Oligosaccharide-debranching enzyme mutants with reduced product inhibition and uses thereof

Abstract

This invention relates to a oligosaccharide debranching enzyme mutant with reduced product inhibition and its applications, belonging to the field of enzyme engineering technology. Through molecular docking, this invention discovered that lysine at position 311 and glutamic acid at position 405 of the oligosaccharide debranching enzyme, as shown in SEQ ID NO.1, are located at key positions in the substrate binding pocket. Based on this, double and triple mutants were constructed, and their enzyme activity was verified by expression in *E. coli*. The triple mutant V219A / K311M / E405A, with the most significant reduction in product inhibition, was selected. This mutant is suitable for the efficient hydrolysis of various oligosaccharides such as maltose, isomaltose, and panose to glucose. When using enzymatic methods to produce glucose, the oligosaccharide debranching enzyme mutant provided by this invention can further hydrolyze oligosaccharides in starch saccharification solutions into glucose, thereby increasing the glucose yield and possessing industrial production potential.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to an oligosaccharide debranching enzyme mutant with weakened product inhibition and its application. Background Technology

[0002] In the enzymatic production process of glucose, starch is usually used as a substrate. After being liquefied into maltodextrin by α-amylase, glucose amylase and debranching enzyme are added for saccharification. Glucose accounts for the majority of the saccharified solution, but it also contains small molecule sugars such as maltose, maltotriose, isomaltose, isomalttriose, and panose.

[0003] Debranching enzymes are a class of specific and highly efficient hydrolases that hydrolyze α-1,6 glycosidic bonds in starch and related polysaccharides. They are mainly classified into pullulanase, isoamylase, and oligosaccharide debranching enzymes. Among them, oligosaccharide debranching enzymes belong to the glycosidic hydrolase family 13 (GH13). Their substrate binding pockets are deeply embedded inside the structure, forming a typical pocket shape. The substrate binding pockets are relatively small, making them suitable for small molecule substrates such as isomaltose and isomalttriose. They can specifically generate glucose, thus making them more suitable for enzymatic glucose production processes.

[0004] Patent CN115851561A discloses a recombinant *E. coli* strain expressing oligosaccharide debranching enzyme (pspOG) derived from *Paenibacillus* sp. STB16 and its application in hydrolyzed maltodextrin products. Patent CN116426447A discloses a recombinant *Bacillus subtilis* strain expressing pspOG extracellularly and its application in the hydrolysis of isomaltooligosaccharides. pspOG exhibits high catalytic activity towards α-1,6 glycosidic bonds, specifically hydrolyzing isomaltooligosaccharides such as isomaltose and isomalttriose, which contain only α-1,6 glycosidic bonds, into glucose, and hydrolyzing panose (containing one α-1,6 glycosidic bond and one α-1,4 glycosidic bond) into glucose and maltose. Patent CN116064456A discloses a pspOG mutant and its application in glucose mother liquor. After modifying the wild-type enzyme, mutant V219A has the ability to simultaneously hydrolyze α-1,4 and α-1,6 glycosidic bonds, and can further convert oligosaccharide impurities containing α-1,4 glycosidic bonds, such as maltose, maltotriose, and panose, into glucose, showing certain application potential. However, wild-type pspOG and its mutant V219A exhibit severe product inhibition. At a glucose concentration of 100 mg / mL, the enzyme activity of wild-type pspOG and its mutant V219A is only 7% and 40% of the initial enzyme activity (glucose concentration of 0 mg / mL), respectively. It is mainly used for the regeneration of glucose in crystalline glucose mother liquor (with relatively low glucose concentration) and is difficult to apply directly to glucose production processes.

[0005] Product inhibition is a common regulatory mechanism in enzyme-catalyzed reactions and a bottleneck that industrial enzymes need to overcome in their applications. It manifests primarily as a significant decrease in enzyme catalytic efficiency, a gradual slowdown or stagnation of the reaction rate, and even the initiation of reverse reactions, with increasing product concentration. Since wild-type pspOG and its mutant V219A both exhibit severe product inhibition, they are unable to further convert oligosaccharide impurities into glucose in saccharification solutions with extremely high glucose concentrations, and may even trigger reverse reactions, resulting in a reduced glucose yield. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention discovered through molecular docking that lysine at position 311 and glutamic acid at position 405 of the oligosaccharide debranching enzyme, as shown in SEQ ID NO.1, are located at key positions in the substrate binding pocket. By combining this with the known oligosaccharide debranching enzyme mutation site V219A, double and triple mutants were constructed, significantly reducing the product inhibition effect of the oligosaccharide debranching enzyme, making it suitable for the efficient hydrolysis of oligosaccharides into glucose.

