An oligo-1,6-glucosidase mutant having enhanced ability to hydrolyze alpha-1,4 glycosidic bonds and use thereof

Abstract

This invention discloses an oligo-1,6-glucosidase mutant with enhanced α-1,4-glycosidic bond hydrolysis activity and its applications, belonging to the fields of genetic engineering and enzyme engineering technology. The oligo-1,6-glucosidase mutant V219A / P227A of this invention retains the original α-1,6-glycosidic bond hydrolysis activity while also possessing α-1,4-glycosidic bond hydrolysis activity not found in the wild-type enzyme, and the α-1,4-glycosidic bond hydrolysis activity is increased by 267% compared to the single mutant V219A. The mutant V219A / P227A was applied to the enzymatic production process of glucose. Using maltodextrin as a substrate, glucosyl amylase and the mutant V219A / P227A were added for enzymatic conversion, achieving a final glucose yield of 98.0%. When the mutant V219A / P227A was applied to the regeneration of glucose from crystalline glucose mother liquor, the final glucose yield reached 95.9%, representing increases of 9.35% and 5.15% compared to the wild type and the single mutant V219A, respectively. This demonstrates that the mutant helps improve substrate conversion rate and product purity in the glucose production process and has significant industrial application value for regenerating glucose from crystalline glucose mother liquor.

Description

Technical Field

[0001] This invention relates to an oligo-1,6-glucosidase mutant with enhanced α-1,4 glycosidic bond hydrolysis ability and its application, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] Starch sugars are a general term for sugars obtained from starch-containing raw materials such as corn, wheat, oats, rice, potatoes, and cassava through acid, acid-enzyme, or total enzymatic reactions. They are the main products of deep starch processing. Industrially produced starch sugars are diverse, mainly including crystalline glucose, fructose syrup, total sugar, and maltose syrup. Starch is gelatinized, liquefied, and saccharified to produce starch sugars. However, liquefying enzymes and saccharifying enzymes are difficult to act on the α-1,6 glycosidic bonds of starch, resulting in low yields of the target product and high proportions of byproducts. Therefore, debranching enzymes are often added to the enzymatic production process of starch sugars to improve production efficiency.

[0003] Debranching enzymes are a class of specific and highly efficient hydrolytic enzymes that break down α-1,6 glycosidic bonds in starch and related polysaccharides. They are mainly classified into pullulanases, isoamylases, and oligo-1,6-glucosidases. Pullulanases tend to act on dextrins and pullulan polysaccharides with relatively small molecular weights, isoamylases tend to act on amylopectin and glycogen and other glucans with higher molecular weights, and oligo-1,6-glucosidases tend to act on small molecule substrates such as isomaltooligosaccharides.

[0004] In the enzymatic production of glucose, starch is typically used as a substrate. After liquefaction into maltodextrin by α-amylase, glucoamylase and debranching enzymes are added for saccharification. The saccharification solution contains glucose as the majority, but also small-molecule sugar impurities such as maltose, maltotriose, isomaltose, isomalttriose, and panose. Wild-type oligo-1,6-glucosidase can completely hydrolyze the α-1,6 glycosidic bonds of maltose, isomalttriose, and panose, but it cannot hydrolyze the α-1,4 glycosidic bonds in panose and has no hydrolytic activity towards maltose and maltotriose. Therefore, it is difficult to convert as many small-molecule sugars in the saccharification solution as possible into glucose to increase yield. Thus, improving the ability of oligo-1,6-glucosidase to hydrolyze α-1,4 glycosidic bonds is an effective strategy to expand its application range and meet the industrial demand for increased glucose production.

