A genetically engineered bacterium expressing oligosaccharide debranching enzyme and its application

Abstract

The present invention discloses a genetically engineered bacterium expressing oligosaccharide debranching enzyme and its application, belonging to the technical field of microbial engineering. The present invention provides an oligosaccharide debranching enzyme with a specific base sequence, and successfully heterologously expresses the oligosaccharide debranching enzyme in Escherichia coli. By using the method of the present invention, the catalytic activity of the crude enzyme solution of the expressed oligosaccharide debranching enzyme can reach 258.83 U / mL. The oligosaccharide debranching enzyme provided by the present invention is a novel oligosaccharide debranching enzyme from a new source, which has not been reported before, has a high specific enzyme activity, and strong substrate specificity. The oligosaccharide debranching enzyme of the present invention can debranch short chain segments with DP<6 in maltodextrin, filling the blank in the substrate specificity of commonly used debranching enzymes at present, and has high potential application prospects in industrial fields such as starch sugar, resistant starch, beer production and ethanol fuel.

Description

Technical Field

[0001] The present invention relates to a genetically engineered bacterium expressing oligosaccharide debranching enzyme and its application, belonging to the technical fields of genetic engineering and enzyme engineering. Background Art

[0002] Starch is a natural macromolecular polymer composed of α-D-glucose monomers, which is divided into amylose and amylopectin. Amylose is a linear polymer formed by glucose units through α-1,4 glycosidic bonds. Amylopectin has a main chain composed of glucose units through α-1,4 glycosidic bonds, and different numbers of glucose units are connected through α-1,6 glycosidic bonds at the branch points to form its side chains, which is a macromolecular polysaccharide with a complex dendritic branched structure. In the starch processing process, generally multiple amylases are required to catalyze the hydrolysis of starch. Enzymes that hydrolyze α-1,4 glycosidic bonds include α-amylase, glucoamylase, and β-amylase. However, these enzymes cannot or can only slowly act on α-1,6 glycosidic bonds, which limits the hydrolysis rate of starch. Starch debranching enzyme can specifically and efficiently hydrolyze α-1,6 glycosidic bonds in amylopectin. By using the synergistic effect of starch debranching enzyme and other saccharifying enzymes, the subsequent enzymatic reaction can be accelerated, the reaction time can be shortened, and the purpose of improving the utilization rate of raw materials and production efficiency can be achieved. Starch debranching enzyme has currently been used in starch processing fields such as glucose syrup, high maltose syrup, cyclodextrin, branched cyclodextrin, baking, beer brewing, and resistant starch.

[0003] According to the difference in its substrate specificity, starch debranching enzyme can be divided into pullulanase, isoamylase, dextrin debranching enzyme, and oligosaccharide debranching enzyme. Pullulanase has high hydrolysis activity towards low molecular weight dextrin, can efficiently hydrolyze pullulan polysaccharide, has low hydrolysis activity towards high molecular weight dextrin and glycogen with dense branches, and the minimum substrate unit for action is maltosylmaltose. Isoamylase is suitable for acting on higher molecular weight dextrin, amylopectin, and glycogen, has lower hydrolysis activity towards small molecular weight branched dextrin, and cannot hydrolyze pullulan polysaccharide. The minimum substrate unit is maltotriosylmaltotetraose. Dextrin debranching enzyme has high hydrolysis activity towards medium molecular weight maltodextrin, and oligosaccharide debranching enzyme has high hydrolysis activity towards smaller chain segment dextrin or oligosaccharide.

[0004] In the starch sugar industry, after starch is slurried and liquefied, glucoamylase is added to produce different starch sugars. Adding debranching enzyme synergistically during the saccharification process can improve the conversion rate. However, there are still certain deficiencies in the current process. For example, the glucoamylase added during the preparation of glucose syrup will catalyze the polymerization of glucose to generate isomaltooligosaccharides such as isomaltose and panose. The β-amylase added during the preparation of maltose syrup cannot act on the α-1,6 glycosidic bond and produces a large amount of β-limit dextrin and other oligosaccharides. Debranching enzymes such as pullulanase and isoamylase have weak hydrolysis ability for such small molecule dextrins and oligosaccharides. Using oligosaccharide debranching enzyme to act on such smaller dextrins and oligosaccharides can improve starch utilization rate, production efficiency, and reduce the generation of by-products.

