Difunctional starch debranching enzyme and gene thereof, recombinant engineering bacterium containing gene and application of recombinant engineering bacterium

Abstract

The invention relates to the technical field of bioengineering (IPC classification number: C12N), in particular to a bifunctional starch debranching enzyme, a gene thereof, a recombinant engineering bacterium containing the gene and application of the bifunctional starch debranching enzyme. The DNA sequence of the gene is as shown in SEQ ID NO: 1. The amino acid sequence of the difunctional starch debranching enzyme is as shown in SEQ ID NO: 2. The invention provides a novel difunctional starch debranching enzyme gene, the full length of the gene is 2139bp, and 712 amino acids are encoded; the starch debranching enzyme has the characteristics of better high temperature resistance and high activity under a slightly acidic pH (Potential of Hydrogen) condition. The specific activity of the bifunctional isoamylase provided by the invention is 1.7 times that of isoamylase (PromozymeD2), and the bifunctional isoamylase can be well applied to the technical fields of grain processing, food industry, brewing, fermentation, textile industry, medicine and the like.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology (IPC classification number: C12N), and in particular to a bifunctional starch debranching enzyme and a gene thereof, a recombinant engineering bacterium containing the gene and an application thereof. Background Art

[0002] Starch is the main energy source for humans and animals, and is also an important raw material in the food, chemical, and pharmaceutical industries. Starch debranching enzymes (SDBEs) are a class of hydrolases that act on the α-1,6-glucosidic bonds of starch. In the starch processing industry, they can be used in conjunction with other amylases that hydrolyze α-1,4-glucosidic bonds to completely hydrolyze starch and improve the raw material conversion rate of starch. According to the differences between the catalytic mode and substrate specificity, starch debranching enzymes can be divided into type I pullulanases, type II pullulanases, and isoamylases (EC. 3.2.1.68). Pullulanases (EC. 3.2.1.41) belong to the glycoside hydrolase (GH13) family and can efficiently hydrolyze pullulan polysaccharides. Amyloplulanase is a debranching multidomain enzyme distributed in the glycoside hydrolase GH13 and glycoside hydrolase GH57 families (with a typical (β / α)7 barrel structure); amyloplulanase is a bifunctional endo-enzyme, which means that it can hydrolyze α-1,4 and α-1,6 glycosidic bonds in linear or branched starch and glycogen. Isoamylase also belongs to the glycoside hydrolase GH13 family, and shows high hydrolysis activity for branched starch and glycogen, but low hydrolysis activity for low molecular weight dextrins. The minimum action unit is maltotriosyl-α-1,6-maltotetraose, and it cannot hydrolyze pullulan. In the starch processing industry, pullulanase plays an important role as a debranching enzyme in the industrial starch saccharification process. The use of starch pullulanase in starch conversion can reduce processing time and cost, and increase the yield of products such as maltose, maltotriose, glucose and maltotetraose. It plays an important role as a preservative in the food, beverage and baking industries, as well as in tableware washing, laundry detergents, chemicals (such as ethanol) production, textiles, pulp and paper industries, etc.

[0003] In 1988, Amemura et al. Pseudomonas amyloderamosaThe gene of the isoamylase from the source was cloned and expressed (https: / / doi.org / 10.1006 / jmbi.1998.1992). After industrial production, the isoamylase was commercially 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 is 70600 U / mg, but the activity of IsoM is measured by iodine staining, and IsoM is expressed in yeast with a very small expression level, which makes mass production and detection difficult. At present, the enzyme has not yet been industrialized.

[0004] Chinese patent CN102533701B provides a method for increasing the yield of starch debranching enzyme by promoting the disaggregation of active protein aggregates, Chinese patent CN102978191B provides a fermentation production process for recombinant starch debranching enzyme, Chinese patent CN113667662A discloses a high-performance starch debranching enzyme chimera strain construction and its production method and application; CN1309701A discloses a starch debranching enzyme, but these prior arts do not solve the technical problems of low enzyme activity and poor stability.

[0005] At present, there are few isoamylases with independent intellectual property rights in China. Due to the low enzyme activity, lack of stability and low expression, the commercial products on the market are mainly imported enzyme preparations. Based on this, providing a starch debranching enzyme with high enzyme activity, high expression and good stability has become a technical problem to be solved in this field. Summary of the invention

[0006] The purpose of the present invention is to provide a bifunctional starch debranching enzyme and its gene, a recombinant engineered bacterium containing the gene and its application. The bifunctional starch debranching enzyme expressed by the gene of the present invention can hydrolyze α-1,4-glycosidic bonds and α-1,6-glycosidic bonds at the same time, and the bifunctional starch debranching enzyme has certain thermal stability and acid resistance, which solves the problem that the existing starch debranching enzyme mainly relies on imports.

