Cellobiose 2-epimerase mutant with high catalytic efficiency and application thereof

Abstract

The invention relates to a cellobiose 2-epimerase mutant with high catalytic efficiency and application of the cellobiose 2-epimerase mutant. The mutant is obtained by changing the specific amino acid site of a wild type enzyme, the double mutant psyCE-FR shows the optimal performance, and the catalytic efficiency (Kcat / Km) of the double mutant is improved by about 40% compared with that of the wild type enzyme. The mutant has excellent catalytic efficiency and substrate affinity at low temperature (4-10 DEG C), and is suitable for low-temperature dairy product processing and functional sugar preparation. Besides, the mutant retains the characteristic that the psychrophilic enzyme is easy to thermally inactivate, can be instantly inactivated at 50 DEG C, is compatible with a pasteurization process in the dairy industry, reduces the downstream treatment cost, and improves the product purity and safety. The invention also provides a gene for coding the mutant, a recombinant expression vector, a recombinant engineering bacterium and a method for producing epilactose by using the mutant enzyme, and the mutant enzyme has wide application prospect and commercial value.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and biocatalysis technology, specifically relating to a mutant based on psychrophilic cellobiose 2-epimerase with high catalytic activity and high substrate affinity, and its application in the enzymatic preparation of ipilactose under low temperature conditions. Background Technology

[0002] Lactose is a widely found and inexpensive carbohydrate in nature. Epilacose, the C-2 epimer of lactose, not only possesses excellent prebiotic properties (such as promoting the growth of bifidobacteria and calcium absorption), but also exhibits physiological functions similar to dietary fiber and good physicochemical stability, making it a promising candidate for applications in dairy products and health foods.

[0003] Cellobiroose 2-epimerase (CE) is the only known biocatalyst that specifically catalyzes the epimerization of lactose at the C-2 position to produce epilactose. Compared to chemical methods, enzymatic conversion offers advantages such as mild reaction conditions, no byproducts, and environmental friendliness. In the dairy industry, low-temperature processing (typically 4–10°C) is the preferred process to maximize the retention of heat-sensitive nutrients and inhibit the growth of spoilage microorganisms. However, most reported CE enzymes are derived from thermophilic or mesophilic microorganisms, with their optimal temperatures usually above 45°C. Their catalytic activity decreases sharply at low temperatures, failing to meet the practical requirements of cold chain processing.

[0004] Existing technology, using metagenomic mining, has yielded a psychrophilic cellobiose 2-epimerase (psyCE) from the glacial environment of the Tibetan Plateau. While this enzyme exhibits good low-temperature adaptability and possesses the highest catalytic conversion number (Kcat) among reported cold-active CEs, it suffers from a low affinity (high Km) for the substrate lactose due to the characteristic structure-stability-affinity trade-off of psychrophilic enzymes. This limitation restricts its overall catalytic efficiency (Kcat / Km) in low substrate concentrations or industrial applications, hindering efficient continuous low-temperature conversion and limiting its industrialization. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies, such as low substrate affinity and limited overall catalytic efficiency of psychrophilic cellobiose 2-epimerase. Through semi-rational design and high-throughput screening, a series of improved psychrophilic cellobiose 2-epimerase mutants, psyCE-FR, and their applications have been obtained.

[0006] The first objective of this invention is to provide a cellobiose 2-epimerase mutant with high catalytic activity and high substrate affinity. This mutant is obtained by mutating the amino acid sequence of the wild-type psychrophilic cellobiose 2-epimerase psyCE (amino acid sequence 1, gene sequence 7) by at least one of the following mutations: The asparagine (Asn) at position 309 is mutated to either phenylalanine (Phe) or histidine (His). The lysine (Lys) at position 189 is mutated to arginine (Arg).