[0005] With the continuous innovation and development of new starch processing products and processes, the modern starch processing industry has put forward new requirements for the performance of starch debranching enzymes, including different substrates, different optimum temperatures, high efficiency, stability, etc. Therefore, it is necessary to develop new starch debranching enzymes to fill the blank in the substrate specificity of commonly used debranching enzymes at present to meet the needs of a wider range of industrial production. Summary of the Invention

[0006] To solve the above-mentioned existing technical problems, the present invention realizes the heterologous expression of oligosaccharide debranching enzyme by introducing the gene encoding oligosaccharide debranching enzyme from Paenibacillus sp. P22 into a genetically engineered bacterium.

[0007] The present invention provides a gene encoding an oligosaccharide debranching enzyme, and the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant vector carrying the above-mentioned oligosaccharide debranching enzyme gene.

[0009] The present invention also provides a recombinant cell carrying the above-mentioned oligosaccharide debranching enzyme gene or the above-mentioned recombinant vector.

[0010] The present invention provides a recombinant Escherichia coli, and the recombinant Escherichia coli expresses an oligosaccharide debranching enzyme derived from Paenibacillus sp. P22, and the amino acid sequence of the oligosaccharide debranching enzyme is as shown in SEQ ID NO.1.

[0011] In one embodiment of the present invention, the nucleotide sequence encoding the oligosaccharide debranching enzyme is as shown in SEQ ID NO.2.

[0012] In one embodiment of the present invention, the recombinant Escherichia coli uses E. coli BL21(DE3) as the expression host.

[0013] In one embodiment of the present invention, the recombinant Escherichia coli uses pET-28a(+) as the expression vector.

[0014] The present invention also provides the application of the above-mentioned recombinant Escherichia coli or the oligosaccharide debranching enzyme with the amino acid sequence shown in SEQ ID NO.1 in hydrolyzing maltodextrin.

[0015] In one embodiment of the present invention, the nucleotide sequence encoding the oligosaccharide debranching enzyme is shown in SEQ ID NO.2.

[0016] The present invention also provides a method for hydrolyzing maltodextrin, and the method is: adding the above-mentioned recombinant Escherichia coli or the recombinant oligosaccharide debranching enzyme prepared by its fermentation into a reaction system containing maltodextrin for hydrolysis; or adding the oligosaccharide debranching enzyme with the amino acid sequence shown in SEQ ID NO.1 into a reaction system containing maltodextrin for hydrolysis.

[0017] In one embodiment of the present invention, the recombinant oligosaccharide debranching enzyme is prepared by inoculating the recombinant Escherichia coli into a seed medium to obtain a seed liquid, and inoculating the seed liquid into a fermentation medium at an inoculation amount of 2% - 4% (v / v), and culturing at 25 - 30 °C for 48 - 96 h to obtain a crude enzyme solution of the recombinant oligosaccharide debranching enzyme.

[0018] In one embodiment of the present invention, the reaction conditions of the reaction system are: reacting at 45 - 55 °C and pH 5.5 - 6.0 for 24 - 36 h.

[0019] In one embodiment of the present invention, the reaction conditions of the reaction system are: reacting at 50 °C and pH 6.0 for 24 h.

[0020] The present invention also provides the application of the above-mentioned recombinant Escherichia coli or the oligosaccharide debranching enzyme with the amino acid sequence shown in SEQ ID NO.1 in preparing products for hydrolyzing maltodextrin.

[0021] In one embodiment of the present invention, the nucleotide sequence encoding the oligosaccharide debranching enzyme is shown in SEQ ID NO.2.

[0022] In one embodiment of the present invention, the crude enzyme solution of the recombinant oligosaccharide debranching enzyme obtained by fermentation or the pure enzyme obtained by purification through a nickel column is used to hydrolyze maltodextrin at 50 °C for a debranching reaction.

[0023] In one embodiment of the present invention, the concentration of the substrate is at least 10 g / L.

[0024] In one embodiment of the present invention, the enzyme addition amount of the oligosaccharide debranching enzyme in the reaction system is at least 50 U / g.

[0025] The present invention also provides the use of the above gene, or the above recombinant vector, or the above recombinant Escherichia coli, or the above method in hydrolyzing maltodextrin.

