Isoamylase and its gene, engineered bacteria containing the gene and its application

Abstract

The present invention discloses an isoamylase, its gene, an engineered bacterium containing the gene and its application; the present invention provides an isoamylase gene, the nucleotide sequence of which is shown in SEQ ID NO.1. The full length of this gene is 2130 bp, the G+C content is 69.11%, encoding 709 amino acids, and the amino acid sequence is shown in SEQ ID NO.2. The isoamylase provided by the present invention has an adaptability to a wide pH range. When reacting at pH 6.0 - 8.0, it has relatively high enzyme activity, and when reacting at 40 °C and pH 7.0, the enzyme activity is the highest. The isoamylase provided by the present invention has a hydrolyzing effect on both α-1,4 and α-1,6 glycosidic bonds. The specific activity of hydrolyzing corn starch is 250.46 U / mg. When used in combination with maltogenic amylase BMAL, it can improve the utilization efficiency of maltogenic amylase BMAL for branched-chain substrates.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to an isoamylase, its gene, an engineered bacterium containing the gene, and their applications. Background Art

[0002] Starch debranching enzymes (SDBEs) are a class of hydrolases 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 increase the starch hydrolysis rate.

[0003] According to the differences in catalytic mode and substrate specificity, starch debranching enzymes are classified into pullulanase (EC. 3. 2.1. 41, type I pullulanase), amylopullulanase (EC. 3. 2. 1. 41, type II pullulanase), and isoamylase (EC.3.2.1.68). Pullulanase has a typical ( β / α )8-barrel structure and belongs to the glycoside hydrolase GH13 family. Pullulanase can efficiently hydrolyze pullulan polysaccharide, and the minimum action unit is maltosyl-α-1,6-maltose, and its hydrolysis activity on large molecular weight amylopectin is relatively low. In the Carbohydrate-Active Enzyme database (CAZY), amylopullulanase is distributed in the glycoside hydrolase GH13 ( β / α )8-barrel) and the glycoside hydrolase GH57 family ( β / α )7-barrel); amylopullulanase is a bifunctional hydrolytic debranching enzyme that can catalyze the hydrolysis of both α-1,4-glycosidic bonds and α-1,6-glycosidic bonds. Isoamylase also belongs to the glycoside hydrolase GH13 family, shows relatively high hydrolysis activity on amylopectin and glycogen, has relatively low hydrolysis activity on low molecular weight dextrin, and the minimum action unit is maltotriosyl-α-1,6-maltotetraose, and it cannot hydrolyze pullulan polysaccharide.

[0004] Since the gene of the isoamylase from Pseudomonas amyloderamosa was first cloned and expressed, the commercial name of this isoamylase after industrial production is Promozyme®D2, but the specific enzyme activity of Promozyme®D2 is only 180 U / mg. The specific enzyme activity of the isoamylase IsoM from Myxococcus xanthus EGB is 70600 U / mg, and it has no hydrolysis effect on amylose. The activity assay method of IsoM is the iodine staining method, and IsoM is expressed in yeast with a very low expression level, and this enzyme has not been industrially produced yet.

[0005] Currently, there are few existing isoamylases, and there are problems such as single function, low enzyme activity, and low expression level. Moreover, the current 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 object of the present invention is to provide an isoamylase, its gene, an engineered bacterium containing the gene and its application; the isoamylase provided by the present invention can simultaneously hydrolyze α-1,4 glycosidic bonds and α-1,6 glycosidic bonds. When used in combination with other amylases, it can improve the starch hydrolysis rate and can be applied to genetic engineering fields such as starch, food, and feed.

[0007] In the first aspect, the present invention provides an isoamylase gene, and the nucleotide sequence of the isoamylase gene is as shown in 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 the second aspect, the present invention provides an isoamylase protein encoded by the isoamylase gene, and the amino acid sequence of the isoamylase protein is as shown in SEQ ID NO.2.

[0012] In the third aspect, the present invention provides a recombinant vector, and the recombinant vector 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 the fourth aspect, the present invention provides a recombinant engineered bacterium, and the recombinant engineered bacterium includes the isoamylase gene, Escherichia coli BL21 DE3.