[0007] The first aspect of the present invention provides a bifunctional starch debranching enzyme gene, the DNA sequence of which is shown in SEQ ID NO: 1. SEQ ID NO: 1 is as follows: In some preferred embodiments, the amino acid sequence encoded by the base sequence in SEQ ID NO: 1 is shown in SEQ ID NO: 2. SEQ ID NO: 2 is as follows: MRRAEVLPGKPFPLGATYDGHGVNFAVFSEHAKKVEVCLYDAQEPTRETRRFPLLETTHQVFHGYVPDLKPGTLYGLRVHGPFEPKKGFRFNPHKLLVDPYARAIHGQVDYRAPIYGHVQGGKDEDLVQDKQDDAAGVPKAVVLEDTFDWEGDTHPRVPWHQTVLYELHVKGFTKLHP RVPEALRGTYAGLGHPATIEHLKKVGVTAVELLPVHHIVDEPFLVERGLTNYWGYSTLGYFAPDARYSTAGAPGAQVDEFKGMVKALHRAGIEVILDVVYNHTCEGNQLGPTLSFKGLDNGAYYRLTEKDPRYYLDVTGTGNSWNATHPYALKLVADSLRYWVEEMHVDGFRFDLATT LGRDRHGYDTRAAFFQIIHQDPVLSRVKLISEPWDVGDFGYQVGNFPVLWSEWNGKYRDTIRRYWKGDDRQAAEIGYRLLTGSSDLYSLSGRKPSASVNFVTAHDGFTLHDLVTYNDKHNEANGEDNRDGGNDNHSWNCGVEGETGDVKINALREQQKRNFLATLFLLSQGVPMLVAGDE MGRTQKGNNNAYCQDNALSWVNWELDDTQRALLDFTCRLTRLRREQPVLRKRRFFRGAHMWDSELKDLAWFRPDGKEMRKDDWEKPYVRSLGILLGGDAIAAPDDEGNRIVGDTLLVLMNAHHEPISFLLPALEWGADWEQVVDTSTAEESQHAHTPAGGKVQVAGRSLMVLRRPSTE The amino acids encoded by the base sequence of SEQ ID NO: 1 correspond to the amino acid sequence shown in SEQ ID NO: 2.

[0008] The present invention innovatively provides a new gene and a bifunctional starch debranching enzyme obtained by expression. The DNA sequence of the gene is shown in SEQ ID NO: 1. The full length of the gene is 2139 bp, and the content of bases (G+C) accounts for 68.26%. The obtained bifunctional starch debranching enzyme encodes a 712 amino acid sequence, and the amino acid sequence is shown in SEQ ID NO: 2.

[0009] The second aspect of the present invention provides a bifunctional starch debranching enzyme expressed by the gene as described above, wherein the bifunctional starch debranching enzyme can hydrolyze α-1,4-glycosidic bonds and α-1,6-glycosidic bonds simultaneously.

[0010] The third aspect of the present invention provides a recombinant vector, wherein the recombinant vector is cloned by the bifunctional starch debranching enzyme gene as described above on an empty vector.

[0011] Preferably, the empty vector is at least one of pET-29a, pUC18, pUC19, and pGEM-T; more preferably, pET-29a.

[0012] The fourth aspect of the present invention provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium is obtained by cloning the above-mentioned bifunctional starch debranching enzyme gene and constructing a host bacterium.

[0013] In some preferred embodiments, the host bacteria is Escherichia coli; further preferably, the host bacteria is specifically Escherichia coli BL21 (DE3).

[0014] The fifth aspect of the present invention provides an application of the gene or bifunctional starch debranching enzyme as described above, which is specifically used in the fields of starch processing, food, feed, textile, medicine, papermaking, and chemical production.

[0015] A sixth aspect of the present invention provides a use of the bifunctional starch debranching enzyme as described above in hydrolyzing starch, wherein the starch is amylopectin and / or amylose.

[0016] In some preferred embodiments, the reaction temperature of the bifunctional starch debranching enzyme during hydrolysis is 45-55° C. and the pH is 5.0-6.8.

[0017] Further preferably, the reaction temperature during hydrolysis by the bifunctional starch debranching enzyme is 50° C. and the pH is 6.0.

