Alpha-1, 4 glucosidic bond hydrolytic ability enhanced oligomerized 1, 6-glucosidase mutant and application of alpha-1, 4 glucosidic bond hydrolytic ability enhanced oligomerized 1, 6-glucosidase mutant

Abstract

The invention discloses an alpha-1, 4 glucosidic bond hydrolytic ability enhanced oligomerized 1, 6-glucosidase mutant and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. The oligomerized 1, 6-glucosidase mutant V219A / P227A disclosed by the invention also has the alpha-1, 4 glucosidic bond hydrolytic activity which is not possessed by a wild type enzyme on the basis of retaining the original alpha-1, 6 glucosidic bond hydrolytic activity, and the alpha-1, 4 glucosidic bond hydrolytic activity is improved by 267% compared with that of a single mutant V219A. The mutant V219A / P227A is applied to an enzymatic production process of glucose, maltodextrin is taken as a substrate, glucamylase and the mutant V219A / P227A are added for enzymatic conversion, and the final glucose yield reaches 98.0%; the mutant V219A / P227A is applied to regeneration of glucose from crystalline glucose mother liquor, and the final glucose yield reaches 95.9%, which is 9.35% and 5.15% higher than those of a wild type V219A and a single mutant V219A respectively. Therefore, the mutant is helpful for improving the substrate conversion rate and the product purity in the glucose production process, can be used for regenerating glucose from crystalline glucose mother liquor, and has higher industrial application value.

Description

Technical Field

[0001] The present invention relates to an oligo-1,6-glucosidase mutant with enhanced α-1,4 glycosidic bond hydrolysis ability and application thereof, belonging to the technical fields of genetic engineering and enzyme engineering. Background Art

[0002] Starch sugar is a general term for sugars obtained from starch-containing raw materials such as corn, wheat, oats, rice, potatoes, and cassava using the acid, acid-enzyme, or whole-enzyme methods. It is the main product of starch deep processing. Industrially produced starch sugars are of various types, primarily crystalline glucose, fructose syrup, whole sugar, and maltose syrup. Starch sugar is produced through gelatinization, liquefaction, and saccharification by heating. However, both liquefaction and saccharification enzymes have difficulty acting on the α-1,6 glycosidic bonds of starch, resulting in low yields of the target product and a high proportion of by-products. Debranching enzymes are often added to the enzymatic production of starch sugars to improve production efficiency.

[0003] Debranching enzymes are a class of enzymes that specifically and efficiently hydrolyze α-1,6-glycosidic bonds in starch and related polysaccharides. They are primarily divided into pullulanases, isoamylases, and oligo-1,6-glucosidases. Pullulanases tend to target relatively small molecular weight dextrins and pullulan, while isoamylases tend to target higher molecular weight glucans such as amylopectin and glycogen. Oligo-1,6-glucosidases tend to target small molecular weight substrates such as isomaltooligosaccharides.

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

[0005] In the patent with authorization announcement number CN116064456B, a mutant of oligosaccharide debranching enzyme derived from Paenibacillus sp. STB16 and its application in glucose mother liquor are disclosed. After the wild-type enzyme is modified, the mutant V219A has a certain ability to hydrolyze α-1,4 glycosidic bonds, but the mutant's ability to hydrolyze α-1,4 glycosidic bonds is weak, and the efficiency of converting maltose, maltotriose, panose, etc. into glucose is low. It is mainly used to regenerate glucose from glucose mother liquor, and does not fundamentally improve the glucose production efficiency and substrate conversion rate, making it difficult to meet the green, low-carbon, energy-saving and consumption-reducing development needs of the starch sugar industry. Summary of the Invention

[0006] In order to solve the problem that the oligosaccharide debranching enzyme mutant V219A has a weak ability to hydrolyze α-1,4 glycosidic bonds, the present invention provides a new oligo-1,6-glucosidase mutant V219A / P227A, which retains the original α-1,6 glycosidic bond hydrolysis activity and has an ability to hydrolyze α-1,4 glycosidic bonds that is 267% higher than that of the mutant V219A. When mutant V219A / P227A was applied to an enzymatic glucose production process, the final glucose yield reached 98.0%, a 2.62% and 1.45% increase compared to using glucoamylase plus wild-type oligo-1,6-glucosidase and glucoamylase plus mutant V219A, respectively. When mutant V219A / P227A was applied to regenerate glucose from crystallized glucose mother liquor, the final glucose yield reached 95.9%, a 9.35% and 5.15% increase compared to using glucoamylase plus wild-type oligo-1,6-glucosidase and glucoamylase plus mutant V219A, respectively. This indicates that mutant V219A / P227A is more adaptable to enzymatic glucose production processes and can be used to treat the byproduct crystallized glucose mother liquor, making it more valuable for industrial applications.