[0016] In the fifth aspect, the present invention provides an isoamylase encoded by the isoamylase gene, and the isoamylase can simultaneously hydrolyze 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 the sixth aspect, the present invention provides an application of an isoamylase in improving the starch hydrolysis rate, and the application includes using the isoamylase in combination with other isoamylases to improve the starch hydrolysis rate.

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

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

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention provides a brand-new isoamylase gene. The nucleotide sequence of the gene is shown in SEQ ID NO.1. The full length of the gene is 2130 bp, the G+C content is 69.11%, and it encodes 709 amino acids in total. The amino acid sequence is shown in SEQ ID NO.2.

[0023] (2) The brand-new isoamylase provided by the present invention is encoded by the isoamylase gene and has bifunctional characteristics. It can simultaneously hydrolyze α-1,4 glycosidic bonds and α-1,6 glycosidic bonds, and has the highest enzyme activity under the conditions of 40 °C and pH 7.0.

[0024] (3) The isoamylase provided by the present invention can improve the starch hydrolysis rate. 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. Moreover, the isoamylase provided by the present invention still retains more than 70-75% of its activity after being placed at 35-40 °C for 12 h. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the PCR amplification electrophoresis pattern of the isoamylase gene;

[0026] Figure 2 It is the SDS-PAGE protein electrophoresis pattern of the isoamylase prepared in Example 1;

[0027] Figure 3 It is the determination result of the optimal temperature of the isoamylase prepared in Example 1;

[0028] Figure 4 It is the determination result of the optimal pH of the isoamylase prepared in Example 1;

[0029] Figure 5 It is the determination result of the thermal stability of the isoamylase prepared in Example 1;

[0030] Figure 6 It is the determination result of the pH stability of the isoamylase prepared in Example 1;

[0031] Figure 7 It is the thin layer chromatography analysis result of the hydrolysis products of different substrates by the isoamylase prepared in Example 1;

[0032] Figure 8 Analysis of the degree of polymerization of the hydrolysis products of amylopectin by the isoamylase prepared in Example 1;

[0033] Figure 9 Results of the combined use of the isoamylase prepared in Example 1 and maltogenic amylase BMAL to improve the utilization efficiency of amylopectin. Detailed implementation mode

[0034] The present invention will be further described through the accompanying drawings of the specification and the following examples.

[0035] 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 as shown in SEQ ID NO.1.

[0036] In some embodiments, the full length of the nucleotide sequence of the isoamylase gene is 2130 bp.

[0037] In some embodiments, the G + C content of the isoamylase gene is 69.11%.

[0038] In some embodiments, the isoamylase gene encodes a total of 709 amino acids.

[0039] In a second aspect, an isoamylase protein encoded by the isoamylase gene is provided in an embodiment of the present invention, and the amino acid sequence of the isoamylase protein is as shown in SEQ ID NO.2.

[0040] In a third aspect, a recombinant vector is provided in an embodiment of the present invention, and the recombinant vector includes the isoamylase gene and an empty vector.

[0041] In some embodiments, the empty vector includes any one of pET-29a, pET-28a, pET-42a, pUC18, and pUC19.

[0042] Specifically, the empty vector is pET-29a.

[0043] In a fourth aspect, a recombinant engineering bacterium is provided in an embodiment of the present invention, and the recombinant engineering bacterium includes the isoamylase gene, Escherichia coli BL21 DE3.

[0044] In a fifth aspect, an isoamylase encoded by the isoamylase gene is provided in an embodiment of the present invention, and the isoamylase can simultaneously hydrolyze α-1,4 glycosidic bonds and α-1,6 glycosidic bonds.

[0045] In some embodiments, the optimal reaction temperature of the isoamylase is 40 °C, and the optimal reaction pH is 7.0.

[0046] In a sixth aspect, an embodiment of the present invention provides an application of isoamylase in improving the starch hydrolysis rate, and the application includes using the isoamylase in combination with other amylases to improve the starch hydrolysis rate.

[0047] In some embodiments, the other amylases include maltogenic amylase.

[0048] In a seventh aspect, an embodiment of the present invention provides an application of an isoamylase gene in the starch processing industry, and the application includes starch hydrolysis, food, and feed processing industries.