[0018] In some preferred embodiments, the yield of oligosaccharides produced by hydrolyzing amylopectin with the bifunctional starch debranching enzyme of the present invention is 10.158% (with glucose as the standard).

[0019] In some preferred embodiments, the yield of oligosaccharides produced by hydrolyzing amylose using the bifunctional starch debranching enzyme of the present invention is 11.703% (with glucose as the standard).

[0020] Beneficial effects: The present invention provides a bifunctional starch debranching enzyme and its gene, an engineered bacterium containing the gene and its application, which have the following advantages: (1) The present invention provides a novel starch debranching enzyme gene, which has a full length of 2139 bp and a base (G+C) content of 68.26%, encoding 712 amino acids. The starch debranching enzyme encoded by the gene has bifunctional characteristics and can hydrolyze α-1,4 and α-1,6 glycosidic bonds simultaneously, significantly improving the hydrolysis efficiency of starch; (2) The starch debranching enzyme expressed by the gene of the present invention effectively solves the problem of low enzyme activity and poor stability of the existing isoamylase, fills the blind spot of domestic isoamylase, and has extremely high commercial value; (3) The starch debranching enzyme of the present invention can maintain an enzyme activity of more than 80% when incubated for 6 h and 12 h in an environment of 40-50°C and pH 4.0-8.0, respectively, and has excellent heat resistance and pH tolerance; (4) It was found that the bifunctional starch debranching enzyme of the present invention had the highest enzyme activity at 50°C and pH 6.0, with the specific activity for hydrolyzing amylopectin reaching 317.31 U / mg and the specific activity for hydrolyzing amylose reaching 55.27 U / mg, which was 1.7 times the specific activity of commercial isoamylase (Promozyme® D2); (5) The starch debranching enzyme of the present invention has a wide range of applications and has high enzyme activity for a variety of starches, especially for the hydrolysis of amylopectin and straight-chain starch, with yields of 11.7% and 10.2%, respectively. This technology has a very high driving effect on the conversion of starch and its application in the downstream end. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the electrophoresis diagram of the PCR amplification of the starch debranching enzyme gene of the embodiment of the present invention (M: DL5000 nucleic acid marker; 1: starch debranching enzyme gene PCR amplification product); Figure 2 This is the SDS-PAGE protein electrophoresis diagram of the bifunctional starch debranching enzyme of the embodiment of the present invention (M: standard protein Marker, the supplier is Beijing Kangrun Chengye Biotechnology Co., Ltd.; 1: vector pET-29a supernatant enzyme solution; 2: starch debranching enzyme supernatant enzyme solution; 3: starch debranching enzyme precipitate; 4: MIsA protein eluted with 50 mM imidazole); Figure 3This is a graph showing the optimum temperature results of the bifunctional starch debranching enzyme according to an embodiment of the present invention; Figure 4 The optimal pH result diagram of the bifunctional starch debranching enzyme in the embodiment of the present invention (the blue line corresponds to ① a citric acid-sodium citrate aqueous solution at pH 3.0-6.0; the red line corresponds to ② a Na2HPO4-NaH2PO4 aqueous solution at pH 6.0-8.0; the green line corresponds to ③ a Tris-HCl aqueous solution at pH 8.0-9.0); Figure 5 This is a graph showing the thermal stability of the bifunctional starch debranching enzyme according to an embodiment of the present invention; Figure 6 The pH stability result diagram of the bifunctional starch debranching enzyme of the embodiment of the present invention (the blue line corresponds to ① citric acid-sodium citrate aqueous solution at pH 3.0-6.0; the red line corresponds to ② Na2HPO4-NaH2PO4 aqueous solution at pH 6.0-8.0; the green line corresponds to ③ Tris-HCl aqueous solution at pH 8.0-9.0; the purple line corresponds to ④ Glycine-NaOH aqueous solution at pH 9.0-10.0); Figure 7 This is the chain length distribution of amylopectin treated with isoamylase from Pseudomonas for 24 h; Figure 8 This is the chain length distribution diagram of amylopectin treated with the bifunctional starch debranching enzyme in the embodiment of the present invention for 0.5 h; Fig. 9 This is the chain length distribution diagram of amylopectin treated with the bifunctional starch debranching enzyme in the embodiment of the present invention for 1 h; Fig.10 This is a TLC (thin layer chromatography) diagram of the hydrolysis products of different substrates treated with the bifunctional starch debranching enzyme in the examples of the present invention. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific embodiments.

[0023] Note: Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be directly obtained from commercial channels; and unless otherwise specified, the solvent in the solution is water.

