at 8000 rpm for 5 min, take 100 μL of competent cells and spread them on LB agar plates containing 50 mg / L kanamycin, and invert the plates in a 37 ℃ incubator for 14 h; S26, extracting multiple single colonies from the transformation plate, and extracting plasmids using a plasmid miniprep kit; S27, double enzyme digestion of the plasmid extracted in step S26; The double enzyme digestion system is: Bm I 1.0 μL 、 HindIII 1.0 μL, 10×H Buffer 2.5 μL, plasmid 10.0 μL, add ddH2O to make the total volume of the system 25.0 μL; S28. Verify and send the obtained recombinant plasmid to Hunan Qingke Biotechnology Co., Ltd. for sequencing; the sequencing results show that the cloned target fragment is inserted into the pET-29a(+) vector, with a nucleotide length of 2130 bp, and then the recombinant expression vector pET-29a- mdbe , mdbe The DNA sequence is shown in SEQ ID NO.1, and the amino acid sequence of the isoamylase protein expressed by it is shown in SEQ ID NO.2; S3. Expression and purification of recombinant isoamylase S31, the pET-29a- mdbe Heat shock transformation into host bacteria E. coli BL21(DE3) to obtain recombinant engineered bacteria containing the recombinant plasmid; S32, inoculate a single colony of the positive clone into 4 mL LB medium (50 μg / mL kanamycin), culture at 37 °C, 180 rpm shaking for 12 h. After the culture is complete, use a plasmid mini-prep kit to extract the plasmid and verify it; S33. After verification, the culture solution of S32 was inoculated into 4 mL LB medium and cultured at 37 °C and 180 rpm for 8 h. After the culture was completed, it was transferred to 400 mL LB medium and cultured at 37 °C and 180 rpm for 3.5 h. S34. After the culture is completed, measure OD 600 When it reached 0.4-0.6, IPTG (final concentration was 2 mmol / L) was added and the expression was induced at 16°C and 180 rpm for 24 h; S35. After the induction expression was completed, the culture solution was centrifuged at 4000 rpm for 0.5 h and the bacteria were collected; S36, resuspend and wash the cells with 20 mmol / L citrate buffer, pH 6.0, and collect the cells by centrifugation at 12000 rpm for 0.25 h; S37, add 20 mmol / L citrate buffer, pH 6.0, in an ice bath, and disrupt the cells using an ultrasonic disruptor; S38. After crushing, centrifuge at 4 °C and 12,000 rpm for 10 min to obtain MDBE crude biological extract; S39. The MDBE crude biological extract was purified using a Ni-NTA affinity chromatography column (from Changzhou Tiandi Renhe Biotechnology Co., Ltd.) to obtain purified recombinant isoamylase. The purity and molecular weight of the recombinant isoamylase were determined by SDS-PAGE. The purity identification results were as follows: Figure 2 As shown, the molecular weight is approximately 80 kDa; Figure 2 Lane 1 is a protein molecular weight standard, and lane 2 is a protein containing the empty vector pET-29a. E. coli BL21(DE3) supernatant, lane 3 is the enzyme solution containing the isostarch gene E. coli BL21(DE3) supernatant, lane 4 is the enzyme solution containing the isostarch gene E. coli Lane 5 is the precipitate of BL21(DE3), and lane 5 is the purified MDBE protein eluted with 100 mmol / L imidazole.

[0042] Performance Verification

[0043] 1. Optimum reaction temperature of MDBE obtained in Example 1 D1, the reaction system is 500 μL: 50 μL purified MDBE, 450 μL pullulan buffer system (final concentration is 0.5% (w / v)); D2, react at different temperatures (30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃) for 10 min at pH 7.0 and 50 mmol / L Tris-HCL; D3. After the reaction is completed, add DNS with the same volume as the reaction system to terminate the reaction, measure the absorbance at 540 nm, set 3 replicates for each temperature, and take the average value; The results showed that at 40 ℃, isoamylase had the highest enzyme activity. Therefore, the enzyme activity of the enzyme activity reaction system at 40 ℃ was set as the relative activity 100%, and the absorbance values ​​of the enzyme activity reaction system at other temperatures and the absorbance value of the highest enzyme activity system were taken as the relative activity. The results are shown in Figure 3 shown.

[0044] 2. Optimal reaction pH value of MDBE prepared in Example 1 F1, reaction system is 500 μL: 50 μL purified MDBE, 450 μL pullulan buffer system (final concentration is 0.5% (w / v)); F2, react at 40 °C for 10 min at different pH conditions (5.0, 6.0, 7.0, 8.0, 9.0); when the pH is 5.0-6.0, it is a citric acid-sodium citrate system; when the pH is 7.0-9.0, it is a Tris-HCl system; F3. After the reaction is completed, add DNS with the same volume as the reaction system to terminate the reaction, measure the absorbance at 540 nm, set 3 replicates under each pH condition, and take the average value; The results showed that isoamylase has a wide range of pH adaptability (6.0-8.0). At pH 7.0, isoamylase has the highest enzyme activity. Therefore, the enzyme activity of the enzyme activity reaction system at pH 7.0 was set as the relative activity 100%, and the absorbance values ​​of the enzyme activity reaction systems at other pH values ​​and the absorbance value of this highest enzyme activity system were taken as relative activities. The results are shown in Figure 4 shown.