[0007] The first object of the present 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 of 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] The 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 as SEQ ID NO.2.

[0011] The third object of the present 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 a backbone, which can be selected according to the host type, and can be a pET series vector.

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

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

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

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

[0017] Furthermore, the oligosaccharide comprises linear maltooligosaccharide or isomaltooligosaccharide. Preferably, the oligosaccharide comprises one or more of maltose, maltotriose, isomaltose, isomaltotriose and panose.

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

[0019] Furthermore, the preparation is carried out using oligosaccharides as substrates.

[0020] The 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 glucoamylase.

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

[0023] Furthermore, the application is: using maltodextrin solution as a substrate, placing it in a constant temperature water bath at 50°C for 15 minutes, adjusting the pH to 6.0, adding glucoamylase at an enzyme dosage of 60U / g and oligo-1,6-glucosidase mutant V219A / P227A at an enzyme dosage of 20-50U / g, and maintaining a constant temperature of 50°C for 72 hours.

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

[0025] The 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 preparing 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 performed at 28-32°C and 180-280 rpm.

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

[0030] A method for improving the glucose yield in an enzymatic production process of glucose, comprising: in a process for producing crystalline glucose using maltodextrin as a raw material, adding glucoamylase, and the oligo-1,6-glucosidase mutant, whole cells or preparations containing the mutant to react during the saccharification reaction stage.

[0031] A method for regenerating glucose from glucose mother liquor comprises the following steps: using the crystallized glucose mother liquor produced by the above process as a raw material, adding glucoamylase, and oligo-1,6-glucosidase mutants, whole cells or preparations containing the mutants to carry out reactions.

[0032] Beneficial effects of the present invention:

[0033] (1) The present invention provides a new oligo-1,6-glucosidase mutant. On the basis of the single oligo-1,6-glucosidase mutant V219A derived from Paenibacillus sp.STB16, the proline at position 227 is mutated to alanine to obtain a double mutant V219A / P227A. While retaining the original α-1,6 glycosidic bond hydrolysis activity, the ability to hydrolyze α-1,4 glycosidic bonds is improved by 267%. It can efficiently and specifically convert common by-products in the glucose production process (including maltose, maltotriose, isomaltose, isomaltotriose, panose, etc.) into glucose, expand the catalytic function of the enzyme and the applicable range of substrates, can better adapt to specific scenarios such as glucose enzymatic production, and has greater industrial application value.

[0034] (2) The present invention provides a method for improving the glucose yield in the enzymatic production process of glucose. In the process of producing crystalline glucose using maltodextrin as raw material, 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 glucose amylase + wild-type oligo-1,6-glucosidase and using glucose amylase + mutant V219A, respectively. It 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 development needs of the starch sugar industry in green, low-carbon, energy-saving and consumption-reducing.

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

[0036] Figure 1 The results of SDS-PAGE analysis of oligo-1,6-glucosidase mutants are shown in Figure 2. DETAILED DESCRIPTION

[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 the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0038] The culture medium involved in the following examples is as follows:

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

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

[0041] TB medium: yeast powder 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 the present invention is as follows:

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

[0044] The activity of the mutants was determined using 10 mM p-nitrophenyl-α-d-glucopyranoside (pNPG) as a substrate. The reaction system consisted of 150 μL of 500 mM phosphate buffer (pH 6.0), 800 μL of pNPG solution, and 50 μL of enzyme. The reaction was incubated at 50°C for 5 minutes, then terminated with 1 mL of 1 M sodium carbonate. The released p-nitrophenyl (pNP) was determined spectrophotometrically by measuring absorbance at 410 nm. One unit (U) of hydrolytic activity was defined as the amount of enzyme that produces 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 mutants was determined using 5 mg / mL maltose as a substrate. The reaction system consisted of 150 μL of phosphate buffer (500 mM, pH 6.0), 800 μL of substrate, and 50 μL of enzyme. The reaction solution was incubated at 50°C for 5 minutes, and the glucose content produced was determined using a glucose assay kit. The glucose content was determined using a reaction system consisting of 1.5 mL of the kit solution and 50 μL of the reaction solution. One unit (U) of hydrolytic activity 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 used in an enzymatic glucose production process, and the components of the product were analyzed by high-performance liquid chromatography (HPLC). The analysis conditions were: a ChromCore normal phase / HILIC column, a 70% acetonitrile-water 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 the present invention is as follows:

[0050] (1) Oligo-1,6-glucosidase mutant V219A / P227A amino acid sequence (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 plasmid containing mutants

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

[0056] Site-directed mutagenesis was performed according to the instructions of the TaKaRa STAR Primer GXL kit. The PCR system was: 5× PrimeSTAR GXL 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, PrimeSTAR GXL DNA Polymerase (1.25 U / μL) 1 μL, and double-distilled water to 50 μL. PCR amplification conditions were: initial denaturation at 98°C for 3 min, followed by 30 cycles (98°C for 10 s, 60°C for 15 s, 68°C for 7 min), and finally incubation at 68°C for 10 min.

[0057] Table 1 Site-directed mutagenesis primers

[0058]

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

[0060] Example 2: Construction of genetically engineered bacteria

[0061] The specific steps are as follows:

[0062] (1) The PCR product obtained in Example 1 was treated with DpnI at 37°C. The DpnI digestion system was as follows: 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 resulting transformation product was then plated onto LB agar solid medium containing 100 μg / mL kanamycin and cultured overnight in a 37°C incubator for 12 hours. A single colony was then selected and inoculated into LB liquid medium containing 100 μg / mL kanamycin and cultured overnight at 37°C and 200 rpm. The plasmid was then extracted and sequenced according to the instructions of the plasmid extraction kit.

[0063] (2) The correctly sequenced plasmid was transformed into the competent expression host E. coli BL21 (DE 3). Finally, the genetically engineered bacteria E. coli BL21 (DE 3) (pET-28a (+) / v219a / p227a) were obtained.

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

[0065] The specific steps are as follows:

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

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

[0068] (3) Obtaining crude enzyme: The fermentation broth was centrifuged at 4°C and 10,000 rpm for 20 min. The supernatant was discarded and the remaining cells were resuspended in 10 mM phosphate buffer (pH 6.0). The cells were then disrupted using an ultrasonic cell disruptor. After centrifugation, the enzyme solution was present in the supernatant.

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

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

[0071] The activity of the mutants was determined using 10 mM pNPG as a substrate. The reaction system consisted of 150 μL of 500 mM phosphate buffer (pH 6.0), 800 μL of pNPG solution, and 50 μL of enzyme. The reaction was incubated at 50°C for 5 minutes, then terminated with 1 mL of 1 M sodium carbonate. The released pNP was determined spectrophotometrically by measuring absorbance at 410 nm.

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

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

[0074]

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

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

[0077] The specific enzymatic activities of the wild-type oligo-1,6-glucosidase and its mutants for 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, and the double mutant V219A / P227A has a 267% higher ability to hydrolyze α-1,4 glycosidic bonds than V219A.

[0078] Table 3 Hydrolysis 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 on different oligosaccharides

[0081] Common byproducts of glucose production, including maltose, maltotriose, isomaltose, isomaltotriose, 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 mutants were added at 10 U / g of enzyme. The reaction was incubated at 50°C for 12 hours, and the resulting glucose content was determined using a glucose assay kit. The reaction system for glucose determination consisted of 1.5 mL of the assay solution and 50 μL of the reaction solution.

[0082] The glucose yields of different oligosaccharides hydrolyzed by the wild-type oligo-1,6-glucosidase and its mutants are shown in Table 4. Among them, maltose is composed of two glucoses linked by α-1,4 glycosidic bonds, maltotriose is composed of three glucoses linked by two α-1,4 glycosidic bonds, isomaltose is composed of two glucoses linked by α-1,6 glycosidic bonds, isomaltotriose is composed of three glucoses linked by two α-1,6 glycosidic bonds, and panose is composed of three glucoses 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 completely converted isomaltose and isomaltotriose into glucose. However, the wild-type enzyme was unable to hydrolyze maltose and maltotriose, with a glucose yield of only 30.7% from panose hydrolysis. The single mutant V219A had some 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 was significantly improved compared to the wild-type enzyme, reaching a glucose yield of 82.5%. The double mutant V219A / P227A was able to nearly completely convert isomaltose, isomaltotriose, and panose into glucose, with a glucose yield approaching 100%. The double mutant V219A / P227A converted most of maltose and maltotriose into glucose, with glucose yields exceeding 90%, significantly higher than both the wild-type enzyme and the single mutant V219A.