[0005] The patent with authorization announcement number CN116064456B discloses an oligosaccharide debranching enzyme mutant derived from Paenibacillus sp. STB16 and its application in glucose mother liquor. After modifying the wild-type enzyme, the mutant V219A has a certain ability to hydrolyze α-1,4 glycosidic bonds. However, the ability of this mutant to hydrolyze α-1,4 glycosidic bonds is weak, and the efficiency of converting maltose, maltotriose, panose, etc. into glucose is low. Moreover, it is mainly used for glucose regeneration from glucose mother liquor, without fundamentally improving glucose production efficiency and substrate conversion rate, which is difficult to meet the development needs of green, low-carbon, energy-saving and consumption-reducing industries in the starch sugar industry. Summary of the Invention

[0006] To address the issue of the weak hydrolysis ability of the oligosaccharide debranching enzyme mutant V219A for α-1,4 glycosidic bonds, this invention provides a novel oligo-1,6-glucosidase mutant V219A / P227A, which, while retaining the original α-1,6 glycosidic bond hydrolysis activity, exhibits a 267% higher ability to hydrolyze α-1,4 glycosidic bonds compared to mutant V219A. When the mutant V219A / P227A was applied to the enzymatic production process of glucose, the final glucose yield reached 98.0%, which was 2.62% and 1.45% higher than that achieved using glucoamylase + wild-type oligo-1,6-glucosidase and glucoamylase + mutant V219A, respectively. When the mutant V219A / P227A was applied to the regeneration of glucose from crystalline glucose mother liquor, the final glucose yield reached 95.9%, which was 9.35% and 5.15% higher than that achieved using glucoamylase + wild-type oligo-1,6-glucosidase and glucoamylase + single mutant V219A, respectively. It is evident that the mutant V219A / P227A is better suited to the enzymatic production process of glucose and can be used to treat the byproduct crystalline glucose mother liquor, thus possessing greater industrial application value.

[0007] The first objective of this invention is to provide an oligo-1,6-glucosidase mutant, the amino acid sequence of which is shown in SEQ ID NO.1. The mutation is a mutation at a specific site of the wild-type oligo-1,6-glucosidase, comprising:

[0008] The valine at position 219 was mutated to alanine, and the proline at position 227 was mutated to alanine.

[0009] A second object of the present invention is to provide a nucleic acid molecule encoding the oligo-1,6-glucosidase mutant.

[0010] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2.

[0011] A third objective of this invention is to provide a gene integration expression cassette or recombinant plasmid carrying the nucleic acid molecule.

[0012] Furthermore, the recombinant plasmid can use any expression vector as its backbone, selected according to the host type, and can be a pET series vector.

[0013] A fourth object of the present invention is to provide recombinant cells containing the oligo-1,6-glucosidase mutant.

[0014] Furthermore, the host cell is a microorganism, such as bacteria or fungi.

[0015] Furthermore, the bacteria are Escherichia coli or Bacillus subtilis, preferably Escherichia coli BL21(DE3).

[0016] A fifth object of the present invention is to provide the use of the aforementioned oligo-1,6-glucosidase mutant, nucleic acid molecule, gene expression cassette, recombinant plasmid, or recombinant cell in the hydrolysis of oligosaccharides.

[0017] Further, the oligosaccharide includes linear maltodextrin or isomaltodextrin. Preferably, the oligosaccharide includes one or more of maltose, maltotriose, isomaltoose, isomaltotriose, and panose.

[0018] A sixth object of the present invention is to provide the use of the aforementioned oligo-1,6-glucosidase mutant, nucleic acid molecule, gene expression cassette, recombinant plasmid, or recombinant cell in the preparation of glucose.

[0019] Furthermore, oligosaccharides were used as substrates for preparation.

[0020] A seventh object of the present invention is to provide an enzyme composition comprising the oligo-1,6-glucosidase mutant.

[0021] Furthermore, the enzyme composition also includes glucosylamylase.

[0022] An eighth object of the present invention is to provide the use of the aforementioned oligo-1,6-glucosidase mutant, nucleic acid molecule, gene expression cassette or recombinant plasmid, recombinant cell or enzyme composition in the regeneration of glucose from crystalline glucose mother liquor.

[0023] Further, the application is as follows: using maltodextrin solution as substrate, equilibrate in a 50°C constant temperature water bath for 15 min, adjust the pH to 6.0, add glucosidase at an enzyme dosage of 60 U / g and oligo-1,6-glucosidase mutant V219A / P227A at an enzyme dosage of 20-50 U / g, and maintain the reaction at a constant temperature of 50°C for 72 h.

[0024] Furthermore, using a primary mother liquor of crystalline glucose with a solid content of 60% as the substrate, the mixture was placed in a 50°C constant temperature water bath for 15 minutes to equilibrate. The pH was adjusted to 6.0, and glucoamylase was added at a dosage of 30 U / g, while oligo-1,6-glucosidase mutant V219A / P227A was added at a dosage of 20–100 U / g. The reaction was maintained at 50°C for 24 hours.