[0045] 3. Thermal stability of MDBE prepared in Example 1 G0, treatment of purified MDBE at different temperatures: the purified MDBE was treated at 20, 30, 35, 40, and 45 °C for 0.5 h, 1 h, 3 h, 6 h, and 12 h; G1, the reaction system is 500 μL: 50 μL of purified MDBE treated at different temperatures, 450 μL of buffer system (50 mmol / L Tris-HCl (pH 7.0), containing a final concentration of pullulan of 0.5% (w / v)); G2, react at 40 ℃ for 10 min; after the reaction, add 500 μL of DNS to terminate the reaction, measure the absorbance at 540 nm, and measure the residual enzyme activity of the enzyme. Set up 3 replicates for each different condition and take the average value. Take the enzyme activity of MDBE stored at 0 ℃ as 100% to calculate the relative enzyme activity of MDBE after treatment at different temperatures; The results showed that after being incubated at 20-40 °C for 12 h, the MDBE provided by the present invention still retained 70-80% of its activity, while incubating at 45 °C for 1 h caused the enzyme activity to drop sharply. Figure 5 As shown, the MDBE provided by the present invention has good stability at 0-40°C.

[0046] 4. pH stability of MDBE prepared in Example 1 H0, purified MDBE treated with different pH values: the purified MDBE was stored at different pH values ​​(5.0, 6.0, 7.0, 8.0, 9.0) for 12 h; when the pH value was 5.0-6.0, it was a citric acid-sodium citrate system; when the pH value was 7.0-9.0, it was a Tris-HCl system; H1, the reaction system is 500 μL: 50 μL purified MDBE treated with different pH values, 450 μL pullulan buffer system (final concentration is 0.5% (w / v)); H2, in 50 mmol / L Tris-HCl (pH 7.0), 40 °C, react for 10 min; H3. After the reaction is completed, add DNS with an equal volume to the reaction system to terminate the reaction, measure the absorbance at 540 nm, and determine the residual enzyme activity of the enzyme. Set up 3 replicates under each different pH treatment condition and take the average value. Take the MDBE enzyme activity without different pH treatment as 100% to calculate the relative enzyme activity of MDBE after different pH treatments. The results show that in the buffer system of pH 6.0, the stability of MDBE provided by the present invention is the best. Figure 6 shown.

[0047] 5. Specificity of MDBE prepared in Example 1 to different substrates. The MDBE obtained in Example 1 of the present invention was respectively acted on cassava starch, β-cyclodextrin, γ-cyclodextrin, potato starch, soluble starch, pullulan, corn starch, amylose and amylopectin, and the enzyme activity was measured. The results are shown in Table 1: Table 1 Specificity of MDBE prepared in Example 1 to different substrates

[0048] As shown in Table 1, the isoamylase provided by the present invention has no hydrolysis effect on pullulan and β-cyclodextrin among the above nine substrates, but has effects on the others, and has the strongest hydrolysis effect on corn starch.

[0049] 6. Analysis of the enzyme reaction products of MDBE prepared in Example 1 on different substrates 250 μL of purified MDBE was added to pullulan, amylopectin, amylose, γ-cyclodextrin, β-cyclodextrin, α-cyclodextrin, maltotetraose, and maltohexaose buffer systems, respectively. The final concentration of the substrates was 1% (w / v). The buffer systems were all prepared with 50 mmol / L Tris-HCl buffer at pH 7.0. After 2 h of reaction at 40 °C, the reaction was terminated in a boiling water bath; centrifuged at 12,000 rpm for 1 min, the supernatant was filtered through a 0.22 μm filter membrane, and detected by thin layer chromatography (TLC); The specific TLC analysis conditions are as follows: the developing agent is n-butanol / methanol / water = 4:2:1 (v / v / v), the color developing agent is concentrated sulfuric acid / methanol = 1:9 (v / v); the concentration of concentrated sulfuric acid is 98%; When the spreading agent reaches 1-2 cm from the top of the thin plate, take out the thin plate, blow dry it, and evenly spray the color developer on it; blow dry it again, and place the thin plate in an oven at 90 °C for color development; TLC test results are as follows Figure 7As shown, Std represents a standard, and Std is a mixture of glucose (G1), maltobiose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), and maltohexaose (G6) standards; Figure 7 The "+" in the table indicates the reaction product of the substrate after adding MDBE in Example 1; "-" indicates the control of the substrate without adding MDBE; "G4+" indicates the reaction of adding MDBE to maltotetraose alone; "G6+" indicates the reaction of adding MDBE to maltohexaose alone; See also Figure 7 The action mode of MDBE on various substrates was studied by TLC analysis, and the hydrolysis products of MDBE on different substrates were obtained. The results showed that the MDBE provided in Example 1 of the present invention did not undergo hydrolysis with pullulan, α-cyclodextrin and β-cyclodextrin; the hydrolysis products of γ-cyclodextrin were maltobiose, maltotriose and maltotetraose; the hydrolysis products of maltotetraose, maltohexaose, amylopectin and amylose included maltooligosaccharides (G2–G6) and linear oligosaccharides.