[0049] In some embodiments, the composition of the LB medium is: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L.

[0050] The materials, reagents, etc. used in the embodiments of the present invention are all commercially available; the vector pET-29a is from Invitrogen; Escherichia coli Escherichia coli BL21(DE3) is from Invitrogen;

[0051] The Cosmosil Sugar-D chromatographic column is from Nacalai Tesuque, Kyoto, Japan; the restriction endonucleases are from Takara; the seamless cloning kit is from Nanjing Novoprotein; the oligosaccharide standard kit is from Sigma.

[0052] Example 1

[0053] Embodiment 1 of the present invention provides a method for cloning an isoamylase gene and constructing an expression vector, including the following steps:

[0054] S1. PCR amplification of the isoamylase gene

[0055] S11. Refer to the genome sequence of Myxococcus Myxococcus sp. V11 for prediction; based on the amino acid sequence of isoamylase (named MDBE), primers are designed according to the restriction sites of the nucleic acid sequence. The upstream primer F sequence is as shown in SEQ ID NO.3, and the downstream primer R sequence is as shown in SEQ ID NO.4; orf

[0056] S12. According to the primers designed in S11, perform PCR amplification of the full length of the isoamylase gene ( mdbe );

[0057] The PCR reaction system is as follows:

[0058] Myxococcus1.0 μL of total DNA of sp.V11, 1.0 μL each of primer F and R, 25.0 μL of PrimeSTAR® Max DNA Polymerase, and ddH2O was added to make the total volume of the system 50.0 μL;

[0059] The PCR amplification conditions were:

[0060] 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 were carried out, and finally extension at 72 °C for 10 min; the full-length sequence was obtained, and the electrophoresis result was as mdbe shown, Figure 1 In Figure 1 lane M was DL5000 nucleic acid Marker, and lane 1 was the PCR amplification product of the isoamylase gene;

[0061] S2, construction and verification of the recombinant vector pET-29a- mdbe

[0062] S21. Double digestion of pET-29a was carried out to obtain the double digestion product;

[0063] The double digestion system was: BamH I 2.0 μL, Hind III 2.0 μL, 10×H Buffer 5.0 μL, pET-29a 20.0 μL, and ddH2O was added to make the total volume of the system 50.0 μL;

[0064] S22. The double digestion product was recovered by a DNA agarose gel recovery kit and ligated with the purified mdbe PCR amplification product;

[0065] The ligation system was: 4.0 μL of purified mdbe PCR amplification product, 2.0 μL of double digestion product, 2.0 μL of 5ⅹCE Buffer, 1.0 μL of ExnaseⅡ, and ddH2O was added to make the total volume of the system 10.0 μL; the ligation system was incubated at 37 °C for 1 h to obtain the ligation product;

[0066] S23. 10 μL of the ligation product was added to 100 μL of E.coli DH5α competent cells and incubated on ice for 30 min; after heat shock in a 42 °C water bath for 90 s, it was quickly transferred to an ice bath for cooling for 5 min;

[0067] S24. Add 800 μL of liquid LB medium to the cooled competent cell system and incubate at 37 °C on a shaker at 110 rpm for 45 min;

[0068] S25. After incubation, centrifuge at 8000 rpm for 5 min. Take 100 μL of competent cells and spread them on an LB agar plate containing 50 mg / L kanamycin. Invert the plate and culture it in an incubator at 37 °C for 14 h;

[0069] S26. Pick multiple single colonies from the transformation plate and extract the plasmid using a plasmid miniprep kit;

[0070] S27. Double-digest the plasmid obtained in step S26;

[0071] The double-digestion system is as follows: BamH I 1.0 μL, Hind III 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;

[0072] 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 expressed isoamylase protein is shown in SEQ ID NO.2;

[0073] S3. Expression and purification of recombinant isoamylase

[0074] S31. Heat-shock transform the pET-29a- mdbe obtained in step S28 into the host bacterium E.coli BL21(DE3) to obtain a recombinant engineering bacterium containing the recombinant plasmid;

[0075] S32. Inoculate a single colony of the positive clone into 4 mL of LB medium (50 μg / mL kanamycin), shake culture at 37 °C and 180 rpm for 12 h. After incubation, use a plasmid miniprep kit to extract the plasmid and verify it;