[0024] Example Provided are a bifunctional starch debranching enzyme and its gene, and a recombinant engineered bacterium containing the gene, as described below.

[0025] Step 1. Gene sequence construction: A bifunctional starch debranching enzyme and its gene The DNA sequence of the bifunctional starch debranching enzyme gene is shown in SEQ ID NO:1.

[0026] The amino acid sequence of the bifunctional starch debranching enzyme is shown in SEQ ID NO:2.

[0027] ORF prediction was performed with reference to the genome sequence of Myxococcus sp. V11. Based on the amino acid sequence of starch debranching enzyme (NCBI No.: MCK8502360.1), the natural signal peptide was removed, and primers were designed based on the restriction site prediction of the nucleic acid sequence to perform PCR amplification of the full length of the starch debranching enzyme gene. 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 50.0 μL, PCR amplification conditions are pre-denaturation at 98 ℃ for 5 min, denaturation at 98 ℃ for 30 s, annealing at 63 ℃ for 22 s, extension at 72 ℃ for 2 min, a total of 30 cycles, and finally extension at 72 ℃ for 10 min. The full-length sequence of the starch debranching enzyme gene was obtained. The primers used were F and R. The PCR amplification electrophoresis of the starch debranching enzyme gene is shown in Figure 1 .

[0028] F: GGTTCCATGGCTGATATC GGATCC ATGAGGAGGGCCGAG (BamH I) R: GTGCTCGAGTGCGGCCGC AAGCTT CTCCGTCGACGGCCG (Hind III) Step 2. Construction and verification of recombinant cloning, expression vector pET-29a-MIsA A recombinant vector is cloned from the above-mentioned bifunctional starch debranching enzyme gene on an empty vector pET-29a.

[0029] Specifically, pET-29a was double-digested with BamH I and Hind III, respectively. The double-digestion system was: 2.0 μL each of BamH I and Hind III, 5.0 μL of 10×H Buffer, 20.0 μL of pET-29a, and ddH2O was added to make up to 50.0 μL. The double-digestion product was recovered using a DNA agarose gel recovery kit (supplier: Beijing Solebow Technology Co., Ltd.). The enzyme-linked system is: 4.0 μL purified PCR product (i.e., PCR amplification product prepared in Step 1), 2.0 μL pET-29a (i.e., double enzyme digestion product prepared in Step 2), 2 μL 5ⅹCE Buffer and 1 μL ExraseⅡ (5ⅹCE Buffer and ExraseⅡ suppliers are Nanjing Novozyme Biotechnology Co., Ltd.), add ddH2O to 10.0 μL, and react at 37℃ for 1 h. Add 10 μL enzyme-linked product to 100 μL E.coli DH5α competent cells melted on ice, ice bath for 30 min, heat shock in a 42℃ water bath for 90 s. Quickly transfer to an ice bath to cool for 5 min, add 800 μL liquid LB medium to each tube, and incubate at 37℃ with a shaker at 100 rpm for 60 min to revive the cells. The plate was centrifuged at 5000 rpm for 5 min, and the remaining 100 μl competent cells were spread on LB agar plates containing 50 mg / L kanamycin. The plates were inverted and cultured in a 37°C incubator for 13 h.

[0030] Multiple single colonies were extracted from the culture product, and plasmids were extracted using a plasmid mini-extraction kit (brand: OMEGA). The obtained plasmid was double-digested (double-digestion system: BamH I and Hind III 2.0 μL each, 10ⅹH Buffer 5.0 μL, plasmid 20.0 μL, ddH2O added to 50.0 μL) for verification, and the obtained recombinant plasmid was sent to Hunan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the cloned target fragment (nucleotide length of 2139 bp) was inserted into the pET-29a(+) vector, and the recombinant expression vector pET-29a(+)-MIsA was obtained. The DNA sequence of its gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein (bifunctional starch debranching enzyme) expressed by it is shown in SEQ ID NO: 2.

