Dexdextrin debranching enzyme mutant with improved debranching performance and application thereof

Abstract

The invention discloses a dextrin debranching enzyme mutant with improved debranching performance and application of the dextrin debranching enzyme mutant, and belongs to the technical field of biology. The dextrin debranching enzyme mutant is obtained by mutation of dextrin debranching enzyme with an amino acid sequence as shown in SEQ ID NO.2. The mutation comprises the following steps: mutating tyrosine at the 323rd site into phenylalanine; or valine at the 375th site is mutated into phenylalanine; or the tyrosine at the 323rd site is mutated into the phenylalanine, the leucine at the 324th site is mutated into the tyrosine, and the phenylalanine at the 326th site is mutated into the asparagine. According to the invention, the specificity and debranching performance of the enzyme substrate are regulated and controlled by changing the distribution of aromatic amino acids in the enzyme substrate binding region, site-directed mutagenesis is carried out on the amino acid sequence, and particularly amino acid residues directly related to the substrate binding region are changed, so that the specificity and catalytic efficiency of the enzyme to the specific substrate are regulated and controlled; the dextrin debranching enzyme mutant with improved debranching performance is obtained.

Description

Technical Field

[0001] The invention relates to a dextrin debranching enzyme mutant with improved debranching performance and application thereof, belonging to the field of biotechnology. Background Art

[0002] Starch debranching enzyme (SDBE) is an important enzyme for starch processing, which can specifically hydrolyze α-1,6 glycosidic bonds in starch and related polysaccharides and remove branching structures in substrates. For general starch debranching enzymes, such as pullulanase, although it can efficiently hydrolyze α-1,6 glycosidic bonds in pullulan and limit dextrin, it has low efficiency in debranching large molecular starch, and the catalytic mode is not necessarily single, which is easily affected by product inhibition in the production of special starch sugars such as cyclodextrin; isoamylase tends to hydrolyze large molecular branched starch, but has low debranching efficiency for small molecular substrates such as maltodextrin. Both enzymes have limitations in substrate specificity and range of action, making it difficult to effectively improve the conversion rate of starch in the production of special starch sugars.

[0003] Selecting a suitable SDBE for the production of starch sugar is the key to improving the conversion rate of starch raw materials and product yield. A dextrin debranching enzyme (Saccharolobus solfataricus STB09 glycogen debranching enzyme, SsGDE) from the thermophilic archaeon Saccharolobus solfataricus STB09 has high debranching efficiency. Its substrate specificity is different from that of pullulanase and isoamylase. SsGDE has high hydrolysis efficiency for short-chain branches with a branching degree of polymerization of 5 to 10, and is more suitable for debranching starch liquefaction products. It can further improve the starch conversion rate by assisting saccharifying enzymes. Compared with pullulanase and isoamylase, it may be more suitable for the production of functional oligosaccharides, cyclodextrins and other starch sugars. In addition, the excellent thermal stability of SsGDE is also more suitable for the production of starch sugars under high temperature environments. For example, in the production of cyclodextrin, dextrin debranching enzyme has good specificity for medium molecular weight substrates. Debranching can increase the proportion of linear segments with degree of polymerization (DP) ≥ 13 in the system, and only produce a small number of segments with DP < 6. In the process of synergistic action with β-CGTase, the proportion of α-1,6 glycosidic bonds in the substrate is reduced by 23.54%, improving the utilization of the substrate by β-CGTase. At the same time, the segments with DP ≥ 13 produced by SsGDE debranching can also be used by β-CGTase to convert into β-cyclodextrin. In addition, oligosaccharides with DP < 6 have an inhibitory effect on β-CGTase, while SsGDE hardly produces such substances when acting on substrates. At the same time, the catalytic activity of SsGDE is not inhibited by the product cyclodextrin, which makes SsGDE superior to other debranching enzymes in promoting β-cyclodextrin production.

[0004] However, the wild-type dextrin debranching enzyme has limited catalytic activity and a narrow range of substrate polymerization degrees, which greatly limits its application in actual production. Therefore, the present invention is proposed. Summary of the invention

[0005] To solve the above problems, the present invention uses site-directed mutagenesis technology to molecularly modify wild-type dextrin debranching enzymes and obtains a series of positive mutants. Compared with the wild enzyme, these mutants significantly improve the efficiency of hydrolyzing branches with a degree of polymerization of 5 to 21 in maltodextrin, and specifically optimize and improve the substrate spectrum and enzyme activity of the wild-type dextrin debranching enzyme, laying the foundation for the industrial application of the enzyme. The mutants of the present invention also overcome the defect that the substrate spectra of existing pullulanase and isoamylase are quite different, enriching the raw materials for starch processing.

[0006] The first object of the present invention is to provide a dextrin debranching enzyme mutant, wherein the dextrin debranching enzyme mutant is obtained by mutating the dextrin debranching enzyme with an amino acid sequence as shown in SEQ ID NO.2, wherein the mutation is:

[0007] mutating tyrosine at position 323 to phenylalanine;

[0008] or mutate valine at position 375 to phenylalanine;

[0009] Or mutate the tyrosine at position 323 to phenylalanine, mutate the leucine at position 324 to tyrosine, and mutate the phenylalanine at position 326 to asparagine.

[0010] Starch is a polysaccharide composed of glucose as the basic unit. It is one of the most abundant carbohydrates in nature and the main source of nutrition for humans and animals. In addition to direct consumption, starch is further processed into starch sugar and other products. The key to starch sugar production is the enzymatic hydrolysis of starch chains. Starch chains are mainly composed of glucose units connected by α-1,4 glycosidic bonds and α-1,6 glycosidic bonds. Although α-1,6 glycosidic bonds only account for about 6% of the total glycosidic bonds, they form a branched structure in the starch chain. At present, the research on amylase that hydrolyzes α-1,4 glycosidic bonds is relatively mature. However, α-1,4 glycosidic bond hydrolases cannot or can only slowly cut α-1,6 glycosidic bonds. Although the content of α-1,6 glycosidic bonds in starch is low, it limits the catalytic efficiency of α-1,4 glycosidic bond hydrolases, forming unusable limit dextrins. Therefore, if an enzyme that can efficiently catalyze α-1,6 glycosidic bonds can be found, it will be of great value to the starch processing industry.