[0007] Preferably, the amino acid sequence of the mutant is selected from any of the following sequences: The mutant N309F contains only the mutation of Asn at position 309 to Phe, and its amino acid sequence is sequence 2. The mutant N309H contains only the mutation of Asn at position 309 to His, and its amino acid sequence is sequence 3. The mutant K189R contains only the Lys mutation at position 189, which is replaced by Arg. The amino acid sequence is sequence 4. The mutant psyCE-FR contains a double mutation, with Asn at position 309 being mutated to Phe and Lys at position 189 being mutated to Arg. The amino acid sequence is shown in sequence 5. The mutant psyCE-HR contains a double mutation, with As at position 309 being mutated to His and Lys at position 189 being mutated to Arg. The amino acid sequence is sequence 6.

[0008] A second object of the present invention is to provide a gene encoding the mutant, the amino acid sequence of which is shown in sequences 2-6.

[0009] A third objective of this invention is to provide a recombinant expression vector containing the above-mentioned genes and a recombinant engineered bacteria.

[0010] A fourth object of the present invention is to provide the application of the mutant enzyme in the preparation of ipilactose, particularly in a low-temperature dairy processing environment.

[0011] Preferably, the low temperature refers to 4~10℃.

[0012] As a preferred option, the low temperature referred to is 8°C.

[0013] The fifth objective of this invention is to provide an enzymatic method for producing ipilactose: using lactose or lactose-containing materials as a substrate, and the cellobiose 2-epimerase mutant as a catalyst, the method catalyzes the epimerization of lactose at the C-2 position to produce ipilactose under low temperature conditions.

[0014] Preferably, the lactose-containing material is milk or whey.

[0015] The mutant enzyme provided by this invention has the following significant beneficial effects: Single-point mutations can significantly improve enzymatic properties: This invention discovered that single-point saturation mutations at positions 309 or 189 of the wild-type enzyme can yield mutants with improved performance. The specific activities of the single mutants N309F, N309H, and K189R reached 134.5 U / mg, 128.6 U / mg, and 124.4 U / mg, respectively, all superior to the wild type (122.2 U / mg). In particular, the N309H mutant showed a significantly reduced Km value to 121.2 mM (compared to 261.9 mM for the wild type), indicating a very high enhancement in substrate affinity; while the K189R mutant also improved affinity while maintaining high activity. This suggests that these two sites are key hotspots for regulating the enzyme's catalytic performance.

[0016] Significant Performance Leap with Dual Mutants: Building upon the single-point mutations described above, this invention further constructs a dual mutant, psyCE-FR (N309F / K189R). This mutant exhibits optimal overall performance, producing a significant synergistic optimization effect. Experiments show that the Kcat value of psyCE-FR is increased to 245.7 s. -1 This sets a new record for the highest reported cold-activity CE enzyme; simultaneously, its Km is reduced to 207.8 mM. Overall, its catalytic efficiency (Kcat / Km) is approximately 40% higher than the wild type, achieving a dual breakthrough of high activity at low temperatures and high affinity, making it the most preferred technical solution of this invention.

[0017] Excellent low-temperature catalytic production capability: This mutant exhibits superior production efficiency at cold chain processing temperatures such as 8°C. In an 8°C reaction system, the psyCE-FR mutant achieves approximately 21% ipilactose conversion in just 3 hours, while the wild-type enzyme requires more than 4 hours to achieve the same conversion rate. Its space-time yield at low temperatures is approximately 20% higher than that of the wild type, significantly shortening the production cycle. This not only helps reduce energy consumption but also minimizes the risk of microbial contamination from prolonged reactions, greatly enhancing its industrial application value in the functional processing of cold chain dairy products, such as pasteurized milk and low-temperature yogurt.