[0031] Step 3. Expression and purification of recombinant starch debranching enzyme Heat shock transformation (the specific steps are the same as those in Step 2) of the recombinant clone and expression vector pET-29a-MIsA (prepared in Step 2) into the host bacteria E. coli BL21 (DE3) to obtain the recombinant engineered bacteria containing the recombinant plasmid. Inoculate the positive clone single colony into 4 mL of Luria-Bertani broth (LB) medium containing 50 μg / mL kanamycin (LB medium formula: yeast powder 5.0 g / L, trypsin 10.0 g / L, NaCl 10.0 g / L (yeast extract powder and trypsin were from Beijing Aoboxing Biotechnology Co., Ltd., NaCl was from Xilong Science Co., Ltd.)), and culture at 37°C with shaking at 180 rpm for 12 h. The plasmid was extracted and verified using a plasmid mini-extraction kit (brand: OMEGA). After verification, the above bacterial solution was inoculated into a 250 mL shake flask containing 50 mL of liquid LB medium, and cultured at 37°C and 180 rpm for 8 h. It was then transferred to a 1000 mL shake flask containing 400 mL of liquid LB medium and cultured at 37°C and 180 rpm for 3.5 h. When the absorbance OD600 reached 0.5±0.1, IPTG (isopropyl-β-D-thiogalactopyranoside) was added at a final concentration of 2 mmol / L, and induced for expression at 16°C and 180 rpm for 27 h.

[0032] The culture solution was centrifuged at 5000 rpm for 0.5 h to collect the cells. The cells were resuspended and washed with 20 mmol / L Tris-HCl buffer at pH 7.0, centrifuged at 5000 rpm for 0.5 h, and the cells were recovered; then the cells were resuspended with 20 mmol / L Tris-HCl buffer at pH 7.0 (1 g of cells plus 10.0 mL of buffer), the cells were disrupted with an ultrasonic cell disruptor in an ice water bath, and centrifuged at 12000 rpm at 4°C for 10 min to obtain a crude biological extract containing starch debranching enzyme MIsA.

[0033] The crude biological extract was purified using a Ni NTA Beads 6FF gravity column to obtain the purified product of recombinant starch debranching enzyme.

[0034] The specific steps for protein purification using Ni NTA Beads 6FF gravity column are as follows: (Refer to the Ni NTA Beads 6FF gravity column instruction manual of Changzhou Tiandi Renhe Biotechnology Co., Ltd.) (1) Column balancing: Fix the Ni NTA Beads 6FF gravity column on an iron stand, remove the lower and upper plugs in turn, and drain the protective liquid of the gravity column; then add 5 column volumes of Lysis Buffer to the column tube for balancing, so that the filler is in the same buffer system as the target protein.

[0035] (2) Loading: Add the sample to the balanced gravity column. The sample retention time is 30 min to ensure that the target protein is fully in contact with the medium and improve the recovery rate of the target protein. Collect the effluent and use it for SDS-PAGE to analyze the binding of the protein.

[0036] (3) Washing: Use 15 times the column volume of Wash Bufer to wash and remove non-specifically adsorbed impurities, and collect the washing solution; use 10 times the column volume of Eluton Bufer to elute the target protein, and collect the purified product in sections (the starch debranching enzyme purification product described below is the 100 mmol imidazole elution product); (4) Elution: Use 3 column volumes of Lysis Bufer and 5 column volumes of deionized water to elute and balance the filler; store the gravity column in an equal volume of 20% (v / v) ethanol solution at 6±2°C to prevent bacterial contamination of the filler.

[0037] Performance Testing The purity and molecular weight of the purified product of recombinant starch debranching enzyme were determined by SDS-PAGE. Figure 2 ; Figure 2 M in the middle: standard protein marker, the supplier is Beijing Kangrun Chengye Biotechnology Co., Ltd.; 1: vector pET-29a supernatant enzyme solution; 2: starch debranching enzyme supernatant enzyme solution; 3: starch debranching enzyme precipitation; 4: MIsA protein eluted with 50 mM imidazole, the mass of MIsA is about 80 kDa. 2. The enzyme activity of the purified bifunctional starch debranching enzyme was determined by 3,5-dinitrosalicylic acid colorimetric method (DNS method).

[0038] The enzyme activity is defined as: one unit of enzyme activity (U) is the amount of substrate hydrolyzed to produce 1 μmol of reducing sugar per minute at 50°C.

[0039] (1) Determination of the optimum temperature The reaction system was set to 500 μL to determine the optimal enzymatic hydrolysis temperature. The purified starch debranching enzyme was added at a volume of 100 μL into a buffer system with a volume concentration of 0.5% amylopectin (the buffer was a Na2HPO4-NaH2PO4 aqueous solution with a pH of 7.0 and 50 mM). The reaction was carried out at 20°C, 30°C, 40°C, 45°C, 50°C, 55°C, and 60°C for 10 min. After the reaction, an equal volume of DNS (dinitrosalicylic acid) was added to terminate the reaction, and the absorbance at 540 nm was measured. Three parallel experiments were designed for each temperature, and the measurement results were averaged.