[0076] S33. After verification, inoculate the bacterial solution obtained after the incubation in step S32 into 4 mL of LB medium, shake culture at 37 °C and 180 rpm for 8 h. After incubation, transfer it to 400 mL of LB medium and shake culture at 37 °C and 180 rpm for 3.5 h;

[0077] S34. After the cultivation is completed, measure the OD 600 When it reaches 0.4 - 0.6, add IPTG (final concentration is 2 mmol / L), and induce expression at 16 °C and 180 rpm for 24 h;

[0078] S35. After the induction expression is completed, centrifuge the culture solution at 4000 rpm for 0.5 h to collect the thalli;

[0079] S36. Resuspend and wash the thalli with a citrate buffer solution with a concentration of 20 mmol / L and a pH of 6.0, then centrifuge at 12000 rpm for 0.25 h to collect the thalli;

[0080] S37. In an ice bath, add a citrate buffer solution with a concentration of 20 mmol / L and a pH of 6.0, and use an ultrasonic crusher to break the cells;

[0081] S38. After the crushing is completed, centrifuge at 4 °C and 12000 rpm for 10 min to obtain the crude MDBE extract;

[0082] S39. Purify the crude MDBE extract using a Ni-NTA affinity chromatography column (from Changzhou Tiandi Renhe Biotechnology Co., Ltd.) to obtain the purified recombinant isoamylase. Determine the purity and molecular weight of the recombinant isoamylase by SDS-PAGE. The purity identification results are as Figure 2 shown. The molecular weight is approximately 80 kDa; Figure 2 In lane 1 of E.coli is the protein molecular weight standard, and lane 2 is the supernatant enzyme solution of BL21(DE3) containing the empty vector pET-29a E.coli BL21(DE3) supernatant enzyme solution, lane 3 is the supernatant enzyme solution of BL21(DE3) containing the isoamylase gene E.coli BL21(DE3) precipitate, and lane 5 is the purified MDBE protein eluted with 100 mmol / L imidazole.

[0083] Performance verification

[0084] 1. The optimal reaction temperature of the MDBE prepared in Example 1

[0085] D1. The reaction system is 500 μL: 50 μL purified MDBE, 450 μL buffer system of amylopectin (final concentration is 0.5% (w / v));

[0086] D2. Under the conditions of pH 7.0 and 50 mmol / L Tris-HCL, react at different temperatures (30 °C, 35 °C, 40 °C, 45 °C, 50 °C) for 10 min;

[0087] D3. After the reaction was completed, an equal volume of DNS was added to the reaction system to terminate the reaction, and the absorbance at 540 nm was measured. Three replicates were set for each temperature, and the average value was taken;

[0088] The results showed that isoamylase had the highest enzyme activity at 40 °C. Therefore, the enzyme activity of the enzyme activity reaction system at 40 °C was set as the relative activity of 100%, and the absorbance values of the enzyme activity reaction systems at other temperatures and that of this highest enzyme activity system were used as the relative activities. The results were as Figure 3 shown.

[0089] 2. Optimal reaction pH value of MDBE prepared in Example 1

[0090] F1. The reaction system was 500 μL: 50 μL of purified MDBE and 450 μL of a buffer system of amylopectin (final concentration 0.5% (w / v));

[0091] F2. React at 40 °C for 10 min under different pH conditions (5.0, 6.0, 7.0, 8.0, 9.0); when the pH was 5.0 - 6.0, it was a citric acid - sodium citrate system; when the pH was 7.0 - 9.0, it was a Tris - HCl system;

[0092] F3. After the reaction was completed, an equal volume of DNS was added to the reaction system to terminate the reaction, and the absorbance at 540 nm was measured. Three replicates were set for each pH condition, and the average value was taken;

[0093] The results showed that isoamylase had a wide pH adaptability (6.0 - 8.0). At pH 7.0, isoamylase had the highest enzyme activity. Therefore, the enzyme activity of the enzyme activity reaction system at pH 7.0 was set as the relative activity of 100%, and the absorbance values of the enzyme activity reaction systems at other pH values and that of this highest enzyme activity system were used as the relative activities. The results were as Figure 4 shown.