[0011] The starch debranching enzymes of the G13H family can specifically and efficiently hydrolyze the α-1,6 glycosidic bonds at the branching sites of starch, thereby effectively improving the utilization rate of starch raw materials. However, although both pullulanase and isoamylase reported so far can hydrolyze α-1,6 glycosidic bonds, their substrate specificity is quite different: pullulanase has low debranching efficiency for macromolecular starch, and isoamylase has low debranching efficiency for small molecule substrates. This leads to the fact that starch debranching enzymes of different properties are usually required to work together to achieve the best hydrolysis effect during starch processing. However, the action conditions of different enzymes are usually very different, which makes the enzymatic hydrolysis process of starch processing more complicated. Therefore, it is necessary to find a new type of debranching enzyme that can hydrolyze starch branches with different degrees of polymerization.

[0012] In addition, as mentioned above, the formation of dextrin is not conducive to starch processing, and the smaller the DE value (Dextrose Equivalent, glucose equivalent, refers to the percentage of reducing sugar (calculated as glucose) in the dry matter of syrup) of dextrin, the more difficult it is to be hydrolyzed by enzymes. Therefore, how to improve the hydrolysis efficiency of low DE value dextrin is also crucial to the processing of amylopectin.

[0013] The present invention finds that the substrate specificity of the dextrin debranching enzyme from Saccharolobus solfataricus is strong, but when it is debranched for maltodextrin, it is limited by the short chain branching degree of polymerization in the substrate, which makes it difficult to meet the application requirements of the diversification of substrate polymerization degree in the production process of different starch sugars. Therefore, after analyzing and comparing the structural differences of SsGDE, PaISO and CrISO using molecular docking and molecular dynamics simulation technology, the present invention finds that the strength of the binding ability of dextrin debranching enzyme to the substrate is related to aromatic amino acids. Therefore, by adjusting the aromatic amino acids in the SsGDE substrate binding region, expanding the substrate binding region or strengthening the affinity of the substrate binding region to the substrate, it is finally verified that the modified partial enzyme molecule substrate spectrum is expanded, and the catalytic effect on low DE value dextrin is significantly improved, which can significantly improve the debranching efficiency of SsGDE to the substrate, thereby strengthening its synergistic effect with saccharifying enzyme in starch sugar production, improving sugar production efficiency and control accuracy, and laying a good foundation for the application of dextrin debranching enzyme in starch industry.

[0014] The second object of the present invention is to provide a nucleic acid molecule encoding the amylin debranching enzyme mutant.

[0015] Furthermore, the sequence of the nucleic acid molecule is any one of SEQ ID NO.3-5.

[0016] The third object of the present invention is to provide a gene integration expression frame carrying the nucleic acid molecule.

[0017] The fourth object of the present invention is to provide a recombinant plasmid carrying the nucleic acid molecule.

[0018] Furthermore, expression vectors include but are not limited to pET series plasmids.

[0019] The fifth object of the present invention is to provide a recombinant cell containing the amylin debranching enzyme mutant.

[0020] Furthermore, the host cell is a microorganism, such as bacteria or fungi.

[0021] The sixth object of the present invention is to provide the use of the dextrin debranching enzyme mutant, nucleic acid molecule, gene expression frame, recombinant plasmid or recombinant cell in starch processing.

[0022] Furthermore, the application includes hydrolyzing α-1,6 glycosidic bonds or hydrolyzing substances containing α-1,6 glycosidic bonds or starch branches.

[0023] Furthermore, substances containing α-1,6 glycosidic bonds or starch branches include amylopectin, dextrin, and the like.

[0024] The seventh object of the present invention is to provide a method for hydrolyzing starch-containing branched-chain substances, comprising the following steps: contacting the starch-containing branched-chain substances with the above-mentioned dextrin debranching enzyme mutant to cause a hydrolysis reaction.

[0025] Furthermore, the starch-containing branched-chain substance includes starch (such as corn starch, wheat starch, tapioca starch, etc.) or dextrin.

[0026] Furthermore, the dextrin includes maltodextrin.

[0027] Furthermore, the maltodextrin includes maltodextrin with a DE value of 2-25, preferably maltodextrin with a DE value of 4. In the present invention, a dextrin debranching enzyme that tends to efficiently debranch maltodextrin is particularly provided. It is a novel dextrin debranching enzyme (Saccharolobus solfataricus STB09 glycogen debranching enzyme, SsGDE) derived from the thermophilic archaeon Saccharolobus solfataricus STB09 mined by bioinformatics means, which can specifically hydrolyze α-1,6 glycosidic bonds in starch liquefaction products or maltodextrin and tends to act on branches with a branch polymerization degree of 5 to 21 in maltodextrin, which is also the innovation of the present invention.

[0028] The eighth object of the present invention is to provide a recombinant Escherichia coli, in which the gene encoding the dextrin debranching enzyme mutant is overexpressed.

[0029] The ninth object of the present invention is to provide a method for constructing the recombinant Escherichia coli, comprising the following steps:

[0030] The recombinant plasmid containing the dextrin debranching enzyme mutant encoding gene is introduced into the starting strain or the dextrin debranching enzyme mutant encoding gene is integrated into the genome of the starting strain to obtain a recombinant strain expressing the dextrin debranching enzyme mutant in a free or integrated manner.

[0031] Preferably, the E.coilBL21 (DE3) strain is used as the starting strain.

[0032] The tenth object of the present invention is to provide a method for producing a dextrin debranching enzyme mutant, comprising the following steps: culturing the recombinant Escherichia coli to express the dextrin debranching enzyme mutant.

[0033] Furthermore, the culture comprises the following steps: activating the recombinant Escherichia coli in a seed culture medium, and then inoculating into a fermentation medium for culture.

