High-activity low-temperature beta-galactosidase mutant as well as coding gene and application thereof

Abstract

The invention discloses a high-activity low-temperature beta-galactosidase mutant as well as a coding gene and application thereof, and belongs to the technical field of functional enzymes. The mutant is characterized in that the mutation is carried out aiming at one or more sites of the 110th site, the 476th site, the 522th site, the 1018th site and the 1023th site of an amino acid sequence shown as SEQ ID No.1. Preferably, the beta-galactosidase OUC-JST is obtained by carrying out R110I and K1023D combined mutation on an amino acid sequence of wild type beta-galactosidase OUC-JST, and has an amino acid sequence as shown in SEQ ID No.3. Compared with a wild type, the mutant has the advantages that the enzyme activity is improved by 2.80 times, the optimal reaction temperature is reduced from 20 DEG C to 15 DEG C, the relative enzyme activity is kept at 50% or above within the range of 15-30 DEG C, 20% of the enzyme activity is still kept at 0 DEG C, and the wide temperature range characteristic is kept.

Description

Technical Field

[0001] This invention relates to a highly active low-temperature β-galactosidase mutant, its encoding gene, and its applications, belonging to the field of functional enzyme technology. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Lactose is widely found in various dairy products. On the one hand, lactose can provide energy after being broken down, and on the other hand, it can promote the growth of beneficial bacteria such as Bifidobacteria in the small intestine. However, people with lactose intolerance may experience diarrhea, bloating, and cramps due to excessive lactose accumulation in the intestines, leading to tissue dehydration and reduced calcium absorption. Lactose intolerance is caused by a deficiency of the enzyme galactosidase. More than 70% of people in Asia suffer from lactose intolerance, while in some African countries, the figure reaches 90% to 100%.

[0004] Currently, the most widely used method to address lactose intolerance is through the hydrolysis of lactose by β-galactosidase, resulting in lactose-free or low-lactose dairy products. The optimal reaction temperature for commercially available β-galactosidases is primarily concentrated between 35 and 65°C. However, many processes in the dairy production industry are carried out at low temperatures. Therefore, there is a need for more β-galactosidases that exhibit high enzymatic activity even at low temperatures.

[0005] Several low-temperature β-galactosidases have been reported in the prior art, for example, those derived from the genus *Alternaria* (…). Alteromonas sp. ), psychrophilic bacteria ( Alkalilactibacillus ikkense Low-temperature enzymes, such as those from natural sources, still have room for improvement in terms of catalytic efficiency and temperature adaptability, making it difficult to fully meet the efficiency and cost requirements of industrial production.

[0006] Modifying enzyme molecules using protein engineering techniques to obtain mutants with higher catalytic activity and better adaptability to low-temperature production conditions is one of the research hotspots in this field. Previous studies have shown that site-directed mutagenesis can improve the performance of refrigerable enzymes, indicating the feasibility of molecular modification of this type of enzyme. However, due to structural differences in enzymes from different sources, the effects of mutations are unpredictable, and the design and validation of mutation sites for specific enzymes still require experimental determination.

[0007] To address the aforementioned problems and existing technologies, this invention relates to a novel low-temperature β-galactosidase, OUC-JST (derived from *Xanthomonas auricula-judae*). Flavobacterium algicolaThrough molecular modification, virtual saturation mutation analysis and experimental verification, a series of mutants with significantly increased enzyme activity and lower optimal reaction temperature were obtained, providing a more efficient enzyme preparation option for low-temperature lactose hydrolysis. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a highly active low-temperature β-galactosidase mutant, its encoding gene, and its applications.

[0009] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a β-galactosidase mutant, said mutation being a mutation made at one or more sites of the amino acid sequence shown in SEQ ID No. 1, namely positions 110, 476, 522, 1018, and 1023.

[0010] In one embodiment of the present invention, the mutation is any one or more of the following combinations: Arginine at position 110 is mutated to isoleucine (R110I); The glutamic acid at position 476 is mutated to tyrosine (E476Y). The lysine at position 522 is mutated to alanine (K522A); The asparagine at position 1018 is mutated to lysine (N1018K). The asparagine at position 1018 is mutated to arginine (N1018R). The lysine at position 1023 is mutated to valine (K1023V). The lysine at position 1023 is mutated to aspartic acid (K1023D). The lysine at position 1023 is mutated to methionine (K1023M).

[0011] In a preferred embodiment of the present invention, the mutation is a combined mutation of R110I and K1023D.

[0012] In a preferred embodiment of the present invention, the mutant has the amino acid sequence shown in SEQ ID No. 3 (corresponding to the R110I + K1023D combination mutation, named OUC-JST-N).

[0013] In one embodiment of the present invention, the mutant has one or more of the following enzymatic properties: The enzyme activity is more than 1.5 times higher than that of the wild type; The optimal reaction temperature is 15-20℃; Within a temperature range of 15℃ to 30℃, the relative enzyme activity is not less than 50%; At 0°C, the relative enzyme activity is not less than 20%.

[0014] A second aspect of the invention provides a gene encoding the β-galactosidase mutant described in the first aspect.

[0015] A third aspect of the present invention provides a recombinant expression vector containing the gene described in the second aspect.

[0016] Preferably, the recombinant expression vector is pET28a-OUC-JST-N, which is obtained by cloning the gene encoding the mutant into the multiple cloning site of the pET-28a(+) vector.

[0017] In a fourth aspect, the present invention provides recombinant engineered bacteria containing the recombinant expression vector described in the third aspect.