[0026] Beneficial effects

[0027] (1) The present invention provides an oligosaccharide debranching enzyme with a specific base sequence, and successfully heterologously expresses the oligosaccharide debranching enzyme using Escherichia coli. By using the method of the present invention, the catalytic activity of the crude enzyme solution of the expressed oligosaccharide debranching enzyme can reach 258.83 U / mL.

[0028] (2) The optimal temperature of the oligosaccharide debranching enzyme of the present invention is 50 °C, and its activity can still remain above 50% after incubation at 50 °C for 60 min. Moreover, the enzyme activity shows an activation phenomenon when incubated at 45 °C, which can meet the requirements for different reaction temperatures in starch processing production.

[0029] (3) The oligosaccharide debranching enzyme provided by the present invention is an oligosaccharide debranching enzyme from a new source, which has not been reported before, has a high specific enzyme activity, and strong substrate specificity.

[0030] (4) The oligosaccharide debranching enzyme of the present invention can debranch short-chain segments with DP < 6 in maltodextrin, filling the blank in the substrate specificity of commonly used debranching enzymes at present, and has high potential application prospects in industrial fields such as starch sugar, resistant starch, beer production, and ethanol fuel. Description of the drawings

[0031] Figure 1 : SDS-PAGE analysis of recombinant oligosaccharide debranching enzyme; wherein, M: protein standard molecular weight; Lane 1: E.coli BL21(DE3) / pET-20b(+)-oga; Lane 2: E.coli BL21(DE3) / pET-22b(+)-oga; Lane 3: E.coli BL21(DE3) / pET-28a(+)-oga; Lane 4: pure enzyme.

[0032] Figure 2 : Relative enzyme activity data of oligosaccharide debranching enzyme at different temperatures.

[0033] Figure 3 : Stability of recombinant oligosaccharide debranching enzyme at different temperatures.

[0034] Figure 4 : High performance anion exchange chromatography (HPAEC-PAD) analysis of recombinant oligosaccharide debranching enzyme hydrolyzing DE 2 maltodextrin. Detailed implementation manners

[0035] The culture media involved in the following examples are as follows:

[0036] LB liquid medium: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) sodium chloride, pH 7.0.

[0037] LB solid medium: Add 1.5 (w / v) agar to the LB liquid medium.

[0038] TB liquid medium: 1.2% (w / v) tryptone, 2.4% (w / v) yeast extract, 0.4% (w / v) glycerol, 17 mM KH2PO4, 72 mM K2HPO4, pH 6.0.

[0039] The detection methods involved in the following examples are as follows:

[0040] The method for determining the activity of recombinant oligosaccharide debranching enzyme is as follows: Pipette 800 μL of the substrate p-nitrophenyl α-D-glucoside (pNPG, 10 mM) into a centrifuge tube, add 150 μL of phosphate buffer (500 mM, pH 6.0), mix well, incubate at 50 °C for 5 min, add 50 μL of the diluted enzyme solution to be tested, react for 5 min, then add 1 mL of Na2CO3 solution (1 M) to terminate the reaction, and measure the absorbance at 410 nm. Use the enzyme solution inactivated at high temperature as the blank control.

[0041] Definition of enzyme activity: Under the above conditions, the hydrolysis of 1 μmol of p-nitrophenol (pNP) per minute is defined as 1 enzyme activity unit.

[0042] The detection method for the degree of polymerization: Dilute the supernatant after the reaction, filter it through a 0.22 μm aqueous filter membrane, and use ICS-5000 high performance anion exchange chromatography-pulsed amperometric detector (HPAEC-PAD) to determine the degree of polymerization distribution of the product after treatment with oligosaccharide debranching enzyme. The mobile phase consists of 250 mM (w / v) sodium hydroxide and 1 M (w / v) sodium acetate, the flow rate is 0.5 mL / min, and the column temperature is maintained at 35 °C.

[0043] Example 1: Construction of a recombinant Escherichia coli secretion expression system

[0044] (1) Using the genomic DNA of Paenibacillus sp. P22 publicly available on NCBI as a template, the target gene SEQ ID NO.2 (oligosaccharide debranching enzyme) with Nco I and Xhol I restriction enzyme cleavage sites at both ends was amplified by PCR.