[0094] 3. Thermal stability of MDBE prepared in Example 1

[0095] G0. Treat purified MDBE at different temperatures: Purified MDBE was treated at 20, 30, 35, 40, 45 °C for 0.5 h, 1 h, 3 h, 6 h, 12 h;

[0096] G1. The reaction system was 500 μL: 50 μL of purified MDBE treated at different temperatures and 450 μL of a buffer system (50 mmol / L Tris - HCl (pH 7.0), containing amylopectin with a final concentration of 0.5% (w / v));

[0097] G2. React at 40 °C for 10 min; after the reaction ends, add 500 μL of DNS to terminate the reaction, measure the absorbance at 540 nm, measure the residual enzyme activity of the enzyme, set 3 replicates for each treatment under different conditions, and take the average value. Take the enzyme activity of MDBE stored at 0 °C as 100%, and calculate the relative enzyme activity of MDBE after different temperature treatments;

[0098] The results show that after incubation at 20 - 40 °C for 12 h, the MDBE provided by the present invention still retains 70 - 80% of its activity. Incubation at 45 °C for 1 h causes a sharp decline in enzyme activity. The results are as Figure 5 shown. The MDBE provided by the present invention has good stability at 0 - 40 °C.

[0099] 4. pH stability of the MDBE prepared in Example 1

[0100] H0. Purify MDBE under different pH treatments: Store the purified MDBE at different pH values (5.0, 6.0, 7.0, 8.0, 9.0) for 12 h; 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;

[0101] H1. The reaction system is 500 μL: 50 μL of the purified MDBE treated under different pH values, 450 μL of the buffer system of amylopectin (final concentration 0.5% (w / v));

[0102] H2. React in 50 mmol / L Tris - HCl (pH 7.0) at 40 °C for 10 min;

[0103] H3. After the reaction ends, add DNS with the same volume as the reaction system to terminate the reaction, measure the absorbance at 540 nm, measure the residual enzyme activity of the enzyme, set 3 replicates for each condition of different pH treatments, and take the average value. Take the enzyme activity of MDBE without different pH treatments as 100%, and calculate the relative enzyme activity of MDBE after different pH treatments;

[0104] The results show that in the buffer system with pH 6.0, the MDBE provided by the present invention has the best stability. The results are as Figure 6 shown.

[0105] 5. Specificity of the MDBE prepared in Example 1 for different substrates. The MDBE obtained in Example 1 of the present invention was respectively applied to 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:

[0106] Table 1 Specificity of MDBE prepared in Example 1 for different substrates

[0107]

[0108] As can be seen from Table 1, the isoamylase provided by the present invention has no hydrolytic effect on pullulan and β-cyclodextrin among the above nine substrates, and has an effect on the others, and has the strongest hydrolytic effect on corn starch.

[0109] 6. Analysis of enzyme reaction products of MDBE prepared in Example 1 for different substrates

[0110] Add 250 μL of purified MDBE to pullulan, amylopectin, amylose, γ-cyclodextrin, β-cyclodextrin, α-cyclodextrin, maltotetraose, and maltohexaose buffer systems respectively. The final concentration of the substrate is 1% (w / v); the buffer system is prepared with 50 mmol / L Tris-HCl buffer solution with a pH of 7.0;

[0111] At 40 °C, after reacting for 2 h, terminate the reaction in a boiling water bath; centrifuge at 12000 rpm for 1 min, take the supernatant and filter it through a 0.22 μm filter membrane, and detect it by thin layer chromatography (TLC);

[0112] The specific TLC analysis conditions are as follows: the developing agent is n-butanol / methanol / water = 4:2:1 (v / v / v), and the coloring agent is concentrated sulfuric acid / methanol = 1:9 (v / v); the concentration of concentrated sulfuric acid is 98%;

[0113] When the developing agent reaches 1-2 cm from the upper end of the thin plate, take out the thin plate, dry it, and evenly spray the coloring agent; dry it again, place the thin plate in an oven at 90 °C for coloring;

[0114] The TLC detection results are as Figure 7 shown, where Std represents the standard product, and Std is a mixture of standard products of glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), and maltohexaose (G6); Figure 7 The "+" in 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;

[0115] See Figure 7, the mode of action 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 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 maltose, maltotriose, and maltotetraose; the hydrolysis products of maltotetraose, maltohexaose, amylopectin, and amylose included maltooligosaccharides (G2–G6) and linear oligosaccharides.