[0018] Excellent process compatibility and safety: Easy heat inactivation. The mutant enzyme of this invention retains the thermoinstability characteristics of its parent psychrophilic enzyme. Experiments show that it is instantly inactivated at 50°C. This characteristic makes it perfectly compatible with standard pasteurization processes in the dairy industry (such as 63°C / 30min or 72°C / 15s), and even under gentler heat treatments, it can achieve complete and rapid inactivation. This allows for precise termination of the reaction through short-time heating in industrial production without the need for chemical terminators or complex physical separation, effectively preventing over-reaction or byproduct formation, thereby significantly improving the purity and food safety of the final product. This characteristic greatly reduces downstream processing costs, making its application in the food industry more flexible and safe.

[0019] In summary, the psyCE-FR and its series of mutants of this invention are cellobiose 2-epimerases with a well-defined origin, excellent performance, high efficiency at low temperatures, high substrate affinity, and good process adaptability. They exhibit significant technical advantages in the bioconversion of lactose to ipilactose, and are particularly suitable for applications such as cold chain dairy processing and functional sugar preparation, showing broad application prospects and commercial value in the food industry and prebiotic production. Attached Figure Description

[0020] Figure 1 The bands of psyCE and its mutants purified in Example 1 on SDS-PAGE are shown.

[0021] Figure 2 This illustrates the effect of temperature on the activity of psyCE and its mutants in Example 3.

[0022] Figure 3 This describes the process of producing lactose from psyCE and its mutants at 8°C in Example 4. Detailed Implementation

[0023] The following are specific embodiments of the present invention to further illustrate the invention, but the invention is not limited thereto. Any equivalent substitutions made to the invention are considered within the scope of protection of the invention. The invention is further illustrated below with reference to specific embodiments.

[0024] The original sequences used in the following examples ， It was obtained by mining and screening from the genome isolated from the Muji Glacier on the Qinghai-Tibet Plateau.

[0025] Unless otherwise specified, all reagents used in the following examples can be purchased commercially.

[0026] Example 1: Construction, expression, and purification of the psychrophilic cellobiose 2-epimerase mutant (psyCE-FR) This embodiment aims to construct and purify psyCE mutant enzymes containing single mutations of N309F, N309H, and K189R, and double mutations of psyCE-FR and psyCE-HR.

[0027] (1) Gene cloning and expression vector construction Using the wild-type psyCE gene (sequence 7) as a template, the gene has been cloned into the pET-20b(+) expression vector (with a C-terminal His tag). Based on structural simulation and substrate docking results, key sites such as K189 and N309 were selected for modification. The asparagine (Asn) at position 309 was mutated to phenylalanine (Phe) or histidine (His); the lysine (Lys) at position 189 was mutated to arginine (Arg), as detailed below: The mutant N309F contains only the mutation of Asn at position 309 to Phe, and its amino acid sequence is sequence 2. The mutant N309H contains only the mutation of Asn at position 309 to His, and its amino acid sequence is sequence 3. The mutant K189R contains only the Lys mutation at position 189, which is replaced by Arg. The amino acid sequence is sequence 4. The mutant psyCE-FR contains a double mutation, with Asn at position 309 being mutated to Phe and Lys at position 189 being mutated to Arg. The amino acid sequence is shown in sequence 5. The mutant psyCE-HR contains a double mutation, with As at position 309 being mutated to His and Lys at position 189 being mutated to Arg. The amino acid sequence is sequence 6.

[0028] Mutation primers containing degenerate codons (NNK) were designed, and site-directed saturation mutagenesis was performed using whole-plasmid PCR. To screen for mutants with high expression levels, a GFP-psyCE fusion expression vector was constructed as a selection template. By detecting the GFP fluorescence intensity to homogenize protein expression levels, single mutants N309F and K189R with significantly enhanced activity were obtained through screening.

[0029] Based on this, the N309F, N309H, K189R, psyCE-FR, and psyCE-HR mutation sites were introduced into the same plasmid using overlap extension PCR or site-directed mutagenesis kits to construct expression plasmids for the N309F, N309H, K189R, psyCE-FR, and psyCE-HR mutants. Sequencing confirmed that the mutation sequences were correct.