[0034] Further, the culture was carried out at 28-32°C and 180-280 rpm.

[0035] Furthermore, the fermentation medium contains the following components: 10-20 g / L tryptone, 20-30 g / L yeast powder, 1-10 g / L glycerol, 1-6 g / L KH2PO4, and 10-20 g / L K2HPO4·3H2O.

[0036] The eleventh object of the present invention is to provide an enzyme composition, wherein the enzyme composition contains the dextrin debranching enzyme mutant. The enzyme composition can be used for the hydrolysis of starch, and different types of enzymes act synergistically to improve the utilization rate of starch.

[0037] Furthermore, the enzyme composition also contains any other enzymes that can hydrolyze starch or dextrin, including but not limited to pullulanase, isoamylase, α-amylase, β-amylase, etc.

[0038] The twelfth object of the present invention is to provide application of the enzyme composition in starch processing.

[0039] Beneficial effects of the present invention:

[0040] (1) The present invention uses dextrin debranching enzyme from Saccharolobus solfataricus as a parent enzyme for molecular modification, mutates at least one of the amino acids at positions 323, 375, and 323 to 326, and obtains a series of mutants with improved enzyme activity.

[0041] (2) The enzymatic activities of a series of dextrin debranching enzyme mutants provided by the present invention are significantly improved compared with the wild enzyme. The single point mutant Y323F increases the hydrolysis activity of corn amylopectin, potato amylopectin and DE4 maltodextrin by 31.73%, 30.19% and 20.63%, respectively; the single point mutant V375F increases the hydrolysis activity of corn amylopectin, potato amylopectin and DE4 maltodextrin by 64.77%, 60.82% and 23.08%, respectively; the combined mutant YLDF / FYDN increases the hydrolysis activity of corn amylopectin, potato amylopectin and DE4 maltodextrin by 6.96%, 4.17% and 5.06%, respectively.

[0042] (3) The substrate ranges of a series of dextrin debranching enzyme mutants provided by the present invention have been significantly expanded, and when DE4 maltodextrin is used as a substrate, the values ​​of the kinetic parameter kcat / Km of Y323F, V375F and YLDF / FYDN are respectively increased by 21.05%, 26.31% and 7.89% compared with the wild-type SsGDE; when amylopectin is used as a substrate, the values ​​of kcat / Km of Y323F, V375F and YLDF / FYDN are higher than those of the wild-type SsGDE; the improvement of the hydrolysis efficiency of amylopectin by Y323F and V375F mutants is higher than that of the hydrolysis efficiency of DE4 maltodextrin. The present invention optimizes and improves the debranching efficiency and substrate specificity of the wild-type dextrin debranching enzyme, creating better conditions for the use of the enzyme in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The substrate specificity of wild-type SsGDE and mutant Y323F.

[0044] Figure 2 This is the alignment of the crystal structures of SsGDE, PaISO and CrISO.

[0045] Figure 3 This is the construction map of the recombinant plasmid.

[0046] Figure 4 SDS-PAGE gel electrophoresis analysis of SsGDE mutants; M: protein molecular weight standard; 1: mutant Y323F after two-step purification; 2: mutant L324Y after two-step purification; 3: mutant F326N after two-step purification; 4: mutant F326Y after two-step purification; 5: mutant V375F after two-step purification; 6: mutant V375P after two-step purification; 7: mutant YLDF / FYDN after two-step purification.

[0047] Figure 5 The hydrolysis activities of wild enzyme and mutants on corn amylopectin, potato amylopectin and DE4 maltodextrin. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0049] The materials and methods involved in the following examples are as follows:

[0050] (1) Sequence:

[0051] The nucleotide sequence of the wild-type SsGDE is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2;

[0052] The gene sequence of mutant Y323F is shown in SEQ ID NO.3;

[0053] The gene sequence of mutant V375F is shown in SEQ ID NO.4;

[0054] The gene sequence of mutant YLDF / FYDN is shown in SEQ ID NO.5.

[0055] The details are as follows:

[0056] SEQ ID NO.1:

[0057] ATGGCATTATTCTTCAGAACTAGAGATAGGCCTCTTCGTCCCGGAGATCCATATCCATTAGGTTCGAATTGGATAGAAGATGATGATGGTGTGAATTTTTCATTATTCTCAGAAAATGCAGAAAAAGTCGAATTACTCCTTTACTCACTGACAAACCAAAAGTACCCAAAGGAGATAATAGAGGTTAAAAACAAAACGGGAGATATTTGGCACGTCTTCGTCCCGGGACTGAGACCCGGGCAACTTTACGCATATAGGGTTTATGGCCCATATAAACCGGAGTTGGGATTAAGGTTTAATCCAAATAAGGTTTTAATAGACCCTTATGCTAAAGCCATAAACGGTAGTGTAATTTGGAACGACGCGGTATTTGGTTATAAGATAGGAGATCAAAATCAAGATTTGACCTATGACGAGAGGGATTCCGGCGAATATGTTCCTAAAAGTGTTGTGATTAATCCATACTTTGAGTGGGACGATGAGGATTTCATTAAGGGAAAGAAAGTTCCATTAAAGGATACAGTAATTTACGAAGTTCACGTAAAAGGGTTTACAAAACTTAGGTTGGATTTGCCGGAAAACATAAGGGGAACTTATGAAGGTCTTGCCTCAGAACAAATGATCAGCTATCTCAAAGAT CTAGGGATTACTACTGTGGAACTAATGCCAGTTTTCCATTTTATCGATCAAAGATTTCTGACAGATAAGGGGCTAACGAATTACTGGGGGTATGACCCAATAAATTTCTTCTCTCCTGAATGTAGATATTCCAGTACTGGCTGTTTAGGAGGGCAAGTATTAAGTTTCAAAAAAATGGTAAACGAATTACATAACGCAGGAATTGAGGTTATAATCGATGTAGTTTATAATCACACTGCTGAGGGGAATCATTTAGGCCCAACGTTAAGTTTCAGAGGTATTGATAATACAGCATATTACATGCTCCAACCAGATAATAAAAGATATTATTTAGACTTTACCGGAACTGGCAATACGTTAAATCTTAGCCATCCTAGAGTCATCCAGATGGTCTTAGATAGCTTAAGATATTGGGTTACAGAGATGCACGTAGATGGCTTTAGA TTTGACTTAGCAGCAGCTTTAGCCAGGGAATTATACAGCGTTAATATGTTAAATACGTTCTTCATTGCTCTACAGCAAGACCCAATATTGTCACAAGTAAAACTGATAGCTGAGCCTTGGGATGTGGGACAAGGGGGATATCAAGTAGGGAATTTCCCATATCAATGGGCAGAGTGGAATGGAAAGTATAGGGATTCGATAAGGAGATTTTGGAGAGGAGAAGCGTTACCCTATAGCGAGATTGCTAACAGATTATTAGGCTCGCCGGACATTTACTTAGGTAATAATAAAACACCATTTGCCAGTATAAATTACGTAACTTCTCATGATGGTTTCACATTAGAGGATTTAGTCAGTTATAATCAAAAACACAATGAAGCGAATGGATTTAACAATCAAGATGGAATGAACGAGAACTATAGTTGGAATTGTGGTGCAGAAGGACCAACAAATGACCAAAACGTGGTAATATGCAGGGAGAAACAAAAAAGGAACTTTATGATAACGTTACTCGTTAGTCAAGGAACCCCTATGATATTGGGAGGAGATGAGCTAAGCAGGACACAAAGAGGAAATAATAACGCGTTTTGTCAAGACAACGAGATAACTTGGTTTGATTGGAATTTAGATGAGAAAATCAAAATTTTTTAGAGTTTGTAAAAAGATGATCCAATTTTAGGCACATCCAGCATTCAGAAGGGAAAGATATTTTTT CAAGGAAAGAAATTATTCGGCATGCCGTTAAAAGATGTGACCTTCTATACTCTAAGGTAGGGAAGTTGATGAAAACATGGAGTTCCCCGACGCAACTAGTTATTTTCGTGTTGG AGGGAAGTGTTATGGACGAGATTAATGTATGGAGAGAGAATGCTGATGATTCCTTCTTAATACTTAACGCAAATCCCAATAACGTAAAGGTGAAATTCCCAAAAGGTAAATG GGAACTAGTTATCAGTTCTTATTTTGAGAGAAATAAAACCAGAAGAGAGAATCATAGAAGGTGAGAAGGAACTGGAAATAGAGGGAAGGACGGCATTAGTTTATAGGAGGAATTA

[0058] SEQ ID NO.2:

[0059] MALFFRTRDRPLRPGDPPYPLGSNWIEDDDGVNFSLFSENAEKVELLLYSLTNQKYPKEIIEVKNKTGDIWHVFPGLRPGQLYAYRVYGPYKPELGLRFNPNKVLIDPYAKAINGSVIWNDAVFGYKIGDQNQDLTYDERDSGEYVPKSVVINPYFEWDDEDFIKGKKVPLKDTVIYEV HVKGFTKLRLDLPENIRGTYEGLASEQMISYLKDLGITTVELMPVFHFIDQRFLTDKGLTNYWGYDPINFFSPECRYSSTGCLGGQVLSFKKMVNELHNAGIEVIIDVVYNHTAEGNHLGPTLSFRGIDNTAYYMLQPDNKRYYLDFTGTGNTNLSHPRVIQMVLDSLRYWVTEMHVDG FRFDLAAALARELYSVNMLNTFFIALQQDPILSQVKLIAEPWDVGQGGYQVGNFPYQWAEWNGKYRDSIRRFWRGEALPYSEIANRLLGSPDIYLGNNKTPFASINYVTSHDGFTLEDLVSYNQKHNEANGFNNQDGMNENYSWNCGAEGPTNDQNVVICREKQKRNFMITLLVSQGTP MILGGDELSRTQRGNNNAFCQDNEITWFDWNLDERKSKFLEFVKKMIQFYRAHPAFRRERYFQGKKLFGMPLKDVTFYLEGREVDEKTWSSPPTQLVIFVLEGSVMDEINMYGERIADDSFLIILNANPNNVKVKFPKGKWELVISSYLREIKPEERIEEGEKELEIEGRTALVYRRIEL

[0060] SEQ ID NO.3:

[0061] MALFFRTRDRPLRPGDPYPLGSNWIEDDDGVNFSLFSENAEKVELLLYSLTNQKYPKEIIEVKNKTGDIWHVFVPGLRPGQLYAYRVYGPYKPELGLRF NPNKVLIDPYAKAINGSVIWNDAVFGYKIGDQNQDLTYDERDSGEYVPKSVVINPYFEWDDEDFIKGKKVPLKDTVIYEVHVKGFTKLRLDLPENIRGTYEGLASEQMISYLKDLGITTVELMPVFHFIDQRFLTDKGLTNYWGYDPINFFSPECRYSSTGCLGGQVLSFKKMVNELHNAGIEVIIDVVYNHTAEGNHLGPTLSFRGIDNTAYYMLQPDNKRYFLDFTGTGNTLNLSHPRVIQMVLDSLRYWVTEMHVDGFRFDLAAALARELYSVNMLNTFFIALQQDPILSQVKLIAEPWDVGQGGYQVGNFPYQWAEWNGKYRDSIRRFWRGEALPYSEIANRLLGSPDIYLGNNKTPFASINYVTSHDGFTLEDLVSYNQKHNEANGFNNQDGMNENYSWNCGAEGPTNDQNVVICREKQKRNFMITLLVSQGTPMILGGDELSRTQRGNNNAFCQDNEITWFDWNLDERKSKFLEFVKKMIQFYRAHPAFRRERYFQGKKLFGMPLKDVTFYTLEGREVDEKTWSSPTQLVIFVLEGSVMDEINMYGERIADDSFLIILNANPNNVKVKFPKGKWELVISSYLREIKPEERIIEGEKELEIEGRTALVYRRIEL