[0040] The results showed that at 50°C, the bifunctional starch debranching enzyme had the highest enzyme activity. The enzyme activity of the enzyme activity reaction system at this temperature 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 relative activities. The results are shown in Figure 3 shown.

[0041] (2) Determination of the optimal pH The reaction system was set to 500 μL to determine the optimal pH. The purified starch debranching enzyme was added in an amount of 100 μL to a buffer system with a final concentration of 0.5% amylopectin. The reaction was carried out in a warm bath at 50°C for 10 min in different pH buffer systems (50 mM ① pH 3.0-6.0 citric acid-sodium citrate aqueous solution; ② pH 6.0-8.0 Na2HPO4-NaH2PO4 aqueous solution; ③ pH 8.0-9.0 Tris-HCl aqueous solution; ④ pH 9.0-10.0 Glycine-NaOH aqueous solution). After the reaction, an equal volume of DNS was added to terminate the reaction, and the absorbance at a wavelength of 540 nm was measured. Three parallel experiments were designed for each pH, ​​and the measurement results were averaged.

[0042] The results showed that at pH = 6.0, the bifunctional starch debranching enzyme had the highest enzyme activity. The enzyme activity of the enzyme activity reaction system at this pH value 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 the highest enzyme activity system were taken as relative activities. The results are shown in Figure 4 As shown (the blue line corresponds to ① citric acid-sodium citrate aqueous solution with a pH of 3.0-6.0; the red line corresponds to ② Na2HPO4-NaH2PO4 aqueous solution with a pH of 6.0-8.0; the green line corresponds to ③ Tris-HCl aqueous solution with a pH of 8.0-9.0;).

[0043] (3) Determination of thermal stability The reaction system was set to 500 μL to determine the thermal stability. The purified starch debranching enzyme was kept at 20℃, 30℃, 40℃, and 50℃ for 1 h, 3 h, 6 h, 9 h, and 12 h, respectively. Then, 100 μL was added to a buffer system with a final concentration of 0.5% amylopectin (the buffer was a Na2HPO4-NaH2PO4 aqueous solution with a pH of 6.0 and 50 mM) and incubated at 50℃ for 10 min. Three parallel experiments were designed for each temperature. After the reaction, an equal volume of DNS was added to terminate the reaction, and the absorbance at 540 nm was measured to determine the residual enzyme activity of the enzyme. The average of the measurement results was taken, and the relative enzyme activity was calculated with the enzyme activity without incubation treatment as 100%.

[0044] The test results showed that temperature had a thermal activation effect on the activity of the starch debranching enzyme. The enzyme activity increased after being placed at 20℃, 30℃, 40℃, and 50℃ for 3 hours. There was a significant heat shock effect after being kept at 30℃ and 50℃ for 3 hours. The relative enzyme activity after being kept at different temperatures for 1 hour was significantly higher than that of the control group. The activity could be tolerated at 50℃ for 9 hours without a significant decrease, but it dropped sharply after 9 hours. The results are as follows: Figure 5 It can be seen that the bifunctional starch debranching enzyme of the present invention has excellent thermal stability below 50°C.

[0045] (4) Determination of pH stability The reaction system was set to 500 μL to determine the pH stability. The purified starch debranching enzyme was added in an amount of 200 μL and placed under different pH conditions (50 mM citric acid-sodium citrate aqueous solution at pH 3.0-6.0; ② Na2HPO4-NaH2PO4 aqueous solution at pH 6.0-8.0; ③ Tris-HCl aqueous solution at pH 8.0-9.0; ④ Glycine-NaOH aqueous solution at pH 9.0-10.0) for 12 h. Then, it was added to a buffer system with a final concentration of 0.5% amylopectin (pH 6.0, 50 mM Na2HPO4-NaH2PO4 aqueous solution) and reacted at 50°C for 10 min. Three parallel tests were designed for each pH. After the reaction, an equal volume of DNS was added to terminate the reaction, and the absorbance at 540 nm was measured to determine the residual enzyme activity of the enzyme, and the average value of the measurement results was taken.

[0046] The relative enzyme activity was calculated with the enzyme activity without pH treatment as 100%. The results showed that the residual enzyme activity remained above 80% in the buffer system of pH 4.0-8.0. Figure 6It can be seen that the bifunctional starch debranching enzyme of the present invention has excellent stability under the conditions of pH 4.0-8.0 (the blue line corresponds to ① citric acid-sodium citrate aqueous solution at pH 3.0-6.0; the red line corresponds to ② Na2HPO4-NaH2PO4 aqueous solution at pH 6.0-8.0; the green line corresponds to ③ Tris-HCl aqueous solution at pH 8.0-9.0; the purple line corresponds to ④ Glycine-NaOH aqueous solution at pH 9.0-10.0).