[0030] (2) Protein expression Escherichia coli BL21(DE3) strain containing the mutant recombinant plasmid was inoculated into LB medium containing ampicillin (100 μg / mL) and cultured at 37°C until the OD600 reached 0.6–0.8. Protein expression was induced by adding 0.5 mM isopropyl-β-D-thiogalactoside (IPTG), and the culture was continued at 18°C ​​for 18 h to promote efficient folding and expression of the soluble protein.

[0031] (3) Cell disruption and crude extraction Collect the induced bacterial cells, centrifuge to remove the supernatant, and resuspend the precipitate in phosphate-buffered saline (PBS, pH 7.5). Disrupt the cells using sonication on ice (3 seconds on, 5 seconds off), centrifuge (12,000 g, 30 min, 4°C) to remove cell debris, and collect the supernatant as the crude enzyme extract.

[0032] (4) Purification by nickel column affinity chromatography The crude enzyme extract was filtered through a 0.45 μm filter and then loaded onto a pre-equilibrated Ni-NTA affinity chromatography column. Capture was achieved by the specific binding of the C-terminal His tag to nickel ions. Gradient elution was performed using elution buffers containing different concentrations of imidazole (50-500 mM), and the elution peaks containing the target protein were collected.

[0033] (5) Anion chromatography purification After dialyzing the nickel column eluent to replace the buffer, it was loaded onto a Q Sepharose anion exchange chromatography column for further purification. Gradient elution was performed using buffers containing different concentrations of sodium chloride (50-800 mM). The target protein eluent was collected, dialyzed, concentrated, and stored at -80°C.

[0034] (6) Protein purity analysis The results showed that the purified mutant enzyme appeared as a single, clear band on the electrophoresis gel, with a molecular weight of approximately 45 kDa, indicating that it reached electrophoretic purity. Protein concentration was determined using the Bradford method or NanoDrop assay.

[0035] Example 2: Determination of Enzymatic Properties of psyCE Mutant This embodiment aims to determine the catalytic kinetic parameters of the mutant and verify its performance improvement.

[0036] (1) Measurement of kinetic parameters Under conditions of 35℃ and pH 7.5, different concentrations of lactose (50–800 mM) were used as substrates, and purified psyCE-FR enzyme (0.01 mg / mL) was added and reacted for 15 minutes. The reaction was terminated by heating or adding acid after completion, and the amount of ipilactose produced was detected by HPLC. Kinetic parameters were calculated based on the data fitted using the Michaelis-Menten equation.

[0037] (2) Product detection and analysis To improve the detection sensitivity and separation efficiency of lactose and ipilactose, pre-column derivatization was performed using p-aminobenzoic acid as the derivatizing reagent. The specific steps were as follows: 100 μL of 2M perchloric acid (HClO4) was added to 200 μL of the reaction solution, and the mixture was cooled in an ice bath for 10 minutes. After centrifugation to remove the precipitate, 150 μL of the supernatant was collected and neutralized with 50 μL of 2M potassium hydroxide (KOH). The neutralized sample was then mixed with an equal volume (1:1, v / v) of derivatizing reagent (0.9M p-aminobenzoic acid + 5% sodium cyanoborohydride in DMSO / acetic acid = 70:30). The mixture was reacted in a 60°C water bath for 30 minutes to complete the derivatization of the reduced sugar ends. After the reaction, the products were analyzed using HPLC (high performance liquid chromatography). Chromatographic column: ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm, Agilent Technologies); mobile phase: 20 mM tetrabutylammonium bisulfate dissolved in 50 mM sodium phosphate buffer (pH 2.0), 20% methanol; detector: photodiode array detector (DAD) 303 nm; flow rate: 0.5 mL / min; column temperature: 30 ℃. The product was identified by comparing its retention time with that of epilactalose standard, and quantification was performed using the external standard method.