[0062] SEQ ID NO.4:

[0063] MALFFRTRDRPLRPGDPPYPLGSNWIEDDDGVNFSLFSENAEKVELLLYSLTNQKYPKEIIEVKNKTGDIWHVFPGLRPGQLYAYRVYGPYKPELGLRFNPNKVLIDPYAKAINGSVIWNDAVFGYKIGDQNQDLTYDERDSGEYVPKSVVINPYFEWDDEDFIKGKKVPLKDTVIYEV HVKGFTKLRLDLPENIRGTYEGLASEQMISYLKDLGITTVELMPVFHFIDQRFLTDKGLTNYWGYDPINFFSPECRYSSTGCLGGQVLSFKKMVNELHNAGIEVIIDVVYNHTAEGNHLGPTLSFRGIDNTAYYMLQPDNKRYYLDFTGTGNTNLSHPRVIQMVLDSLRYWVTEMHVDG FRFDLAAALARELYSFNMLNTFFIALQQDPILSQVKLIAEPWDVGQGGYQVGNFPYQWAEWNGKYRDSIRRFWRGEALPYSEIANRLLGSPDIYLGNNKTPFASINYVTSHDGFTLEDLVSYNQKHNEANGFNNQDGMNENYSWNCGAEGPTNDQNVVICREKQKRNFMITLLVSQGTP MILGGDELSRTQRGNNNAFCQDNEITWFDWNLDERKSKFLEFVKKMIQFYRAHPAFRRERYFQGKKLFGMPLKDVTFYLEGREVDEKTWSSPPTQLVIFVLEGSVMDEINMYGERIADDSFLIILNANPNNVKVKFPKGKWELVISSYLREIKPEERIEEGEKELEIEGRTALVYRRIEL

[0064] SEQ ID NO.5:

[0065] MALFFRTRDRPLRPGDPYPLGSNWIEDDDGVNFSLFSENAEKVELLLYSLTNQKYPKEIIEVKNKTGDIWHVFVPGLRPGQLYAYRVYGPYKPELGLRFNPNKVLIDPYAKAINGSVIWNDAVFGYKIGDQNQDLTYDERDSGEYVPKSVVINPYFEWDDEDFIKGKKVPLKDTVIYEV HVKGFTKLRLDLPENIRGTYEGLASEQMISYLKDLGITTVELMPVFHFIDQRFLTDKGLTNYWGYDPINFFSPECRYSSTGCLGGQVLSFKKMVNELHNAGIEVIIDVVYNHTAEGNHLGPTLSFRGIDNTAYYMLQPDNKRYFYDNTGTGNTLNLSHPRVIQMVLDSLRYWVTEMHVDG FRFDLAAALARELYSVNMLNTFFIALQQDPILSQVKLIAEPWDVGQGGYQVGNFPYQWAEWNGKYRDSIRRFWRGEALPYSEIANRLLGSPDIYLGNNKTPFASINYVTSHDGFTLEDLVSYNQKHNEANGFNNQDGMNENYSWNCGAEGPTNDQNVVICREKQKRNFMITLLVSQGTP MILGGDELSRTQRGNNNAFCQDNEITWFDWNLDERKSKFLEFVKKMIQFYRAHPAFRRERYFQGKKLFGMPLKDVTFYTLEGREVDEKTWSSPTQLVIFVLEGSVMDEINMYGERIADDSFLIILNANPNNVKVKFPKGKWELVISSYLREIKPEERIIEGEKELEIEGRTALVYRRIEL

[0066] (2) Materials and reagents

[0067] The restriction endonucleases, Dpn I enzymes, PCR reagents, etc. used were purchased from Takara Biotechnology Co., Ltd.; primers were purchased from Anshengda Biotechnology Co., Ltd.; plasmid extraction kits, genome extraction kits, agarose purification kits, and E.coilBL21 (DE3) strains were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; other reagents were analytical grade reagents purchased domestically or abroad.

[0068] (3) Culture medium

[0069] The culture medium was prepared using dd H2O and sterilized at 121°C for 15-20 min.

[0070] LB liquid culture medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L.

[0071] LB solid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L.

[0072] TB liquid culture medium: tryptone 12 g / L, yeast powder 24 g / L, glycerol 5 g / L, KH2PO4 2.31 g / L, K2HPO4·3H2O 16.43 g / L.

[0073] (4) Purification buffer

[0074] Solution A: 20 mM CH3COONa, pH 6.0;

[0075] Solution B: 400 mM NaCl, 20 mM CH3COONa, pH 6.0;

[0076] Solution C: 20% saturation (NH4)2SO4, 20 mM Tris, pH 7.0;

[0077] Solution D: 20 mM Tris, pH 7.0.

[0078] (5) Detection method of dextrin debranching enzyme activity

[0079] Enzyme activity assay conditions: The reaction system includes 150 μL of purified fermentation broth or supernatant, 700 μL of 1% (w / v) DE 4 maltodextrin and 150 μL of 500 mM sodium acetate buffer (pH 5.0). The reaction is carried out at 70°C for 15 min and the reaction is terminated by boiling in a water bath for 15 min. After cooling, 100 μL of the reaction solution and 100 μL of 0.01 M I2-0.1 M KI are aspirated and allowed to stand for color development for 15 min. After dilution by a certain multiple, the absorbance is measured at 610 nm.

[0080] Definition of enzyme activity: Under the above reaction conditions, the crude enzyme solution inactivated by high temperature was used as blank control. 610 At 300 nm absorbance, an increase of 0.1 per hour with 1% DE4 maltodextrin as substrate was considered as 1 enzyme activity unit (U).