[0047] 3. Purified bifunctional starch debranching enzyme was used to study the specificity of various types of starch Specifically, 0.414 mg of the recombinant starch debranching enzyme was reacted with amylopectin, soluble starch, potato starch, cassava starch, corn starch, and amylose at 50 °C for 10 min, and the enzyme activity was measured. The test results are recorded in Table 1. The Km value is equal to the substrate concentration corresponding to the enzyme reaction rate reaching half of the maximum reaction rate.

[0048] Table 1

[0049] Among them, - means not detected.

[0050] As can be seen from Table 1, the bifunctional starch debranching enzyme of the present invention has a hydrolysis effect on the above six substrates (branched starch, soluble starch, potato starch, cassava starch, corn starch, and amylose), especially the strongest hydrolysis effect on branched starch, with a specific enzyme activity of 317.31±2.34U / mg.

[0051] 4. Distribution of degree of polymerization of bifunctional starch debranching enzyme hydrolysis products The purified starch debranching enzyme was added in an amount of 40.0 μL to a 0.5% amylopectin buffer system (the buffer was a Na2HPO4-NaH2PO4 aqueous solution at pH 6.0), reacted at 50°C for 0.5 h and 1 h, inactivated by centrifugation, and the supernatant was freeze-dried.

[0052] Preparation of standard: Weigh 5 mg of DP4-DP7 (from Sigma) of the oligosaccharide standard set, resuspend in 5 mL double distilled water, boil in a water bath for 60 min, and vortex intermittently; add 50 μL of sodium acetate (0.6 M, pH 4.4), NaN3 aqueous solution (10 μL, 2% w / v) and 10 μL of isoamylase (1400 U), and incubate at 37°C for 24 h. Add 0.5% (w / v) sodium borohydride aqueous solution, vortex mix and place for 20 h. Take 600 μL in a centrifuge tube and dry it with nitrogen at room temperature (25°C). Dissolve in 30 μL of 1M NaOH aqueous solution for 60 min, then add 570 μL of water to dilute, centrifuge at 12000 rpm for 5 min, take the supernatant and load it, and measure it by ion chromatography.

[0053] Sample pretreatment: Weigh 10 mg of purified amylopectin, resuspend in 5 mL of water, boil in a water bath for 60 min, and vortex intermittently; add 50 μL of sodium acetate (0.6 M, pH 4.4), NaN3 (10 μL, 2% w / v) aqueous solution and 10 μL of isoamylase (1400U), and incubate at 37°C for 24 h. Add 0.5% (w / v) sodium borohydride aqueous solution, vortex mix and place for 20 h. Take 600 μL in a centrifuge tube and dry it with nitrogen at room temperature (25°C). Dissolve in 30 μL of 1 M NaOH aqueous solution for 60 min, then add 570 μL of water to dilute, centrifuge at 12000 rpm for 5 min, take the supernatant and load it, and measure it by ion chromatography.

[0054] Chromatographic conditions: Thermo ICS5000 ion chromatography system (ICS5000+, Thermo Fisher Scientific, USA) was used to analyze starch using an electrochemical detector. A Dionex™ CarboPac™ PA200 (250*4.0mm, 10 um) liquid chromatography column was used with an injection volume of 5 μL. Mobile phase A: 0.2 M NaOH aqueous solution, phase B: 0.2 M NaOH / 0.2 M NaAC aqueous solution; column temperature was 30℃, flow rate was 0.4 mL / min; elution gradient: 0 min A / B (90:10 V / V), 10 min A / B (90:10V / 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 components were analyzed and detected using an electrochemical detector.

[0055] The ion chromatography results of the hydrolyzate of amylopectin treated with isoamylase from Pseudomonas for 24 h are shown in Figure 7 The ion chromatography test results of the hydrolyzate of amylopectin after treatment with starch debranching enzyme MISA for 0.5 h and 1 h are shown in Figure 8 , Fig. 9 . These data results all show that the number of chains in DP 6-13 increased significantly, while the number of chains in DP 14-50 decreased. From the measurement results, it can be seen that the shorter the reaction time between starch debranching enzyme MIsA and amylopectin, the more short chains appear. As the reaction time increases, starch debranching enzyme MIsA mainly acts on short chains, and the product is less polymerized.