[0050] 7. The debranching degree of amylopectin by MDBE prepared in Example 1 The reaction system volume was 500 μL: 25 μL of MDBE prepared in Example 1, 475 μL of 50 mmol / L Tris-HCl buffer with a pH of 7.0 (containing amylopectin with a final concentration of 0.2% (w / v)); 40 ℃, react for 0.25 h, 0.5 h, 1 h, 2 h, 3 h and 6 h respectively. After the reaction, 500 μL of DNS was added to terminate the reaction. The amount of reducing sugar produced was determined by DNS method, with glucose as the standard curve. The results are shown in Table 2: Table 2 Debranching degree of MDBE prepared in Example 1

[0051] As shown in Table 2, the debranching degree of MDBE prepared in Example 1 of the present invention reaches 71.35% after 0.5 h of reaction, 91.48% after 2 h of reaction, and 100% after 6 h of reaction; The equation for calculating the degree of starch debranching is: [(Rs–Ra) / (Rd–Ra)]×100; Among them, Rs represents the reducing sugar in the sample after adding MDBE debranching treatment for different time periods, Rd represents the reducing sugar in the amylopectin at the end point of the reaction (6h), and Ra represents the reducing sugar in the untreated amylopectin; the standard curve of reducing sugar content was prepared using glucose as the standard.

[0052] 8. Determination of chain length distribution of the hydrolyzate of MDBE obtained in Example 1 Preparation of standard products: Weigh DP4 to DP7 from the oligosaccharide standard set, weigh 5 mg of each oligosaccharide, resuspend in 5 mL of double distilled water, and boil in a boiling water bath for 60 min. Vortex mix every 10 min. After the water bath, add 50 μL of 0.6 mol / L sodium acetate with a pH of 4.4 and 10 μL of 2% (w / v) NaN3 aqueous solution, and incubate at 37 °C for 24 h. After incubation, 0.5% (w / v) sodium borohydride aqueous solution was added, vortexed and allowed to stand for 20 h; After standing, 600 μL was taken out and dried by nitrogen blowing at 25 °C; After nitrogen blowing, it was dissolved in 30 μL of 1 mol / L NaOH aqueous solution for 60 min. Add 570 μL of water, centrifuge at 12,000 rpm for 5 min, take the supernatant, and measure the standard by ion chromatography; Sample preparation and pretreatment: Add 50 μL of purified MDBE to a 1% pullulan buffer system (final concentration is 1% (w / v), buffer is pH 7.0, 50 mmol / L Tris-HCL aqueous solution); React at 40 °C for 0.5 h, inactivate, centrifuge, take the supernatant and freeze-dry; Weigh 10 mg of the above lyophilized powder, resuspend in 5 mL of water, and boil in a boiling water bath for 60 min. Vortex mix every 10 min. After the water bath, add 50 μL of 0.6 mol / L sodium acetate with a pH of 4.4, 10 μL of 2% (w / v) NaN3 aqueous solution, and 0.5% (w / v) sodium borohydride aqueous solution, vortex mix, and let stand for 20 h; After standing, 600 μL was taken out and dried by nitrogen blowing at 25 °C; After nitrogen blowing, it was dissolved in 30 μL of 1 mol / L NaOH aqueous solution for 60 min. Add 570 μL of water, centrifuge at 12000 rpm for 5 min, take the supernatant, and measure the sample by ion chromatography; The ion chromatography parameters were as follows: Thermo ICS5000 ion chromatography system was used to analyze and detect starch using an electrochemical detector; Mobile phase A: 0.2 mol / L NaOH aqueous solution; Mobile phase B: 0.2 mol / L NaOH - 0.2 mol / L NaAC aqueous solution; The chromatographic column was a Dionex CarboPac PA200 (250*4.0mm, 10 μm) liquid chromatography column, the column temperature was 30 °C, the flow rate was 0.4 mL / min, and the injection volume was 5 μL; Elution gradient: 0 min A / B (90:10 V / V), 10 min A / B (90:10 V / V), 30 min A / B (40:60 V / V), 50 min A / B (40:60 V / V); 50.1 min A / B (90:10 V / V); 60 min A / B (90:10 V / V); the detection results are as follows: Figure 8 As shown; See also Figure 8 The free short chains of the amylopectin not treated with MDBE prepared in Example 1 were very small and the chain length polymerization degree (DP value) was concentrated in 12-18. The short chains of the amylopectin product treated with MDBE increased significantly, and the polymerization degree distribution was mainly concentrated in the DP value between 6-40, indicating that MDBE has a debranching function and can hydrolyze the side chains of amylopectin from the branching point; The branching degree of the product of amylopectin after the reaction for 30 minutes is determined, comprising the following steps: Take 5 mg of amylopectin after 30 min reaction in a centrifuge tube, add 1 mL of deuterated dimethyl sulfoxide, and react at 80 °C for 12 h; After the reaction, centrifuge at 12000 rpm for 10 min, and take the supernatant into a nuclear magnetic resonance tube for detection; The branching degree of the product was determined using a control of amylopectin not treated with MDBE; This experiment was performed using a Bruker BioSpin GmbH nuclear magnetic resonance instrument. 1 The H NMR analysis method was used for inspection, and the parameters were: scan number 32, resonance radio frequency 500.23 MHz, and NMR spectrum 1 H; The measured data were analyzed by MestReNova software, and the peak range was selected according to the sample peak time, and the final results were shown in Table 3. The following formula was used for calculation: ; Wherein, DB in the formula represents the branching degree; A represents the peak area of ​​α-1,6 glycosidic bond; B represents the peak area of ​​α-1,4 glycosidic bond; the calculation results are shown in Table 3; Table 3 Branching degree of products after MDBE treatment of amylopectin