[0025] A ninth object of the present invention is to provide a recombinant Escherichia coli in which the oligo-1,6-glucosidase mutant is overexpressed.

[0026] The tenth object of the present invention is to provide the use of the recombinant Escherichia coli in the preparation of oligo-1,6-glucosidase mutants.

[0027] The eleventh object of the present invention is to provide a method for producing oligo-1,6-glucosidase mutants, comprising the step of fermentation production using the recombinant Escherichia coli.

[0028] Further fermentation was carried out at 28-32℃ and 180-280rpm.

[0029] Furthermore, the fermentation medium contains the following components: yeast extract 20-30 g / L, tryptone 10-15 g / L, KH2PO4 1-5 g / L, K2HPO4·3H2O 15-20 g / L, and glycerol 1-10 g / L.

[0030] A method for improving glucose yield in an enzymatic glucose production process, wherein in the process of producing crystalline glucose from maltodextrin, glucosidase is added during the saccharification reaction stage, along with the aforementioned oligo-1,6-glucosidase mutant, whole cells or preparations containing the mutant, to carry out the reaction.

[0031] A method for regenerating glucose from glucose mother liquor involves using crystalline glucose mother liquor produced by the above process as raw material, adding glucosidase, an oligo-1,6-glucosidase mutant, whole cells or preparations containing the mutant, and reacting.

[0032] The beneficial effects of this invention are:

[0033] (1) This invention provides a novel oligo-1,6-glucosidase mutant. Based on the single mutant V219A of oligo-1,6-glucosidase derived from Paenibacillus sp. STB16, the double mutant V219A / P227A is obtained by mutating proline at position 227 to alanine. While retaining the original α-1,6 glycosidic bond hydrolysis activity, the ability to hydrolyze α-1,4 glycosidic bonds is increased by 267%. It can efficiently and specifically convert common by-products in glucose production (including maltose, maltotriose, isomaltose, isomalttriose, panose, etc.) into glucose, expanding the enzyme's catalytic function and substrate applicability range. It can better adapt to specific scenarios such as glucose enzymatic production and has greater industrial application value.

[0034] (2) This invention provides a method to improve the glucose yield in the glucose enzymatic production process. In the process of producing crystalline glucose from maltodextrin, the mutant V219A / P227A is applied to the saccharification reaction stage, and the final glucose yield reaches 98.0%, which is 2.62% and 1.45% higher than that of using glucoamylase + wild-type oligo-1,6-glucosidase and using glucoamylase + mutant V219A, respectively. This can effectively improve the glucose production efficiency and substrate conversion rate, reduce the generation of mother liquor in the subsequent glucose crystallization process, and help meet the green, low-carbon, energy-saving and consumption-reducing development needs of the starch sugar industry.

[0035] (3) This invention provides a method for regenerating glucose from crystalline glucose mother liquor. The crystalline glucose mother liquor is treated with glucoamylase + mutant V219A / P227A. The final glucose yield reaches 95.9%, which is 9.35% and 5.15% higher than that of glucoamylase + wild-type oligo-1,6-glucosidase and glucoamylase + mutant V219A, respectively. This helps to realize the recycling of by-products of glucose production and reduce wastewater discharge. Attached Figure Description

[0036] Figure 1 SDS-PAGE analysis results for oligo-1,6-glucosidase mutants. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0038] The culture media involved in the following examples are as follows:

[0039] LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0040] LB solid medium: 1.5% (w / v) agar powder is added to LB medium.

[0041] TB medium: yeast extract 24 g / L, tryptone 12 g / L, KH2PO4 2.32 g / L, K2HPO4·3H2O 16.43 g / L, glycerol 5 g / L, pH 7.0.