[0116] 7. The debranching degree of amylopectin by MDBE prepared in Example 1

[0117] The volume of the reaction system was 500 μL: 25 μL of MDBE prepared in Example 1, 475 μL of 50 mmol / L Tris-HCl buffer solution with pH 7.0 (containing amylopectin with a final concentration of 0.2% (w / v));

[0118] At 40 °C, the reaction was carried out for 0.25 h, 0.5 h, 1 h, 2 h, 3 h, and 6 h respectively. After the reaction was completed, 500 μL of DNS was added to terminate the reaction, and the amount of reducing sugar produced was measured by the DNS method. Using glucose as the standard curve, the results are shown in Table 2:

[0119] Table 2 The debranching degree of MDBE prepared in Example 1

[0120]

[0121] As can be seen from Table 2, the debranching degree of MDBE prepared in Example 1 of the present invention reached 71.35% after 0.5 h of reaction, 91.48% after 2 h of reaction, and 100% after 6 h of reaction;

[0122] The equation for calculating the debranching degree of starch is:

[0123] [(Rs–Ra) / (Rd–Ra)]×100;

[0124] Where Rs represents the reducing sugar in the sample after debranching treatment with MDBE for different times, Rd represents the reducing sugar in amylopectin at the reaction end point (6 h), and Ra represents the reducing sugar in untreated amylopectin; the standard curve of the reducing sugar content was made using glucose as the standard.

[0125] 8. Determination of the chain length distribution of the hydrolysis products of MDBE prepared in Example 1

[0126] Preparation of standard products:

[0127] Weigh 5 mg of each of the DP4 to DP7 in the oligosaccharide standard set, resuspend in 5 mL of double-distilled water, place in a boiling water bath for 60 min, and vortex every 10 min;

[0128] After the water bath, add 50 μL of sodium acetate with a concentration of 0.6 mol / L and a pH of 4.4, 10 μL of 2% (w / v) aqueous NaN3 solution, and incubate at 37 °C for 24 h;

[0129] After incubation, add 0.5% (w / v) aqueous sodium borohydride solution, vortex and let stand for 20 h;

[0130] After standing, take 600 μL, dry under nitrogen at 25 °C;

[0131] After nitrogen drying, dissolve in 30 μL of 1 mol / L aqueous NaOH solution and react for 60 min;

[0132] Add 570 μL of water, centrifuge at 12000 rpm for 5 min, take the supernatant, and determine the standard by ion chromatography;

[0133] Sample preparation and pretreatment:

[0134] Add 50 μL of purified MDBE to a 1% amylopectin buffer system (final concentration 1% (w / v), buffer is 50 mmol / L Tris-HCL aqueous solution with pH 7.0);

[0135] React at 40 °C for 0.5 h, inactivate, centrifuge, take the supernatant and lyophilize;

[0136] Weigh 10 mg of the above lyophilized powder, resuspend in 5 mL of water, place in a boiling water bath for 60 min, and vortex every 10 min;

[0137] After the water bath, add 50 μL of sodium acetate with a concentration of 0.6 mol / L and a pH of 4.4, 10 μL of 2% (w / v) aqueous NaN3 solution, 0.5% (w / v) aqueous sodium borohydride solution, vortex and let stand for 20 h;

[0138] After standing, take 600 μL, dry under nitrogen at 25 °C;

[0139] After nitrogen drying, dissolve in 30 μL of 1 mol / L aqueous NaOH solution and react for 60 min;

[0140] Add 570 μL of water, centrifuge at 12000 rpm for 5 min, take the supernatant, and determine the sample by ion chromatography;

[0141] The ion chromatography parameters are as follows: using a Thermo ICS5000 ion chromatography system, and analyzing and detecting starch by an electrochemical detector;

[0142] Mobile phase A: 0.2 mol / L aqueous NaOH solution;

[0143] Mobile phase B: 0.2 mol / L NaOH - 0.2 mol / L NaAC aqueous solution;