[0038] (3) Results of reaction conditions The results showed that the catalytic performance of the mutant was significantly improved compared with that of the wild type (WT): Wild type (WT): Enzyme activity: 122.2 U / mg, Transformation number (Kcat): 219.9 s -1 Michaelis constant (Km): 261.9 mM, catalytic efficiency (Kcat / Km): 0.84 s⁻¹ -1 mM -1 Single mutant manifestations: N309F: Specific enzyme activity increased to 134.5 U / mg, Kcat maintained at 215.7 s. -1The Km level decreased to 220.6 mM, indicating that the mutation at this site effectively improved affinity.

[0039] N309H: Specific enzyme activity was 128.6 U / mg, Km significantly decreased to 121.2 mM (significantly improved affinity), and catalytic efficiency increased to 1.29 ss. -1 mM -1 .

[0040] K189R: Specific enzyme activity is 124.4 U / mg, and Km is reduced to 240.6 mM.

[0041] Double mutant behavior (best practice): psyCE-FR (N309F / K189R): Exhibits the best overall performance. Specific enzyme activity reaches 153.3 U / mg, and Kcat is increased to 245.7 s. -1 The Km decreased to 207.8 mM. Its catalytic efficiency (Kcat / Km) reached 1.18 s2. -1 mM -1 It is about 40% higher than the wild type.

[0042] psyCE-HR (N309H / K189R): Kcat is 166.6 s -1 However, the Km is extremely low (124.9 mM), and the highest catalytic efficiency reaches 1.33 s⁻¹. -1 mM -1 .

[0043] Experimental results show that the mutations at positions 309 and 189 involved in this invention can independently increase the substrate affinity or activity of the enzyme. In particular, the combination of N309F and K189R (psyCE-FR) produces a significant synergistic effect, achieving a simultaneous increase in Kcat and affinity, which is the most preferred technical solution; while other mutants (such as N309H and its combinations) have slightly decreased Kcat, but extremely high affinity, and also have industrial application value, all of which are within the protection scope of this invention.

[0044] Example 3: Effect of temperature on the activity of psyCE mutant This embodiment aims to evaluate the effect of temperature on the activity of the psyCE mutant, in order to assess the high low-temperature activity of the psyCE mutant.

[0045] (1) Reaction system and conditions Prepare a phosphate buffer solution (50 mM, pH 7.5) containing 200 mM lactose, and add purified psyCE mutant or wild-type enzyme to a final concentration of 0.02 mg / mL. React at 4–50 °C for 15 min, then detect the amount of product generated. The activity of the wild-type enzyme at 35 °C is defined as 100%. (2) Results The results are as follows Figure 2 As shown, the psyCE-FR mutant exhibits superior catalytic efficiency compared to the wild type at various temperatures. At 35-45℃, the psyCE-FR mutant demonstrates a 30-75% higher conversion efficiency than the wild type. In the low-temperature range of 4-20℃, the psyCE-FR mutant shows a 10-20% higher conversion efficiency than the wild type. This indicates that the psyCE-FR mutant significantly enhances catalytic activity under low-temperature conditions, maintaining high activity in cold environments, making it particularly suitable for low-temperature processing or industrial applications requiring catalytic reactions at low temperatures.

[0046] Example 4: Application of psyCE mutant in low-temperature lactose conversion This embodiment aims to evaluate the practical application potential of the psyCE mutant in catalyzing the production of epiprolose from lactose under low temperature (8°C) conditions.

[0047] (1) Reaction system and conditions Prepare a phosphate buffer solution (50 mM, pH 7.5) containing 200 mM lactose, and add purified psyCE mutant or wild-type enzyme to a final concentration of 0.02 mg / mL. The reaction is carried out at an isothermal temperature of 8℃, and samples are taken at different time points for analysis.