[0042] The detection method involved in this invention is as follows:

[0043] (1) Determination of α-1,6 glycosidic bond hydrolysis activity

[0044] The activity of the mutant was determined using 10 mM p-nitrophenyl-α-d-glucopyranoside (pNPG) as a substrate. The reaction system consisted of 150 μL phosphate buffer (500 mM, pH 6.0), 800 μL pNPG solution, and 50 μL enzyme. After reacting at 50 °C for 5 min, 1 mL sodium carbonate (1 M) was added to terminate the reaction, and the absorbance at 410 nm was measured. The released p-nitrophenyl (pNP) was determined spectrophotometrically. One unit of hydrolytic activity (U) was defined as the amount of enzyme producing 1 μmol of p-nitrophenyl (pNP) per minute under the assay conditions.

[0045] (2) Determination of α-1,4 glycosidic bond hydrolysis activity

[0046] The activity of the mutant was determined using 5 mg / mL maltose as a substrate. The reaction system consisted of 150 μL phosphate buffer (500 mM, pH 6.0), 800 μL substrate, and 50 μL enzyme. After incubating at 50 °C for 5 min, the glucose content produced was determined using a glucose assay kit. The reaction system for determining glucose content consisted of 1.5 mL of the kit solution and 50 μL of the reaction solution. One unit of hydrolytic activity (U) was defined as the amount of enzyme required to produce 1 μmol of glucose per minute under the assay conditions.

[0047] (3) Analysis of saccharification reaction products

[0048] The oligo-1,6-glucosidase mutant V219A / P227A was applied to the glucose enzymatic production process, and the content of each component in the product was analyzed by high performance liquid chromatography (HPLC). The analytical conditions were as follows: a ChromCore normal phase / HILIC column was used, with 70% acetonitrile-water solution as the mobile phase, a flow rate of 1.0 mL / min, a column temperature of 30 °C, and an injection volume of 10 μL.

[0049] The sequence involved in this invention is as follows:

[0050] (1) Amino acid sequence of oligo-1,6-glucosidase mutant V219A / P227A (SEQ ID NO.1):

[0051] MLLFPFESRSRSIPTGGWQMKRAWWKESVVYQIYPRSFQDSNGDGIGDIPGIVSRLDYLQELGVDVVWLCPVYDSPNDDNGYDIRDYRRIMDEFGTLEDWERLLEDLHARGMKLIMDLVVNHSSDEHAWFSESRKSRDGEHRDY YIWRDGKGGAEPNNWSSFFSGSAWKYDGETDQYYLHLFSSKQPDLNWENGKVRREVYNMMAWWLDKGIDGFRMDAINLISKVAGLPDAPGEGRYRSGADYFMNGPRVHEYLQEMNREVLSRYDIMTVGETPGVTPEQAALYVGE DRGELNMVFQFEHMDIDSGPGGKWDVQPWRLTDFKRVMGKWQRELQDRGWNSLYLNNHDQPRMVSRFGDDKNFRKQSAKMLGTLLHTLQGTPYIYQGEELGMTNVRFGSIEDYRDIETLNMYKEATGAGRPAEAVMASVYSKGR DNARTPMQWDGSAHGGFTTGTPWIASNPNYTEINAEDARRDPDSIFHYYRRLIALRKQHDVIVYGRYEALLEEDERIYAYTRMLDGERLLVVLNFFGEEADCSLPEKIRFESAEPLIGNYGNGADRDWRSLKLRPYEALVLRLQG

[0052] (2) Nucleotide sequence of oligo-1,6-glucosidase mutant V219A / P227A (SEQ ID NO.2):

[0053]

[0054] Example 1: Construction of recombinant plasmids containing mutants

[0055] Complementary primer chains were designed using the expression vector pET-28a(+) as a template (see Table 1).

[0056] Site-directed mutagenesis was performed according to the instructions for the TaKaRa STAR Primer GXL kit. The PCR system consisted of 5×PrimeSTAR GXL Buffer (Mg... 2+ 10 μL of dNTP primer (2.5 mM each), 4 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 1 μL of template DNA, 1 μL of PrimeSTAR GXL DNA Polymerase (1.25 U / μL), and double-distilled water were added to a final volume of 50 μL. PCR amplification conditions were: 98℃ pre-denaturation for 3 min; followed by 30 cycles (98℃ for 10 s, 60℃ for 15 s, 68℃ for 7 min); and finally, incubation at 68℃ for 10 min.

[0057] Table 1. Site-directed mutagenesis primers

[0058]

[0059] Note: 1 The underlined bases correspond to the mutated amino acids.