[0007] The first objective of this invention is to provide an oligosaccharide debranching enzyme mutant, which makes the following mutations to the parent amino acid sequence as shown in SEQ ID NO.1:

[0008] (1) Valine at position 219 is mutated to alanine;

[0009] (2) The lysine at position 311 is mutated to methionine and / or the glutamic acid at position 405 is mutated to alanine.

[0010] Furthermore, the nucleotide sequence of the parent oligosaccharide debranching enzyme is shown in SEQ ID NO.2.

[0011] Furthermore, the oligosaccharide debranching enzyme mutant mutates valine at position 219 to alanine, lysine at position 311 to methionine, and glutamic acid at position 405 to alanine in the parent sequence shown in SEQ ID NO.1.

[0012] A second objective of this invention is to provide a gene encoding the aforementioned oligosaccharide debranching enzyme mutant.

[0013] A third objective of this invention is to provide an expression vector containing the aforementioned genes.

[0014] Furthermore, the expression vector uses pET-28a(+) as its backbone.

[0015] A fourth objective of the present invention is to provide a host cell comprising the above-mentioned oligosaccharide debranching enzyme mutant, the above-mentioned gene, or the above-mentioned expression vector.

[0016] In one embodiment of the present invention, the host cell is Escherichia coli.

[0017] A fifth object of the present invention is to provide a formulation comprising the aforementioned host cells.

[0018] The sixth objective of this invention is to provide a method for reducing the inhibition of oligosaccharide debranching enzyme products by mutating valine at position 219 to alanine, lysine at position 311 to methionine, and glutamic acid at position 405 to alanine in the oligosaccharide debranching enzyme nucleotide sequence as shown in SEQ ID NO.1.

[0019] A seventh object of the present invention is to provide the use of the above-mentioned oligosaccharide debranching enzyme mutant, the above-mentioned host cell, or the above-mentioned preparation in the hydrolysis of oligosaccharides into glucose.

[0020] Furthermore, the oligosaccharide includes one or more of maltose, maltotriose, isomaltose, isomalttriose, and panose.

[0021] An eighth object of the present invention is to provide a method for hydrolyzing oligosaccharides into glucose, wherein the above-mentioned oligosaccharide debranching enzyme mutant, the above-mentioned host cell, or the above-mentioned preparation is added to a reaction system with oligosaccharides as a substrate.

[0022] Furthermore, the oligosaccharide includes one or more of maltose, maltotriose, isomaltose, isomalttriose, and panose.

[0023] The ninth object of the present invention is to provide a method for producing glucose, comprising the following steps:

[0024] Step S1: Prepare starch slurry;

[0025] Step S2: Add amylase to the starch milk to obtain a liquefied solution;

[0026] Step S3: Add glucoamylase to the liquefied solution to obtain the saccharified solution;

[0027] Step S4: Add the above-mentioned oligosaccharide debranching enzyme mutant, the above-mentioned host cells, or the above-mentioned preparation to the saccharification solution.

[0028] Further, in step S4, the amount added is 10-500U of the oligosaccharide debranching enzyme mutant per gram of dry substrate.

[0029] In one embodiment of the present invention, in step S4, the amount added is 200U of the oligosaccharide debranching enzyme mutant per gram of dry substrate.

[0030] Furthermore, in step S4, the reaction time is 12-48 hours.

[0031] In one embodiment of the present invention, the reaction time in step S4 is 24 hours.