[0056] 5. Product analysis of different substrates hydrolyzed by bifunctional starch debranching enzyme The purified starch debranching enzyme was added in an amount of 50.0 μL to a 1% final concentration of amylopectin, pullulan, amylose and β-cyclodextrin buffer system (all prepared with Na2HPO4-NaH2PO4 buffer at pH 6.0), and reacted at 50°C for 9 h, 1 h, 9 h, and 1 h, respectively. After the reaction was completed, the reaction was terminated in boiling water, and the supernatant was filtered through a 0.2 μm filter membrane to remove impurities after centrifugation at 12000 rpm for 2 min, and then TLC was performed.

[0057] A mixture of glucose and maltooligosaccharide standards (G2–G6) was used as the standard. The TLC analysis conditions were as follows: the developing agent for TLC analysis was n-butanol / methanol / water = 4:2:1 (V / V / V), and the color developer was concentrated sulfuric acid / methanol = 1:9 (V / V); the concentration of concentrated sulfuric acid was 98%. When the developing agent reached 1-2 cm from the top of the thin plate, the thin plate was taken out, blown dry, and then sprayed with the color developer evenly. After blowing dry again, the thin plate was placed in an oven at 90°C for color development. The results are as follows: Fig.10 shown.

[0058] Where Std represents glucose and maltooligosaccharide standards (G2–G6); reactions on substrates with (+) and without (-) enzyme.

[0059] The action mode of starch debranching enzyme on various substrates was studied by thin layer chromatography (TLC) analysis. The hydrolysis product results were as follows Fig.10 As shown; the results showed that the bifunctional starch debranching enzyme hydrolyzed pullulan into maltotriose and did not hydrolyze β-cyclodextrin; the hydrolysis products of amylopectin and straight-chain starch produced linear oligosaccharides in addition to glucose and maltooligosaccharides (G2-G6). The substrate spectrum results showed that MIsA could hydrolyze α-1,4-glycosidic bonds and α-1,6-glycosidic bonds at the same time.

[0060] 6. Analysis of the yield of amylopectin and amylose hydrolyzed by bifunctional starch debranching enzyme 2.5 mg of amylopectin and amylose were respectively added to the reaction system (the reaction system was a Na2HPO4-NaH2PO4 aqueous solution with a pH of 6.0), 50.0 μL of purified starch debranching enzyme was added, and the mixture was incubated at 50°C for 9 h. Three parallel experiments were designed for each experiment. After the reaction was completed, an equal volume of DNS was added to terminate the reaction, and the absorbance at 540 nm was measured. The absorbance was substituted into the DNS standard curve to calculate the amount of oligosaccharide produced. The measurement results are recorded in Table 2. It can be seen that the starch debranching enzyme provided by the present invention has a high hydrolysis efficiency, with a yield of 10.158% on amylose and 11.703% on amylopectin.

[0061] Table 2

Claims

1. A bifunctional starch debranching enzyme gene, characterized in that The DNA sequence of the gene is shown in SEQ ID NO:

1.

2. A bifunctional starch debranching enzyme expressed by the gene according to claim 1, characterized in that: The amino acid sequence of the bifunctional starch debranching enzyme is shown in SEQ ID NO:

2.

3. The bifunctional starch debranching enzyme expressed by the gene according to claim 2, characterized in that The bifunctional starch debranching enzyme can hydrolyze α-1,4-glycosidic bonds and α-1,6-glycosidic bonds simultaneously.

4. A recombinant vector, characterized in that: The recombinant vector is cloned by the bifunctional starch debranching enzyme gene according to claim 1 on an empty vector.

5. A recombinant vector according to claim 4, characterized in that: The empty vector is at least one of pET-29a, pUC18, pUC19, and pGEM-T; preferably pET-29a.

6. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria is obtained by cloning the bifunctional starch debranching enzyme gene as claimed in claim 1 and constructing a host bacteria.

7. The recombinant engineered bacterium according to claim 6, characterized in that The host bacteria is Escherichia coli; preferably, the host bacteria is Escherichia coli BL21(DE3) (supplier: Invitrogen).

8. Use of the gene according to claim 1 or the bifunctional starch debranching enzyme according to claim 2, characterized in that: It is specifically used in starch processing, food, feed, textile, medicine, papermaking, and chemical production.

9. Use of the bifunctional starch debranching enzyme according to claim 2 in hydrolyzing starch, characterized in that: The starch is amylopectin and / or amylose.

10. Use of the bifunctional starch debranching enzyme according to claim 9 in hydrolyzing starch, characterized in that: The reaction temperature of the bifunctional starch debranching enzyme during hydrolysis is 45-55° C. and the pH is 5.0-6.8.

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