[0060] Example 2: Construction of genetically engineered bacteria

[0061] The specific steps are as follows:

[0062] (1) At 37°C, the PCR product obtained in Example 1 was treated with DpnI. The DpnI digestion system consisted of 10 μL of PCR product, 7 μL of double-distilled water or Milli-Q water, 2 μL of 10X Buffer Y, and 1 μL of DpnI. The treated PCR product was then transformed into E. coli JM109 competent cells. The transformed product was then plated onto LB agar solid medium containing 100 μg / mL kanamycin and cultured overnight at 37°C for 12 h. Single colonies were then selected and inoculated into LB liquid medium containing 100 μg / mL kanamycin and cultured overnight at 37°C and 200 r / min. Plasmids were then extracted, identified, and sequenced according to the instructions of the plasmid extraction kit.

[0063] (2) The correctly sequenced plasmid was transferred into the competent cells of the expression host E.coli BL21(DE 3). The final result was the genetically engineered strain E.coli BL21(DE 3)(pET-28a(+) / v219a / p227a).

[0064] Example 3: Expression of oligo-1,6-glucosidase mutant

[0065] The specific steps are as follows:

[0066] (1) Seed culture: 100 μL of the genetically engineered bacteria E.coli BL21(DE3)(pET-28a(+) / v219a / p227a) was inoculated into LB medium containing the corresponding antibiotics and cultured at 37℃ and 200 r / min for 8–10 h.

[0067] (2) Fermentation culture: The seed culture solution was inoculated into the fermentation medium containing the corresponding antibiotic at an inoculation amount of 4% (v / v) and cultured in a shaker at 37℃ and 200r / min. When the OD600 value of the culture solution reached 0.4-0.6, IPTG was added to make the final concentration 0.05mM and expression was induced for 48h at 25℃ and 200r / min.

[0068] (3) Crude enzyme acquisition: Centrifuge the fermented bacterial solution at 4℃ and 10000r / min for 20min, discard the supernatant, resuspend the remaining cells in 10mM phosphate buffer (pH 6.0), and then break the cells with an ultrasonic cell disruptor. After centrifugation, the enzyme solution is present in the supernatant.

[0069] (4) Enzyme purification: Oligo-1,6-glucosidase was purified using a nickel column. The equilibration buffer (Solution A, pH 7.5) consisted of 500 mM NaCl, 50 mM Tris-HCl, and 20 mM imidazole; the elution buffer (Solution B, pH 7.5) consisted of 500 mM NaCl, 50 mM Tris-HCl, and 500 mM imidazole. The crude enzyme solution was filtered through a 0.45 μm aqueous membrane. The nickel column was first equilibrated with buffer A at a flow rate of 2 mL / min, and then the sample was loaded at a rate of 1.5 mL / min to bind the target protein to the nickel column. After loading, the nickel column was equilibrated again with buffer A. After equilibration, the sample was eluted with 60% elution buffer B at a flow rate of 1 mL / min, and the corresponding eluent was collected based on the elution peak.

[0070] Example 4: Detection of the hydrolytic activity of oligo-1,6-glucosidase mutants on α-1,6 glycosidic bonds

[0071] The activity of the mutant was determined using 10 mM pNPG as a substrate. The reaction system consisted of 150 μL phosphate buffer (500 mM, pH 6.0), 800 μL pNPG solution, and 50 μL enzyme. After reacting at 50 °C for 5 min, 1 mL sodium carbonate (1 M) was added to terminate the reaction, and the absorbance at 410 nm was measured. The released pNPs were determined spectrophotometrically.

[0072] The specific enzyme activities of wild-type oligo-1,6-glucosidase and its mutants in hydrolyzing α-1,6 glycosidic bonds are shown in Table 2. The wild-type enzyme has strong hydrolytic activity of α-1,6 glycosidic bonds. The single mutant V219A and the double mutant V219A / P227A still retain strong hydrolytic activity of α-1,6 glycosidic bonds, which are 90% and 94.67% of that of the wild-type enzyme, respectively.

[0073] Table 2 Hydrolytic activity of oligo-1,6-glucosidase mutants on α-1,6 glycosidic bonds.