[0032] The beneficial effects of this invention are:

[0033] The present invention

[0034] This invention significantly reduces the product inhibition effect of oligosaccharide debranching enzymes by using three-point mutations in V219A, K311M, and E405A, thereby improving the enzyme's hydrolysis efficiency for oligosaccharides such as maltose and isomaltose. The resulting oligosaccharide debranching enzyme mutant can be directly added after the saccharification stage in enzymatic glucose production, compatible with traditional processes. It can improve glucose yield, shorten reaction time, or reduce enzyme dosage without altering the existing starch liquefaction-saccharification process. The mutant enzyme is efficiently expressed using engineered host cells (such as *E. coli*) based on the pET-28a(+) vector, ensuring the feasibility of industrial production. The formulation stability meets the requirements for large-scale application, overcoming the limitations of traditional glucoamylases on α-1,6 glycosidic bonds, reducing residual recalcitrant oligosaccharides in the saccharification solution, and improving the adaptability of oligosaccharide debranching enzymes in glucose production processes, demonstrating strong industrial application value. Attached Figure Description

[0035] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0036] Figure 1 This is a diagram showing the molecular docking results in an embodiment of the present invention;

[0037] Figure 2 This invention illustrates the effect of glucose concentration on the activity of oligosaccharide debranching enzymes and their mutants. Detailed Implementation

[0038] 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.

[0039] The culture media involved in the following examples are shown below:

[0040] LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0041] LB solid medium: 1.5% (w / v) agar powder is added to LB medium.

[0042] 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.

[0043] The detection methods involved in the following embodiments are as follows:

[0044] (1) Determination of oligosaccharide debranching enzyme activity

[0045] 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 of sodium carbonate (1 M) was added to terminate the reaction. The absorbance at 410 nm was measured, and 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 pNP per minute under the assay conditions.

[0046] (2) Effect of product concentration on oligosaccharide debranching enzyme activity

[0047] Using 10 mM pNPG as a substrate, different amounts of glucose (0-500 mg / mL) were added to the substrate, and enzyme activity was determined according to detection method (1). The inhibitory effect of glucose on the enzyme was evaluated by the changes in enzyme activity. The relative activity under different glucose addition amounts was calculated with the enzyme activity without the addition of exogenous glucose as 100%.

[0048] (3) Analysis of saccharification reaction products

[0049] The oligosaccharide debranching enzyme mutant V219A / K311M / E405A 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.

[0050] Example 1: Construction of recombinant plasmids containing mutants

[0051] Discovery through molecular docking ( Figure 1The glucose molecule produced will form stable hydrogen bonds with lysine at position 311 and glutamic acid at position 405 of the oligosaccharide debranching enzyme (nucleotide sequence shown in SEQ ID NO. 2), as shown in SEQ ID NO. 1, thereby binding to these two amino acids. Since these two amino acids are located at key positions in the substrate binding pocket and have relatively large side chains, they not only bind glucose through hydrogen bonds, hindering glucose release, but also impede glucose release due to steric hindrance. To reduce steric hindrance and weaken hydrogen bond interactions while minimizing impact on enzyme stability, based on FoldX simulated mutation results, this invention mutates lysine at position 311 to methionine and glutamic acid at position 405 to alanine, obtaining oligosaccharide debranching enzyme mutants V219A / K311M, V219A / E405A, and V219A / K311M / E405A, respectively.

[0052] Using the expression vector pET-28a(+) as a template, complementary primer chains were designed, and the primer sequences are shown in Table 1.

[0053] 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 Mixture (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 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.

[0054] Table 1. Site-directed mutagenesis primers

[0055]

[0056] Note: Underlined bases correspond to the corresponding mutant amino acids.

[0057] Example 2: Construction of genetically engineered bacteria

[0058] The PCR product obtained in Example 1 was treated with Dpn I at 37°C. The Dpn I 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 Dpn I. 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 incubated overnight at 37°C for 12 h. Single colonies were then selected and inoculated into LB liquid medium containing 100 μg / mL kanamycin and incubated overnight at 37°C and 200 rpm. Plasmids were then extracted, identified, and sequenced according to the instructions of the plasmid extraction kit.

[0059] The correctly sequenced plasmids were transferred into competent cells of the expression host E. coli BL21(DE3). This resulted in the genetically engineered bacteria E. coli-V219A / K311M, E. coli-V219A / E405A, and E. coli-V219A / K311M / E405A.

[0060] Example 3: Expression of oligosaccharide debranching enzyme mutant

[0061] Seed culture: 100 μL of the genetically engineered bacteria E. coli-V219A / K311M, E. coli-V219A / E405A and E. coli-V219A / K311M / E405A were inoculated into LB medium containing the corresponding antibiotics and cultured at 37℃ and 200 r / min for 8-10 h.