[0053] As shown in Table 3, the branching degree of amylopectin after MDBE treatment decreased from 6.53% to 3.65%, indicating that MDBE has a debranching function.

[0054] 9. The MDBE prepared in Example 1 was combined with maltogenic amylase (BMAL) to verify the effect of promoting substrate utilization Pullulan (final concentration 0.2% (w / v)) and enzyme complex (MDBE:BMAL = 3:2, (w:w)) were added to 50 mmol / L Na2HPO4-NaH2PO4 buffer at pH 6.0; BMAL was prepared as described in reference Bacillus sp. B110 (Food Industry Science and Technology, 2023, 44(10):123-129); After incubation at 40 °C for 7 h, the reaction was terminated in a boiling water bath; centrifuged at 12,000 rpm for 2 min, and the supernatant was filtered through a 0.22 μm filter membrane; High performance liquid chromatography (HPLC) was performed using a Cosmosil Sugar-D column and a refractive index detector (RID) at 30 °C; HPLC conditions were as follows: mobile phase was acetonitrile and water (65:35, v / v), flow rate was 1 mL / min; HPLC test results were as follows Fig. 9 As shown; See also Fig. 9 The glucose (G1) and maltobiose (G2) produced by the dual enzyme combined method were 2.0 mg / mL and 1.76 mg / mL, respectively. The G1 and G2 produced by using BMAL alone were 1.82 mg / mL and 0.96 mg / mL, respectively. The glucose and maltobiose yields produced by the dual enzyme combined method were 1.1 times and 1.84 times of those when using BMAL alone, respectively. The MDBE provided by the present invention can significantly improve the hydrolysis rate of amylopectin.

[0055] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. An isoamylase gene, characterized in that The nucleotide sequence of the isoamylase gene is shown in SEQ ID NO.

1.

2. An isoamylase protein encoded by the isoamylase gene according to claim 1, characterized in that: The amino acid sequence of the isoamylase protein is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that: The recombinant vector comprises the isoamylase gene as claimed in claim 1 and an empty vector.

4. The recombinant vector according to claim 3, characterized in that The empty vector includes any one of pET-29a, pET-28a, pET-42a, pUC18, and pUC19.

5. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria is a Escherichia coli BL21DE3.

6. An isoamylase encoded by the isostarch gene according to claim 1, characterized in that: The isoamylase is capable of hydrolyzing α-1,4 glycosidic bonds and α-1,6 glycosidic bonds simultaneously.

7. The isoamylase according to claim 6, characterized in that The starch substrates hydrolyzed by the isoamylase include amylose, amylopectin, corn starch, potato starch, cassava starch, soluble starch and gamma-cyclodextrin.

8. Use of the isoamylase according to claim 6 in improving starch hydrolysis rate, characterized in that: The application comprises using the isoamylase in combination with other amylases, thereby improving the starch hydrolysis rate.

9. Use of the isoamylase according to claim 6 in starch processing, characterized in that: The applications include starch hydrolysis, food, and feed.

Images