[0045] The PCR amplification program involved is: Taq Buffer (Mg 2+Plus) 10 μL, dNTP Mixture (2.5 mM each) 4 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template DNA 1 μL, Taq DNA Polymerase (1.25 U / μL) 1 μL, add double-distilled water to 50 μL. The PCR amplification conditions were as follows: pre-denaturation at 98°C for 3 min; then 30 cycles (98°C for 10 s, 60°C for 15 s, 68°C for 2 min); finally, incubation at 68°C for 10 min.

[0046] (2) The oligosaccharide debranching enzyme gene (SEQ ID NO. 2) amplified by PCR was inserted into the pMD 18-T simple plasmid to obtain the cloning vector pMD 18-T simple / oga. After the vector was digested with double enzymes, the target gene fragment with sticky ends was recovered and inserted into the pET-28a(+) plasmid treated with the same restriction enzyme to obtain the recombinant expression vector pET-28a(+)-oga.

[0047] According to the above steps (1)-(2), the difference is that the plasmid pET-28a(+) was adjusted to pET-22b(+) and pET-20b(+) respectively to obtain the recombinant expression vectors pET-22b(+)-oga and pET-20b(+)-oga.

[0048] (3) The recombinant expression vectors prepared in step (2) were respectively transformed into Escherichia coli E. coli JM109, spread on LB plates containing kanamycin, and transformants were picked for sequencing and colony PCR verification. The recombinant plasmids containing the oligosaccharide debranching enzyme gene were respectively extracted.

[0049] The recombinant plasmids were respectively transformed into E. coli BL21(DE3) to obtain recombinant genetically engineered bacteria: E. coli BL21(DE3) / pET-28a(+)-oga, E. coli BL21(DE3) / pET-22b(+)-oga, E. coli BL21(DE3) / pET-20b(+)-oga.

[0050] Example 2: Expression, isolation, and purification of recombinant oligosaccharide debranching enzyme

[0051] The specific steps are as follows:

[0052] 1. Expression of recombinant oligosaccharide debranching enzyme

[0053] (1) Inoculate the single colonies of E. coli BL21(DE3) / pET-28a(+)-oga, E. coli BL21(DE3) / pET-22b(+)-oga, and E. coli BL21(DE3) / pET-20b(+)-oga prepared in Example 1 into 50 mL of LB liquid medium containing 20 μg / mL kanamycin, and culture them in a shaking flask at 37 °C and 200 rpm for 8 - 12 h to prepare seed solutions respectively;

[0054] (2) Transfer the above-prepared seed solutions to 50 mL of TB liquid medium containing 20 μg / mL kanamycin at an inoculation amount of 2% - 4% (v / v). When OD 600 = 0.6, add IPTG with a final concentration of 0.01 mM, and culture in a shaking flask at 25 - 30 °C and 200 rpm for 48 - 96 h to prepare fermentation broths respectively;

[0055] (3) Centrifuge the fermentation broths at 4 °C and 10,000 rpm for 20 min, discard the supernatant, resuspend the cells with 50 mM sodium acetate buffer at pH 6.0, and place them on ice for ultrasonic disruption for 15 min under the program of 200 W, on for 1 s, off for 2 s. Finally, centrifuge at 4 °C and 10,000 rpm for 20 min, and take the supernatant, which is the crude intracellular enzyme solution.

[0056] Detect the enzyme activity data of the crude intracellular enzyme solutions prepared from E. coli BL21(DE3) / pET-28a(+)-oga, E. coli BL21(DE3) / pET-22b(+)-oga, and E. coli BL21(DE3) / pET-20b(+)-oga respectively, and verify by SDS-PAGE protein electrophoresis. The results are shown in Table 1 and Figure 1 as follows.

[0057] Table 1: Enzyme activity data of recombinant oligosaccharide debranching enzyme prepared from different recombinant strains

[0058]

[0059] The results show that when using pET-22b(+) and pET-20b(+) as expression vectors, no enzyme activity was detected, and protein electrophoresis showed that the recombinant oligosaccharide debranching enzyme could not be expressed. Only when using pET-28a(+) as the expression vector, the recombinant oligosaccharide debranching enzyme was detected to be expressed. After detection, its crude enzyme activity was 258.83 U / mL.