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

[0085]

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

[0087] A 35% maltodextrin (DE value 6) substrate was used as the substrate. The mixture was equilibrated in a 50°C water bath for 15 minutes. The pH was adjusted to 6.0. Glucoamylase (from Norwegian) was added at 60 U / g and the oligo-1,6-glucosidase mutant V219A / P227A was added at 30 U / g. The reaction was maintained at 50°C for 72 hours. Addition of glucoamylase plus wild-type oligo-1,6-glucosidase or glucoamylase plus the single mutant V219A served as controls.

[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 double mutants of oligo-1,6-glucosidase V219A / P227A resulted in a glucose content of 98.0% after saccharification for 72 h, which was 2.62% and 1.45% higher than those using glucoamylase + wild-type oligo-1,6-glucosidase and glucoamylase + single mutant of oligo-1,6-glucosidase V219A, respectively. In addition, the proportions of by-products such as maltose, maltotriose, isomaltotriose, and panose were significantly reduced, which helped to improve the efficiency of the subsequent glucose crystallization process and reduce the production of glucose mother liquor.

[0089] Table 5 Analysis of saccharification reaction products

[0090]

[0091] Example 8: Application of oligo-1,6-glucosidase mutants in regenerating glucose from crystallized glucose mother liquor

[0092] A 60% solids content, crystallized glucose mother liquor, was used as the substrate. The solution was equilibrated in a 50°C water bath for 15 minutes. The pH was adjusted to 6.0. Glucoamylase was added at 30 U / g and the oligo-1,6-glucosidase mutant V219A / P227A at 60 U / g. The reaction was maintained at 50°C for 24 hours. Controls included additions of glucoamylase plus wild-type oligo-1,6-glucosidase or glucoamylase plus the single mutant V219A.

[0093] The product analysis results are shown in Table 6. When glucoamylase and oligo-1,6-glucosidase double mutant V219A / P227A were added to the crystallized glucose mother liquor, the glucose proportion reached 95.9% after 24 hours of reaction, which was 9.35% and 5.15% higher than those using glucoamylase + oligo-1,6-glucosidase and glucoamylase + oligo-1,6-glucosidase mutant, respectively. In addition, the proportions of by-products such as maltose, maltotriose, isomaltotriose, and panose were significantly reduced, which helped to improve the efficiency of the subsequent glucose crystallization process and reduce the production of glucose mother liquor.

[0094] Table 6 Analysis of saccharification reaction products

[0095]

[0096] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present 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 according to 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 according to claim 2 or 3.

5. A recombinant cell expressing the oligo-1,6-glucosidase mutant according to claim 1, characterized in that: The host cell is a microorganism.

6. Use of the oligo-1,6-glucosidase mutant according to claim 1, the nucleic acid molecule according to claim 2 or 3, the gene integration expression cassette or recombinant plasmid according to claim 4, or the recombinant cell according to claim 5 in hydrolyzing oligosaccharides or maltodextrin.

7. The use according to claim 6, characterized in that The oligosaccharide comprises linear maltooligosaccharide or isomaltooligosaccharide; and the DE value of the maltodextrin is 5-20.

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

9. The use according to claim 8, characterized in that Contain at least one of the following characteristics: (1) Preparation using oligosaccharides or maltodextrin as substrate; (2) The application includes regenerating glucose using crystallized glucose mother liquor.

10. The use according to claim 9, characterized in that The method uses maltodextrin solution or crystallized glucose primary mother liquor as substrate, adds oligo-1,6-glucosidase mutant, whole cells or preparation containing the mutant, and reacts for a period of time to generate glucose.

11. An enzyme composition, characterized in that The enzyme composition comprises the oligo-1,6-glucosidase mutant according to claim 1.

12. The enzyme composition according to claim 11, characterized in that Glucoamylase is also included in the enzyme composition.

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

14. Use of the recombinant Escherichia coli according to claim 13 in preparing oligo-1,6-glucosidase mutants.

15. A method for producing an oligo-1,6-glucosidase mutant, characterized in that: The method comprises the step of using the recombinant Escherichia coli according to claim 13 to carry out fermentation production.

Images