[0081] Among them, the calculation formula of the enzyme activity of dextrin debranching enzyme is as follows:

[0082]

[0083] Calculate the specific enzyme activity, where the specific enzyme activity of dextrin debranching enzyme is calculated as follows

[0084]

[0085] Example 1: Substrate specificity of wild-type dextrin debranching enzyme SsGDE

[0086] In order to analyze the substrate specificity and hydrolysis pattern of SsGDE, different substrates were selected to measure the hydrolysis activity of the enzyme. In this example, 1% (w / v) of potato amylose, pullulan, potato amylopectin, corn amylopectin, DE2 maltodextrin, DE4 maltodextrin, DE9 maltodextrin, DE16 maltodextrin, DE19 maltodextrin, DE21 maltodextrin and DE25 maltodextrin were used as substrates, and the DNS method was used to measure the hydrolysis activity of SsGDE, with the highest enzyme activity defined as 100%. The measurement results are shown in FIG. Figure 1 shown.

[0087] It can be seen that the hydrolysis activity of SsGDE on different types of substrates varies greatly. SsGDE has no hydrolysis activity on potato amylose, indicating that it cannot hydrolyze the α-1,4 glycosidic bonds in the starch substrate; in addition, SsGDE also has difficulty acting on pullulan, which is different from the hydrolysis mode of pullulanase; SsGDE can act on large molecular amylopectin and maltodextrin and has a higher hydrolysis efficiency on maltodextrin. When DE4 maltodextrin is used as a substrate, SsGDE has the highest hydrolysis activity, which is 22 times that of corn amylopectin and potato amylopectin; when high DE value (such as DE16 and DE19) maltodextrin is used as a substrate, the hydrolysis activity of SsGDE is 70% of the hydrolysis activity when DE4 maltodextrin is used as a substrate. This shows that SsGDE tends to act on maltodextrins with a certain DE value, and its hydrolysis ability on large molecular amylopectin and maltodextrins with high DE values ​​is reduced. In order to further explore and optimize the performance of dextrin debranching enzyme (SsGDE) in practical applications, the following experiments were designed to further improve its substrate specificity and catalytic efficiency through molecular modification.

[0088] Example 2: Design of dextrin mutation sites

[0089] According to the classification of the Carbohydrate active enzymes database (CAZY), SsGDE (PDB ID: 7EAV) and isoamylase PaISO (PDB ID: 1BF2) and CrISO (PDB ID: 4OKD) all belong to the GH13 family.

[0090] The structures of SsGDE, PaISO and CrISO were aligned and it was found that there were four loop regions in the catalytic cavity of the three enzymes. Except for loop region I, the amino acid residues exposed in the catalytic cavity in the other three loop regions are highly conserved. The loop region I of SsGDE is composed of amino acid residues Phe232, Leu233, Thr234, Asp235 and Lys236, forming an α-helical structure. Among them, the side chain of Phe232 faces the inside of the catalytic cavity and may form a π-π stacking interaction with the substrate during the catalytic process. Compared with SsGDE, the region I sequences of PaISO and CrISO are flexible loop sequences. The difference in the structure of region I in the catalytic cavity may be the reason why the substrate specificity of SsGDE is different from that of isoamylase. In region II, the side chain of Phe557 residue of SsGDE overlaps with the side chain of Tyr595 of PaISO and the side chain of Tyr706 of CrISO in spatial conformation, and the aromatic ring side chain of this residue faces the catalytic cavity, which can form π-π stacking interactions with the sugar unit of the substrate. In region IV, Trp401 of SsGDE is conserved in PaISO and CrISO, and the aromatic ring side chain also faces the catalytic cavity, which is speculated to be related to substrate recognition and substrate conformation adjustment.

[0091] In addition, molecular dynamics simulation results showed that the distribution of aromatic amino acids in the substrate binding region of SsGDE was different from that of PaISO and CrISO. Multiple sequence alignment results showed that the amino acid sequence similarity between SsGDE and CrISO and PaISO was low, and most of the amino acid residues in the substrate binding region of SsGDE were not conserved in PaISO and CrISO. The interaction force between residue Val375 in the substrate binding region of SsGDE and the G10 ligand was about -9.65 kJ·mol -1 ; However, in PaISO and CrISO structures, the amino acid residues at the corresponding positions are Phe403 and Pro503, respectively. Residues Tyr323, Leu324, and Phe326 in SsGDE are also not conserved in PaISO (Phe335, Tyr336, and Asn338) and CrISO (Tyr412, Tyr413, and Tyr415).

[0092] Therefore, we tried to adjust the aromatic amino acids in the substrate binding region of SsGDE and select several potential active sites for site-directed mutagenesis in order to improve the enzyme's ability to hydrolyze specific substrates. Specifically: Based on the structural analysis of the three debranching enzymes and the study of the substrate specificity of the wild-type SsGDE, the mutation sites were selected by multiple sequence alignment combined with an online server: the SWISS-MODEL software was used to simulate the protein structure of the amylin debranching enzyme to obtain the tertiary structure model of the amylin debranching enzyme. The amino acid sites to be mutated were determined to be tyrosine at position 323, leucine at position 324, phenylalanine at position 326, valine at position 375, and the loop structure "Tyrosine Y-Leucine L-Aspartic Acid D-Phenylalanine F" ( Figure 2 ).

[0093] Example 3: Site-directed mutagenesis of dextrin debranching enzyme and construction of recombinant plasmid and recombinant Escherichia coli

[0094] (1) Construction of wild-type plasmid

[0095] The accession number (PDBID) of the dextrin debranching enzyme gene from the microorganism Saccharolobus solfataricus STB09 is 7EAV, the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0096] The dextrin debranching enzyme encoding gene with a nucleotide sequence as shown in SEQ ID NO.1 was constructed between the NcoI and XhoI restriction sites of the plasmid vector pET-20b(+) to construct a recombinant plasmid, which was named E. coli BL21(DE3)(Ssgde / pET-20b(+)).