[0062] Fermentation culture: The seed culture was inoculated into a fermentation medium containing the appropriate antibiotic at an inoculation rate of 4% (v / v), and cultured on a shaker at 37°C and 200 rpm until the OD of the culture was measured. 600 When the value reached 0.6, IPTG was added to make the final concentration 0.05mM, and expression was induced for another 48h at 25℃ and 200r / min.

[0063] 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.

[0064] Enzyme purification: Oligosaccharide debranching enzymes were 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, then loaded with the target protein at a flow rate of 1.5 mL / min. After loading, the nickel column was equilibrated again with buffer A. Following 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 peaks.

[0065] Example 4: Product inhibition analysis of oligosaccharide debranching enzyme mutant

[0066] 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. The absorbance at 410 nm was measured, and the released p-nitrophenyl (pNP) was determined spectrophotometrically.

[0067] Using pNPG as a substrate, different amounts of glucose (0-500 mg / mL) were added to the substrate, and enzyme activity was measured according to the enzyme activity assay method. The inhibitory effect of glucose on the enzyme was evaluated by the changes in enzyme activity. The relative activity at different glucose addition levels was calculated with the enzyme activity without the addition of exogenous glucose as 100%.

[0068] The results are as follows Figure 2 As shown, when the glucose concentration is 100 mg / mL, the relative enzyme activity of the wild-type enzyme is 7%, the relative enzyme activity of the single mutant V219A is about 40%, the relative enzyme activity of the double mutant V219A / E405A is about 80%, the relative enzyme activity of the double mutant V219A / K311M is about 73%, and the relative enzyme activity of the triple mutant V219A / K311M / E405A is about 90%.

[0069] When the glucose concentration is 500 mg / mL, the wild-type enzyme is basically inactivated. The relative enzyme activity of the single mutant V219A is about 12%, the relative enzyme activity of the double mutant V219A / E405A is about 50%, the relative enzyme activity of the double mutant V219A / K311M is about 40%, and the relative enzyme activity of the triple mutant V219A / K311M / E405A is about 60%.

[0070] Example 5: Effect of glucose on the hydrolysis efficiency of different oligosaccharides by oligosaccharide debranching enzyme mutants

[0071] Common byproducts in glucose production, including maltose, maltotriose, isomaltose, isomalttriose, and panose, were selected and prepared into standard solutions containing 100 mg / mL glucose (500 mM phosphate buffer, pH 6.0, oligosaccharide concentration 5 mg / mL). Wild-type oligosaccharide debranching enzyme or its mutant was added at a dosage of 10 U / g. After reacting at 50°C for 12 h, the glucose content produced in the reaction was determined using a glucose kit, and the original 100 mg / mL glucose in the substrate was subtracted. The reaction system for determining glucose content consisted of 1.5 mL of kit solution and 50 μL of reaction solution.

[0072] The glucose yields of wild-type oligosaccharide debranching enzymes and their mutants after hydrolyzing different oligosaccharides are shown in Table 2. In the presence of 100 mg / mL glucose in the system, the wild-type enzyme and the single mutant V219A showed very low hydrolytic abilities for maltose, maltotriose, isomaltose, isomalttriose, and panose, with a final glucose yield of less than 30%. In contrast, V219A / K311M / E405A can almost completely convert isomaltose, isomalttriose, and panose into glucose, with glucose yields all exceeding 80% and approaching 100%. It can convert most of the maltose and maltotriose into glucose, which is significantly improved compared to the wild-type enzyme and the single mutant V219A.

[0073] Table 2. Effect of glucose on the hydrolysis efficiency of different oligosaccharides by oligosaccharide debranching enzyme mutants.

[0074]

[0075] Example 6: Application of mutants in the enzymatic production process of glucose

[0076] (1) Preparation of slurry: Using corn starch milk with a mass fraction of 30% as substrate, place it at 60℃ for 15 min, adjust the pH to 6.0, and add heat-resistant α-amylase at an enzyme addition rate of 12 U / g.

[0077] (2) Liquefaction: The starch milk is liquefied twice by spray liquefaction device: the first spray is at 110°C and the second spray is at 140°C. The liquefied liquid is kept at the temperature in the laminar flow tank for 30 minutes so that the DE value (glucose value, which characterizes the degree of starch hydrolysis) reaches 15.