[0060] 2. Purification of recombinant oligosaccharide debranching enzyme

[0061] The crude enzyme solution of recombinant oligosaccharide debranching enzyme prepared from the recombinant strain E. coli BL21(DE3) / pET-28a(+)-oga in step 1 was purified using a nickel column. The equilibration buffer (solution A, pH 7.5) was 500 mM NaCl + 50 mM Tris-HCl + 20 mM imidazole, and the elution buffer (solution B, pH 7.5) was 500 mM NaCl + 50 mM Tris-HCl + 500 mM imidazole. After the crude fermentation enzyme solution was filtered through a 0.45 μm aqueous membrane, the nickel column was equilibrated with 5 - 6 column volumes of solution A; the sample was loaded at a flow rate of 2 mL / min; then the ion column was first equilibrated with solution A, and then the target protein was eluted with 60% of solution B. The corresponding eluate was collected according to the elution peak, placed in a dialysis bag, and dialyzed overnight at 4°C using 50 mM sodium acetate buffer with pH 6.0. Subsequently, it was identified by SDS-PAGE protein electrophoresis (the results are as shown in Figure 1 ) and enzyme activity determination. After detection, the enzyme activity of the pure enzyme was 407.65 U / mg.

[0062] Example 3: Optimal temperature and thermal stability of recombinant oligosaccharide debranching enzyme

[0063] The optimal temperature and thermal stability of the purified recombinant oligosaccharide debranching enzyme prepared in Example 2 were detected respectively, and the specific steps were as follows:

[0064] (1) The method for determining the optimal temperature of recombinant oligosaccharide debranching enzyme was as follows: According to the enzyme activity determination method, the enzyme activities of the enzyme solution at 30°C - 80°C were measured respectively, and the one with the highest enzyme activity was taken as 100%, and the results are shown in Table 2 and Figure 2 .

[0065] Table 2: Relative enzyme activity data at different temperatures

[0066]

[0067] (2) The method for determining the thermal stability of recombinant oligosaccharide debranching enzyme was as follows: The enzyme solution was incubated at different temperatures (45°C - 60°C), and at regular intervals, the enzyme solution was taken out, quickly cooled, and the residual enzyme activity was measured. The activity of the unincubated enzyme solution was taken as 100%, and the results are shown in Table 3 and Figure 3 .

[0068] Table 3: Relative enzyme activity data of incubation at different temperatures for different times

[0069]

[0070]

[0071] The results showed that the optimal temperature and thermal stability of the recombinant oligosaccharide debranching enzyme were as shown in Figure 2 andFigure 3 As shown. The enzyme reaches its optimal activity at 50 °C. When the temperature is further increased above 50 °C, the activity of the enzyme drops sharply and the enzyme is basically inactivated at 70 °C. The enzyme activity is stable when incubated at 45 °C and slightly increases with the prolongation of time. After incubation at 50 °C for 60 min, its activity still remains above 50%.

[0072] Example 4: Application of the recombinant oligosaccharide debranching enzyme

[0073] In this example, maltodextrin was hydrolyzed using the oligosaccharide debranching enzyme; the specific steps were as follows:

[0074] (1) Prepare 100 g of a 1% (w / w) DE 2 maltodextrin solution and adjust the pH to 6.0.

[0075] (2) Place the 1% (w / w) DE 2 maltodextrin solution in a 50 °C water bath shaker and preheat it for 10 min. Subsequently, add the pure oligosaccharide debranching enzyme prepared in Example 2 at an enzyme addition amount of 50 U / g, react in a 50 °C water bath shaker for 24 h, and then transfer it to boiling water to inactivate the enzyme for 20 min to terminate the reaction; among them, an equal amount of sodium acetate buffer solution was used to replace the enzyme solution as a blank control.

[0076] (3) After the enzyme inactivation treatment, centrifuge the reaction solution at 10,000 rpm for 10 min, take the supernatant, pass it through a 0.22 μm aqueous membrane to remove impurities, and then the distribution of different degrees of polymerization in the reaction solution can be analyzed by ion chromatography. To explore the substrate specificity of the oligosaccharide debranching enzyme in hydrolyzing maltodextrin, the peak areas of oligosaccharide chains with different DP values in the system before and after the reaction were measured, and their contents were represented by the peak areas, so as to characterize the hydrolysis of the α-1,6 glycosidic bond of the substrate by the oligosaccharide debranching enzyme. The chromatographic analysis results are shown in Table 4 and Figure 4 as shown.