[0097] (2) Construction of mutant plasmid

[0098] Primers were designed based on the gene encoding SsGDE from Saccharolobus solfataricus STB09 as shown in SEQ ID NO.1, and site-directed mutagenesis was performed using the E. coli BL21 (DE3) (Ssgde / pET-20b (+)) constructed in step (1) as a template to construct a mutant plasmid:

[0099] Y323F_F1:CCCAGCCGGCGATGGCC;

[0100] Y323F_R1: AATATCTTTTATTATCTGGTTGGAGCATGTAATATGC;

[0101] Y323F_F2:

[0102] ATAATAAAAGATAT TTT TTAGACTTTACCGGAACTGGCAATACG;

[0103] Y323F_R2: GTGGTGGTGGTGGTGCTC;

[0104] V375F_F1:CCCAGCCGGCGATGGCC;

[0105] V375F_R1: GCTGTATAATTCCCTGGCTAAAGCTG;

[0106] V375F_F2:

[0107] AGGGAATTATACAGC TTT AATATGTTAAATACGTTCTTCATTGTCCTAC AG;

[0108] V375F_R2: GTGGTGGTGGTGGTGCTC;

[0109] YLDF / FYDN_F1:CCCAGCCGGCGATGGCC;

[0110] YLDF / FYDN_R1:

[0111] AATATCTTTTATTATCTGGTTGGAGCATGTAATATG;

[0112] YLDF / FYDN_F2:

[0113] ATAATAAAAGATAT TTTTATGACAAT ACCGGAACTGGCAATACGTTAA ATC;

[0114] YLDF / FYDN_R2:GTGGTGGTGGTGGTGCTC.

[0115] The underlined parts represent the codons corresponding to the 323rd tyrosine, the 375th valine, and the 323rd to 326th loop structure "tyrosine Y-leucine L-aspartic acid D-phenylalanine F" encoded by the mutant gene.

[0116] The composition of the PCR reaction solution and the PCR amplification procedure were carried out according to the method provided in the instruction manual of Takara Ex Taq DNA Polymerase. After PCR amplification, the DNA fragments were purified and recovered by DNA gel electrophoresis. The empty pET-20b(+) vector was double-digested with NcoI and XhoI restriction endonucleases, and the linear vector was purified and recovered by DNA gel electrophoresis to obtain a linear pET-20b(+) vector. The DNA fragments were connected and transformed into E.coli JM109 competent cells using a seamless cloning kit, LB plates were coated, single colonies were picked into LB liquid culture medium, plasmids were extracted, and plasmids with completely correct sequences were selected after DNA sequencing and transformed into E.coli BL21(DE3) competent cells for mutant protein production, and the correct mutants Y323F, V375F and YLDF / FYDN were obtained.

[0117] The mutants L324Y, F326N, F326Y, and V375P were constructed according to the above method. The primer sequences involved are:

[0118] L324Y_F1 CCCAGCCGGCGATGGCC

[0119] L324Y_R1 AATAATATCTTTTATTATCTGGTTGGAGCATGTAATATG L324Y_F2:

[0120] ATAAAAGATATTAT TAC GACTTTACCGGAACTGGCAATAC

[0121] L324Y_R2:GTGGTGGTGGTGGTGCTC

[0122] F326N_F1 CCCAGCCGGCGATGGCC

[0123] F326N_R1 GTCTAAAATAATATCTTTTATTATCTGGTTGGAGC

[0124] F326N_F2:

[0125] AGATATTATTTAGAC AAT ACCGGAACTGGCAATACGTTAAATC

[0126] F326N_R2: GTGGTGGTGGTGGTGCTC

[0127] F326Y_F1 CCCAGCCGGCGATGGCC

[0128] F326Y_R1 GTCTAAAATAATATCTTTTATTATCTGGTTGGAGC

[0129] F326Y_F2:

[0130] AGATATTATTTAGAC TAT ACCGGAACTGGCAATACGTTAAATC

[0131] F326Y_R2: GTGGTGGTGGTGGTGGTGCTC

[0132] V375P_F1 CCCAGCCGGCGATGGCC

[0133] V375P_R1 GCTGTATAATTCCCTGGCTAAAGCTG

[0134] V375P_F2:

[0135] AGGGAATTATACAGC CCT AATATGTTAAATACGTTCTTCATTGCTCTAC

[0136] AG

[0137] V375P_R2: GTGGTGGTGGTGGTGCTC

[0138] Example 4: Expression and purification of wild-type enzymes and mutant enzymes

[0139] E. coli BL21 (DE3) containing the recombinant plasmid or mutant plasmid was inoculated into LB medium and cultured at 37°C until OD 600 The final concentration of IPTG was about 0.6, and the culture was continued for 4 hours to induce protein expression. The cells were collected, ultrasonically disrupted, and the supernatant and precipitate were separated by centrifugation. The expression of SsGDE and its mutants was analyzed by SDS-PAGE. The results are shown in Figure 4 .

[0140] SsGDE and mutants were purified using anion exchange chromatography column and hydrophobic interaction chromatography column, and the elution buffer contained appropriate concentration of Tris-HCl.

[0141] The specific activities of the obtained pure enzyme solutions were detected respectively, and the results of evaluating the enzyme activity are shown in Table 1.

[0142] Table 1 Specific enzyme activity of wild-type enzyme and pure enzyme solution of mutants

[0143]

[0144] Compared with the specific activity of the wild-type enzyme, the specific activity of the single point mutant V375F increased by 375.7%, the specific activity of Y323F increased by 306.2%, and the specific activity of the combined mutant YLDF / FYDN increased by 273.8%. The specific activity of the mutants L324Y, F326N, F326Y, and V375P did not increase compared with the wild-type. This shows that mutating some amino acids in the substrate binding region of SsGDE to phenylalanine, which can form a stronger interaction with the substrate, can enhance the enzyme's ability to bind to the substrate.