[0078] (3) Saccharification: Cool the liquefied liquid at 60℃ for 15 min, adjust the pH to 5.0, add glucoamylase at an enzyme dosage of 60 U / g, and maintain the reaction at a constant temperature of 60℃ for 48 h.

[0079] (4) Oligosaccharide debranching enzyme conversion: After the liquefaction and saccharification reactions are completed, the saccharification solution is cooled at 60℃ for 15 min, the pH is adjusted to 6.0, and oligosaccharide debranching enzyme mutant V219A / K311M / E405A is added at an enzyme dosage of 200 U / g, and the reaction continues for 24 h.

[0080] The product analysis results of the saccharification reaction are shown in Table 3. In the enzymatic production process of glucose, after liquefaction and saccharification, the glucose yield in the saccharification solution was 95.0%. Adding the triple mutant V219A / K311M / E405A for further conversion further increased the glucose yield, reaching a final yield of 98.1%. However, the wild-type enzyme and the single mutant V219A showed significant product inhibition and reverse reaction, resulting in glucose yields of 88.2% and 90.6%, respectively. Therefore, the triple mutant V219A / K311M / E405A is better suited for the enzymatic production process of glucose, helping to improve substrate conversion and product purity, thereby reducing energy consumption and waste generation in the subsequent glucose crystallization process.

[0081] Table 3 Analysis of saccharification reaction products

[0082]

[0083] 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 oligosaccharide debranching enzyme mutant, characterized in that: The oligosaccharide debranching enzyme mutant makes the following mutations to the parent amino acid sequence shown in SEQ ID NO.1: (1) Valine at position 219 is mutated to alanine; and (2) The lysine at position 311 is mutated to methionine and / or the glutamic acid at position 405 is mutated to alanine.

2. The oligosaccharide debranching enzyme mutant according to claim 1, characterized in that: The oligosaccharide debranching enzyme mutant mutates valine at position 219 to alanine, lysine at position 311 to methionine, and glutamic acid at position 405 to alanine from the parent sequence shown in SEQ ID NO.

1.

3. A gene encoding the oligosaccharide debranching enzyme mutant of claim 1 or 2.

4. An expression vector comprising the gene of claim 3.

5. The expression vector according to claim 4, characterized in that: The expression vector uses pET-28a(+) as its backbone.

6. A host cell comprising the oligosaccharide debranching enzyme mutant of claim 1 or 2, the gene of claim 3, or the expression vector of claim 4 or 5.

7. A formulation comprising the host cell of claim 6.

8. A method for reducing the inhibition of oligosaccharide debranching enzyme products, characterized in that: The oligosaccharide debranching enzyme with the amino acid sequence shown in SEQ ID NO.1 was modified by mutating valine at position 219 to alanine, lysine at position 311 to methionine, and glutamic acid at position 405 to alanine.

9. The use of the oligosaccharide debranching enzyme mutant of claim 1 or 2, the host cell of claim 6, or the formulation of claim 7 in the hydrolysis of oligosaccharides to glucose, characterized in that: The oligosaccharides include one or more of maltose, maltotriose, isomaltose, isomalttriose, and panose.

10. A method for hydrolyzing oligosaccharides into glucose, characterized in that: The oligosaccharide debranching enzyme mutant of claim 1 or 2, the host cell of claim 6, or the preparation of claim 7 are added to the reaction system with oligosaccharides as the substrate, wherein the oligosaccharides include one or more of maltose, maltotriose, isomaltose, isomalttriose, and panose.

11. A method for producing glucose, characterized in that, Includes the following steps: Step S1: Prepare starch milk; Step S2: Add amylase to the starch milk to obtain a liquefied solution; Step S3: Add glucoamylase to the liquefied solution to obtain the saccharified solution; Step S4: Add the oligosaccharide debranching enzyme mutant of claim 1 or 2, the host cell of claim 6, or the preparation of claim 7 to the saccharification solution.

12. The method according to claim 11, characterized in that: In step S4, the amount added is 10-500 U of the oligosaccharide debranching enzyme mutant per gram of dry substrate.

13. The method according to claim 11, characterized in that: In step S4, the reaction time is 12-48 hours.

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