[0144] The chromatographic column is a Dionex CarboPac PA200 (250*4.0 mm, 10 μm) liquid chromatographic column, the column temperature is 30 °C, the flow rate is 0.4 mL / min, and the injection volume is 5 μL;

[0145] 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 Figure 8 shown;

[0146] See Figure 8 , the number of free short chains of amylopectin without being treated with MDBE prepared in Example 1 is very small and the chain length polymerization degree (DP value) is concentrated in 12 - 18. After being treated with MDBE, the short chains of the amylopectin product are significantly increased, and the polymerization degree distribution is mainly concentrated between DP values of 6 - 40, indicating that MDBE has a debranching function and can hydrolyze the side chains of amylopectin from the branch points;

[0147] For the determination of the branching degree of the product of amylopectin after reacting for 30 min, the following steps are included:

[0148] Take 5 mg of amylopectin after reacting for 30 min into a centrifuge tube, add 1 mL of deuterated dimethyl sulfoxide, and react at 80 °C for 12 h;

[0149] After the reaction is completed, centrifuge at 12000 rpm for 10 min, and take the supernatant into a nuclear magnetic tube for on-machine detection;

[0150] Using the amylopectin without being treated with MDBE as a control, the branching degree of the product is determined;

[0151] In this experiment, a Bruker BioSpin GmbH nuclear magnetic resonance spectrometer is used, and 1Examination was carried out by the 1H nuclear magnetic resonance analysis method, and the parameters were as follows: the number of scans was 32, the resonance radio frequency was 500.23 MHz, and the nuclear magnetic spectrum was 1 1H;

[0152] The measured data were analyzed by MestReNova software. According to the peak emergence time of the sample, the peak emergence range was selected, and the final results are shown in Table 3. The following formula was used for calculation:

[0153] ;

[0154] Among them, DB in the formula represents the degree of branching; 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;

[0155] Table 3 Degree of branching of the product after treating amylopectin with MDBE

[0156]

[0157] Referring to Table 3, the degree of branching of amylopectin after treatment with MDBE decreased from 6.53% to 3.65%, indicating that MDBE has a debranching function.

[0158] 9. The MDBE prepared in Example 1 was combined with maltogenic amylase (BMAL) to verify the promoting effect on substrate utilization

[0159] Amylopectin (final concentration of 0.2% (w / v)) and the enzyme complex (MDBE: BMAL = 3:2, (w:w)) were added to 50 mmol / L Na2HPO4-NaH2PO4 buffer solution with a pH of 6.0; the preparation of BMAL refers to the method in Bacillus sp. B110 (Science and Technology of Food Industry, 2023, 44(10): 123-129);

[0160] After incubation at 40 °C for 7 h, the reaction was terminated by boiling water bath; centrifuged at 12000 rpm for 2 min, and the supernatant was filtered through a 0.22 μm filter membrane;

[0161] High performance liquid chromatography (HPLC) analysis was carried out using a Cosmosil Sugar-D chromatographic column and a refractive index detector (RID) at 30 °C;

[0162] The HPLC conditions were as follows: the mobile phase was acetonitrile and water (65:35, v / v), and the flow rate was 1 mL / min; the HPLC detection results are as Figure 9 shown;

[0163] Referring to Figure 9, the glucose (G1) produced by the double-enzyme combined method was 2.0 mg / mL, and maltose (G2) was 1.76 mg / mL; the G1 and G2 produced by using BMAL alone were 1.82 mg / mL and 0.96 mg / mL respectively; the yields of glucose and maltose produced by the double-enzyme combined method were 1.1 times and 1.84 times the yields when using BMAL alone respectively; the MDBE provided by the present invention can significantly improve the hydrolysis rate of amylopectin.

[0164] Although the embodiments of the present invention have been described in detail above, it is obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes are all within the scope and spirit of the present invention described in the claims. Moreover, the present invention described herein can have other embodiments and can be implemented or realized in various ways.

Claims

1. Use of an isoamylase in the simultaneous hydrolysis of α-1,4 glycosidic bonds and α-1,6 glycosidic bonds, characterized in that: The nucleotide sequence of the isoamylase is shown in SEQ ID NO.1.

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