[0145] Example 5: Hydrolysis activity of wild enzyme and mutant on corn amylopectin, potato amylopectin and DE4 maltodextrin

[0146] The catalytic activity of the purified mutants was measured. Figure 5 As shown in the figure, the mutants Y323F, V375F and YLDF / FYDN showed improved hydrolysis activity on corn amylopectin, potato amylopectin and DE4 maltodextrin. When Tyr323 in SsGDE mutated to Phe323, the hydrolysis activity of mutant Y323F on corn amylopectin, potato amylopectin and DE4 maltodextrin increased by 31.73%, 30.19% and 20.63%, respectively; the V375F mutant also significantly improved the hydrolysis activity of amylopectin and DE4 maltodextrin. Compared with the wild type, the hydrolysis activity of mutant V375F on corn amylopectin, potato amylopectin and DE4 maltodextrin increased by 64.77%, 60.82% and 23.08%, respectively; in addition, the hydrolysis activity of mutant YLDF / FYDN on amylopectin and DE4 maltodextrin was also improved, increasing the hydrolysis activity of corn amylopectin, potato amylopectin and DE4 maltodextrin by 6.96%, 4.17% and 5.06%, respectively. The hydrolysis activities of mutants L324Y, F326N, F326Y, and V375P on corn amylopectin, potato amylopectin, and DE4 maltodextrin were all reduced, indicating that these mutants had a negative impact on the enzyme activity.

[0147] Using corn amylopectin, potato amylopectin and DE4 maltodextrin as substrates, the kinetic parameters of mutants Y323F, V375F and YLDF / FYDN were determined, and the results are shown in Table 2. When DE4 maltodextrin was used as a substrate, the kcat / Km values ​​of Y323F, V375F and YLDF / FYDN were 21.05%, 26.31% and 7.89% higher than those of wild-type SsGDE, respectively; when corn amylopectin and potato amylopectin were used as substrates, the kcat / Km values ​​of Y323F, V375F and YLDF / FYDN were also higher than those of wild-type SsGDE, indicating that the hydrolysis of macromolecular amylopectin by SsGDE mutants was also improved to a certain extent, among which the improvement of the hydrolysis efficiency of amylopectin by Y323F and V375F was significantly higher than that of DE4 maltodextrin. This indicates that the mutant effectively enhances the catalytic efficiency of SsGDE on macromolecular branched starch by expanding the substrate binding region or improving the binding ability of amino acid residues in the substrate binding region with the substrate.

[0148] Table 2 Kinetic parameters of mutants Y323F, V375F and YLDF / FYDN

[0149]

[0150] Note: Data are mean ± standard deviation; different letters in the same column indicate significant differences (p<0.05)

[0151] Example 6

[0152] The hydrolysis activity of the purified mutant Y323F on different substrates was measured. Figure 1 As shown. The wild-type SsGDE has high hydrolysis efficiency for short-chain branches with a degree of polymerization (DP) of 4 to 10 in the substrate, and low hydrolysis efficiency for long-chain branches (DP11 to 20 and DP>21) and short-chain branches with DP<4. The mutant Y323F has different degrees of improvement in the hydrolysis activity of amylopectin and maltodextrins with different DE values, indicating that by adjusting the aromatic amino acids in the SsGDE substrate binding region, expanding the substrate binding region or strengthening the affinity of the substrate binding region with the substrate, the hydrolysis efficiency of SsGDE for macromolecular starch and can be significantly improved, which lays a certain foundation for the subsequent selection of suitable dextrin debranching enzymes for use in starch sugar production.

[0153] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A dextrin debranching enzyme mutant, characterized in that: The dextrin debranching enzyme mutant is obtained by mutating the dextrin debranching enzyme with an amino acid sequence as shown in SEQ ID NO.2, and the mutation includes: mutating tyrosine at position 323 to phenylalanine; or mutate valine at position 375 to phenylalanine; Or mutate the tyrosine at position 323 to phenylalanine, mutate the leucine at position 324 to tyrosine, and mutate the phenylalanine at position 326 to asparagine.

2. A nucleic acid molecule encoding the dextrin debranching enzyme mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The sequence of the nucleic acid molecule is any one of SEQ ID NO.3-5.

4. A gene integration expression cassette carrying the nucleic acid molecule of claim 2 or 3.

5. A recombinant plasmid carrying the nucleic acid molecule according to claim 2 or 3.

6. A recombinant cell expressing the amylin debranching enzyme mutant according to claim 1.

7. Use of the dextrin debranching enzyme mutant according to claim 1, the nucleic acid molecule according to claim 2 or 3, the gene integration expression frame according to claim 4, the recombinant plasmid according to claim 5 or the recombinant cell according to claim 6 in starch processing.

8. The use according to claim 7, characterized in that: Contain at least one of the following characteristics: (1) The application includes hydrolyzing α-1,6 glycosidic bonds or hydrolyzing substances containing α-1,6 glycosidic bonds or starch branches; (2) The application includes hydrolyzing pullulan or dextrin.

9. A method for hydrolyzing starch-containing branched-chain substances, characterized in that: The method comprises the following steps: contacting starch branch-containing material with the dextrin debranching enzyme mutant according to claim 1 to cause a hydrolysis reaction.

10. The method according to claim 9, characterized in that The starch-containing branched-chain substances include starch or dextrin.

11. The method according to claim 10, characterized in that Contain at least one of the following characteristics: (1) The starch comprises at least one of corn starch, wheat starch and tapioca starch; (2) The dextrin includes maltodextrin. (3) The dextrin includes maltodextrin with a DE value of 2-25.

12. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli overexpresses the gene encoding the dextrin debranching enzyme mutant according to claim 1.

13. The method for constructing the recombinant Escherichia coli according to claim 12, characterized in that: The following steps are involved: The recombinant plasmid containing the dextrin debranching enzyme mutant encoding gene is introduced into the starting strain or the dextrin debranching enzyme mutant encoding gene is integrated into the genome of the starting strain to obtain a recombinant strain expressing the dextrin debranching enzyme mutant in a free or integrated manner.

14. A method for producing a dextrin debranching enzyme mutant, characterized in that: The method comprises the following steps: culturing the recombinant Escherichia coli according to claim 12 to make it express a dextrin debranching enzyme mutant.

15. An enzyme composition, characterized in that The enzyme composition contains the dextrin debranching enzyme mutant according to claim 1.

16. Use of the enzyme composition according to claim 15 in starch processing.

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