[0074]

[0075] Example 5: Detection of the hydrolytic activity of oligo-1,6-glucosidase mutants on α-1,4 glycosidic bonds

[0076] The activity of the mutant was determined using 5 mg / mL maltose as a substrate. The reaction system consisted of 150 μL phosphate buffer (500 mM, pH 6.0), 800 μL substrate, and 50 μL enzyme. After incubating at 50 °C for 5 min, the glucose content was determined using a glucose assay kit. The reaction system for determining glucose content consisted of 1.5 mL of the kit solution and 50 μL of the reaction solution.

[0077] The specific enzyme activities of wild-type oligo-1,6-glucosidase and its mutants in hydrolyzing α-1,4 glycosidic bonds are shown in Table 3. The wild-type enzyme does not have the ability to hydrolyze α-1,4 glycosidic bonds. The single mutant V219A has a certain ability to hydrolyze α-1,4 glycosidic bonds. The ability of the double mutant V219A / P227A to hydrolyze α-1,4 glycosidic bonds is increased by 267% compared with V219A.

[0078] Table 3 Hydrolytic activity of oligo-1,6-glucosidase mutants on α-1,4 glycosidic bonds.

[0079]

[0080] Example 6: Hydrolysis efficiency of oligo-1,6-glucosidase mutants for different oligosaccharides

[0081] Common byproducts in glucose production, including maltose, maltotriose, isomaltose, isomalttriose, and panose, were prepared into 5 mg / mL standard solutions (500 mM phosphate buffer, pH 6.0). Wild-type oligo-1,6-glucosidase or its mutant was added at a dosage of 10 U / g, and the mixture was reacted at 50°C for 12 h. The glucose content produced was then determined using a glucose assay kit. The reaction system for determining glucose content consisted of 1.5 mL of the kit solution and 50 μL of the reaction solution.

[0082] The glucose yields of different oligosaccharides hydrolyzed by wild-type oligo-1,6-glucosidase and its mutants are shown in Table 4. Maltose is composed of two glucose molecules linked by α-1,4 glycosidic bonds; maltotriose is composed of three glucose molecules linked by two α-1,4 glycosidic bonds; isomaltose is composed of two glucose molecules linked by α-1,6 glycosidic bonds; isomalttriose is composed of three glucose molecules linked by two α-1,6 glycosidic bonds; and panose is composed of three glucose molecules linked by one α-1,4 glycosidic bond and one α-1,6 glycosidic bond.

[0083] As shown in Table 4, both the wild-type oligo-1,6-glucosidase and its mutants can completely convert isomaltose and isomalttriose into glucose. However, the wild-type enzyme lacks the ability to hydrolyze maltose and maltotriose, and the glucose yield from panose hydrolysis is only 30.7%. The single mutant V219A has a certain ability to hydrolyze maltose and maltotriose, with glucose yields of 60.7% and 42.2%, respectively. The efficiency of panose hydrolysis by the single mutant V219A is significantly higher than that of the wild-type enzyme, with a glucose yield of 82.5%. The double mutant V219A / P227A can almost completely convert isomaltose, isomalttriose, and panose into glucose, with a glucose yield close to 100%. The double mutant V219A / P227A can convert most of the maltose and maltotriose into glucose, with glucose yields exceeding 90%, which is significantly higher than both the wild-type enzyme and the single mutant V219A.

[0084] Table 4. Hydrolysis efficiency of oligo-1,6-glucosidase mutants for different oligosaccharides.

[0085]

[0086] Example 7: Application of oligo-1,6-glucosidase mutant in enzymatic production of glucose

[0087] Using 35% maltodextrin (DE 6) as the substrate, the mixture was equilibrated in a 50°C water bath for 15 min, and the pH was adjusted to 6.0. Glucoamylase (from Novosib) was added at a dose of 60 U / g, and oligo-1,6-glucosidase mutant V219A / P227A was added at a dose of 30 U / g. The reaction was maintained at 50°C for 72 h. Controls were provided by adding glucamylase + wild-type oligo-1,6-glucosidase or glucamylase + single mutant V219A.