[0077] Table 4: Distribution of different degrees of polymerization

[0078]

[0079] The results showed that using DE 2 maltodextrin as the substrate, after hydrolysis by the oligosaccharide debranching enzyme, the content of the chain segments with DP < 6 in the system all increased, indicating that the oligosaccharide debranching enzyme acts on the α-1,6 glycosidic bond of maltodextrin, hydrolyzing the branch points of the short branched chains and generating a series of oligosaccharide chains with DP < 6. Therefore, the oligosaccharide debranching enzyme tends to hydrolyze the branch points with DP < 6 in hydrolyzing maltodextrin.

[0080] Example 5: Application of the recombinant oligosaccharide debranching enzyme

[0081] The specific implementation is the same as that of Example 4, except that in step (1), the substrates are respectively replaced with 100 g of DE 4 maltodextrin solution with a concentration of 1% (w / w) and 100 g of DE 6 maltodextrin solution with a concentration of 1% (w / w).

[0082] According to the method of Example 4, the content changes of different degrees of polymerization in the system before and after the reaction were measured. The results are shown in Table 5.

[0083] Table 5: Distribution of different degrees of polymerization

[0084]

[0085] The results show that when the oligosaccharide debranching enzyme hydrolyzes maltodextrins with different DE values, it can specifically hydrolyze the short branches in maltodextrin to generate a series of oligosaccharide chains with DP < 6.

[0086] In addition, with the increase of the DE value of maltodextrin, the content of oligosaccharide chains with DP < 6 generated by the hydrolysis of the oligosaccharide debranching enzyme increases, indicating that the oligosaccharide debranching enzyme has a stronger affinity for maltodextrin with a higher DE value.

[0087] In summary, the oligosaccharide debranching enzyme has a certain hydrolysis effect on maltodextrin, can hydrolyze the α-1,6 glycosidic bond in the substrate, and has high substrate specificity, specifically hydrolyzing the branch points with DP < 6. Its specificity for short branched chains fills the blank in the substrate specificity of commonly used debranching enzymes at present, and has high application potential in industrial fields such as starch sugar, resistant starch, beer production, and ethanol fuel.

[0088] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for hydrolyzing maltodextrin, characterized in that, The method is as follows: adding recombinant Escherichia coli or recombinant oligosaccharide debranching enzyme prepared by fermenting the same into a reaction system containing maltodextrin for hydrolysis. The recombinant Escherichia coli expresses an oligosaccharide debranching enzyme derived from Paenibacillus sp. P22, and the amino acid sequence of the oligosaccharide debranching enzyme is as shown in SEQ ID NO.1; or adding an oligosaccharide debranching enzyme with the amino acid sequence as shown in SEQ ID NO.1 into a reaction system containing maltodextrin for hydrolysis to generate oligosaccharide chains with DP < 6.

2. The method according to claim 1, wherein The nucleotide sequence encoding the oligosaccharide debranching enzyme is shown as SEQ ID NO.

2.

3. The method according to claim 2, wherein The recombinant Escherichia coli uses E. coli BL21(DE3) as the expression host.

4. The method according to claim 3, wherein The recombinant E. coli uses pET-28a(+) as the expression vector.

5. The method according to claim 1, characterized in that, The recombinant oligosaccharide debranching enzyme is prepared by inoculating the recombinant E. coli into a seed medium to obtain a seed solution, and then inoculating the seed solution into a fermentation medium at an inoculation amount of 2% - 4% for fermentation.

6. The method according to claim 1 or 5, characterized in that, The reaction conditions of the reaction system are: reacting at 45 - 55 °C and pH 5.5 - 6.0 for 24 - 36 h.

7. Use of recombinant Escherichia coli or oligosaccharide debranching enzyme with an amino acid sequence shown in SEQ ID NO.1 in the preparation of a product for hydrolyzing maltodextrin, characterized in that, The recombinant Escherichia coli expresses a oligosaccharide debranching enzyme derived from Paenibacillus sp. P22, and the amino acid sequence of the oligosaccharide debranching enzyme is as shown in SEQ ID NO.1.

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