[0048] (2) Results The results showed that the psyCE-FR mutant exhibited superior catalytic efficiency at low temperatures: after 3 hours of reaction at 8°C, the conversion rate of epilactose in the psyCE-FR system reached 21%; in contrast, the wild-type enzyme required 4 hours to achieve a 20% conversion rate. This indicates that the space-time productivity of the mutant at low temperatures is approximately 20% higher than that of the wild-type, significantly shortening the production time and helping to reduce energy consumption and the risk of microbial contamination. Figure 3 ).

[0049] Example 5: Thermostability and Inactivation Characteristics of psyCE Mutant Enzyme This embodiment aims to evaluate the thermal stability (Tm value) and inactivation at high temperatures of each mutant described in this invention, and to verify its industrial suitability.

[0050] (1) Determination of melting temperature (Tm) The Tm values ​​of wild-type and mutant proteins were determined using protein thermal shift analysis.

[0051] (2) Results The measurement results are shown in the table below: Wild type (WT): 40.97 °C; N309F: 42.75 °C; N309H: 42.08 °C; K189R: 40.80 °C; psyCE-FR: 42.81 °C; psyCE-HR: 41.92 °C.

[0052] The results showed that all mutants involved in this invention exhibited thermal stability (Tm) superior to or equivalent to the wild type. This indicates that the introduction of mutations at positions 309 and 189, while enhancing catalytic activity, did not disrupt the overall folding stability of the protein. Instead, it slightly improved structural rigidity by strengthening local hydrogen bond networks (such as N309F and psyCE-FR).

[0053] (3) Inactivation characteristics at 50°C Further testing showed that, despite a slight increase in Tm values, all mutants (including psyCE-FR) exhibited transient inactivation characteristics at 50°C. This confirms that the mutant family of this invention fully retains the technological advantage of psychrophilic enzymes being "easy to heat inactivate," and all are suitable for rapid termination reactions in pasteurization processes. Figure 2 ).

Claims

1. A cellobiose 2-epimerase mutant with high catalytic efficiency, characterized in that... The mutant is obtained by mutating Asn at position 309 of the amino acid sequence of wild-type psychrophilic cellobiose 2-epimerase with amino acid sequence 1 to Phe or His, or by mutating Lys at position 189 to Arg.

2. A cellobiose 2-epimerase mutant with high catalytic efficiency, characterized in that... The mutant was obtained by mutating Asn at position 309 of the amino acid sequence of wild-type psychrophilic cellobiose 2-epimerase with amino acid sequence 1 to Phe or His, and mutating Lys at position 189 to Arg.

3. The cellobiose 2-epimerase mutant according to claim 1 or 2, characterized in that... The amino acid sequences of the mutants are as follows: mutant N309F, with Asn at position 309 mutated to Phe, amino acid sequence is sequence 2; mutant N309H, with Asn at position 309 mutated to His, amino acid sequence is sequence 3; mutant K189R, with Lys at position 189 mutated to Arg, amino acid sequence is sequence 4; mutant psyCE-FR, a double mutation with Asn at position 309 mutated to Phe and Lys at position 189 mutated to Arg, amino acid sequence is sequence 5; mutant psyCE-HR, a double mutation with Asn at position 309 mutated to His and Lys at position 189 mutated to Arg, amino acid sequence is sequence 6.

4. The cellobiose 2-epimerase mutant according to claim 3, characterized in that... The mutant enzyme is used to prepare epiprolose in a low-temperature environment.

5. The cellobiose 2-epimerase mutant according to claim 3, characterized in that... The low-temperature environment refers to 4~10℃.

6. The cellobiose 2-epimerase mutant according to claim 3, characterized in that... The low-temperature environment refers to 8°C.

7. A method for producing ipilactose using the cellobiose 2-epimerase mutant of claim 3: using lactose or lactose-containing material as a substrate, and the cellobiose 2-epimerase mutant as a catalyst, catalyzing the epimerization of lactose at the C-2 position to generate ipilactose under low temperature conditions.

8. A recombinant expression vector comprising the cellobiose 2-epimerase mutant gene of claim 3 and a recombinant engineered bacterium.

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