[0088] The product analysis results of the saccharification reaction are shown in Table 5. In the enzymatic production process of glucose, the addition of the oligo-1,6-glucosidase double mutant V219A / P227A resulted in a glucose content of 98.0% after saccharification for 72 hours. This is 2.62% and 1.45% higher than that obtained by using glucoamylase + wild-type oligo-1,6-glucosidase and simultaneously using glucoamylase + oligo-1,6-glucosidase single mutant V219A, respectively. Furthermore, the proportions of byproducts such as maltose, maltotriose, isomalttriose, and panose were significantly reduced, which helps to improve the efficiency of the subsequent glucose crystallization process and reduce the generation of glucose mother liquor.

[0089] Table 5. Analysis of saccharification reaction products

[0090]

[0091] Example 8: Application of oligo-1,6-glucosidase mutant in the regeneration of glucose from crystalline glucose mother liquor

[0092] Using a 60% solids-to-volume crystalline glucose mother liquor as the substrate, the mixture was equilibrated in a 50°C water bath for 15 min, and the pH was adjusted to 6.0. Glucoamylase was added at a dosage of 30 U / g, and oligo-1,6-glucosidase mutant V219A / P227A was added at a dosage of 60 U / g. The reaction was maintained at 50°C for 24 h. Controls were provided by adding glucamylase + wild-type oligo-1,6-glucosidase or glucamylase + single mutant V219A.

[0093] The product analysis results are shown in Table 6. In the primary mother liquor of glucose crystallization, the addition of the double mutant V219A / P227A of glucosidase and oligo-1,6-glucosidase resulted in a glucose content of 95.9% after 24 hours of reaction. This was 9.35% and 5.15% higher than that achieved by using glucosidase + oligo-1,6-glucosidase and simultaneously using the glucosidase + oligo-1,6-glucosidase mutant, respectively. Furthermore, the proportions of byproducts such as maltose, maltotriose, isomalttriose, and panose were significantly reduced, which helps to improve the efficiency of subsequent glucose crystallization processes and reduce the generation of glucose mother liquor.

[0094] Table 6. Analysis of Saccharification Reaction Products

[0095]

[0096] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An oligo-1,6-glucosidase mutant, characterized in that, The amino acid sequence of the oligo-1,6-glucosidase mutant is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the oligo-1,6-glucosidase mutant of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

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

5. Recombinant cells expressing the oligo-1,6-glucosidase mutant of claim 1, characterized in that, The host cell is a microorganism.

6. The use of the oligo-1,6-glucosidase mutant of claim 1, the nucleic acid molecule of claim 2 or 3, the gene integration expression cassette or recombinant plasmid of claim 4, or the recombinant cell of claim 5 in hydrolyzing oligosaccharides or maltodextrin, characterized in that, The oligosaccharides include one or more of maltose, maltotriose, isomaltose, isomalttriose, and panose.

7. The application according to claim 6, characterized in that, The DE value of the maltodextrin is 5-20.

8. The use of the oligo-1,6-glucosidase mutant of claim 1, the nucleic acid molecule of claim 2 or 3, the gene integration expression cassette or recombinant plasmid of claim 4, or the recombinant cell of claim 5 in the preparation of glucose.

9. The application according to claim 8, characterized in that, It must contain at least one of the following characteristics: (1) Prepared using oligosaccharides or maltodextrin as substrates, wherein the oligosaccharides include one or more of maltose, maltotriose, isomaltose, isomalttriose, and panose; (2) The application includes regenerating glucose using crystallized glucose mother liquor.

10. The application according to claim 9, characterized in that, Using maltodextrin solution or primary mother liquor of crystalline glucose as substrates, an oligo-1,6-glucosidase mutant, whole cells containing the mutant, or formulations are added to react and glucose is generated.

11. An enzyme composition, characterized in that, The enzyme composition includes the oligo-1,6-glucosidase mutant of claim 1.

12. The enzyme composition according to claim 11, characterized in that, The enzyme composition also includes glucosylamylase.

13. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli overexpressed the oligo-1,6-glucosidase mutant of claim 1.

14. The use of the recombinant Escherichia coli of claim 13 in the preparation of oligo-1,6-glucosidase mutants.

15. A method for producing oligo-1,6-glucosidase mutants, characterized in that, The step includes fermentation production using the recombinant Escherichia coli as described in claim 13.

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