[0018] Preferably, the recombinant engineered bacteria is Escherichia coli BL21(DE3) / pET28a-OUC-JST-N.

[0019] A fifth aspect of the present invention provides a method for preparing the β-galactosidase mutant, comprising the following steps: (1) Using a plasmid containing the gene shown in SEQ ID No.2 as a template, specific primers were designed for the mutation site, and the plasmid containing the mutation site was obtained by PCR amplification. (2) Transform the mutant plasmid obtained in step (1) into the host cell to obtain the recombinant engineered bacteria expressing the mutant; (3) Cultivate the recombinant engineered bacteria and induce the expression of the β-galactosidase mutant; (4) Purify the β-galactosidase mutant expressed in step (3).

[0020] Preferably, the induction condition in step (3) is: in OD 600 When the value is 0.8, IPTG with a final concentration of 0.1 mM is added, and induction is carried out at 37℃ and 220 rpm for 20 hours.

[0021] Preferably, the purification in step (4) includes: ultrasonically disrupting the bacterial cells, centrifuging to collect the supernatant, and then performing affinity chromatography purification using a Ni-NTA column.

[0022] A sixth aspect of the present invention provides the use of the β-galactosidase mutant described in the first aspect in the hydrolysis of lactose at low temperature.

[0023] Furthermore, the application involves adding the β-galactosidase mutant to the dairy product to be treated and carrying out a hydrolysis reaction under low temperature conditions.

[0024] Furthermore, the low temperature condition is 0-30℃, preferably 10-15℃, and most preferably 15℃.

[0025] Furthermore, the dairy product is cow's milk.

[0026] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) Significantly increased enzyme activity This invention successfully screened and obtained several mutants with significantly increased enzyme activity through virtual saturation mutation analysis and experimental verification: the enzyme activity of the single-point mutant K1023D was 3.07 times higher than that of the wild type OUC-JST; the enzyme activities of mutants R110I, K522A, N1018R, and K1023V were all more than 1.5 times higher; and the highest enzyme activity of the combined mutant R110I + K1023D (OUC-JST-N) was 2.80 times higher than that of the wild type.

[0027] This significant increase in enzyme activity means that the hydrolysis time can be significantly shortened with the same amount of enzyme added, or the amount of enzyme used can be reduced within the same hydrolysis time, thereby reducing production costs.

[0028] (2) Better low temperature adaptability The present invention unexpectedly discovered that some mutants not only have increased enzyme activity, but also shift their optimal reaction temperature to lower temperatures: the optimal reaction temperature of mutant R110I decreased from 20℃ in the wild type to 15℃; the optimal reaction temperature of the combined mutant OUC-JST-N remained at 15℃; OUC-JST-N retained more than 50% of its enzyme activity in the temperature range of 15-30℃, and at 0℃ it still retained 20% of its highest enzyme activity, maintaining the wide temperature range characteristics of the wild type.

[0029] This combination of higher activity and lower temperature makes it better suited to the actual needs of low-temperature dairy processing.

[0030] (3) Excellent practical application effect Adding 1 U / mL OUC-JST-N to milk at 10°C resulted in a lactose hydrolysis rate of 90.86% within 6 hours and 98.37% after 48 hours, with residual lactose at only 812.25 mg / L, which is better than the requirements of Chinese and EU lactose-free product standards (≤1000 mg / L).

[0031] Compared with wild-type OUC-JST (89.64% hydrolysis rate in 12 hours), OUC-JST-N shortens the hydrolysis time by more than 50% with the same amount of enzyme, significantly improving production efficiency.

[0032] (4) Unpredictability of mutation sites This invention utilizes virtual saturation mutagenesis analysis to systematically screen amino acid residues within the lactose-binding pocket, and experimentally verified the identification of multiple effective mutation sites. The effects of these site modifications are unpredictable—not all predicted site mutations enhance enzyme activity; only specific mutations such as R110I, E476Y, K522A, N1018K, N1018R, K1023V, K1023D, and K1023M exhibit increased enzyme activity. This further confirms the inventiveness of this invention. Attached Figure Description

[0033] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0034] Figure 1 Image showing the results of OUC-JST molecular docking.

[0035] Figure 2 : Analysis of virtual saturation mutation results; A represents the change in binding energy, and B represents the change in stability energy. The more blue the color, the more negative the energy (favorable mutation), and the more red the color, the more positive the energy (unfavorable mutation).

[0036] Figure 3 Comparison of relative enzyme activities of OUC-JST and its mutants.

[0037] Figure 4 : The optimal reaction temperature of OUC-JST and its mutants.

[0038] Figure 5 Comparison of enzyme activities between OUC-JST and OUC-JST-N.

[0039] Figure 6 : Optimal reaction temperature of OUC-JST-N.

[0040] Figure 7 Analysis of the effects of OUC-JST and OUC-JST-N on lactose hydrolysis in cow's milk. Detailed Implementation

[0041] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0044] The *Xanthomonas auricula-judae* mentioned in this invention ( Flavobacterium algicola This belongs to existing technology, and it has been described in the academic literature "Characterization of a GH20 β-N-Acetylhexosaminidase from..." Flavobacterium algicola The strain is disclosed in detail in *Suitable to Synthesize Lacto-N-triose II* (Li C, Cao Z, Jiang H, Secundo F, Mao XJ Agric. Food Chem. 2024, 72, 4849-4857). The source of this strain is clear, and those skilled in the art can reproduce it using the methods described in that document. Furthermore, the applicant undertakes to release samples of this strain to the public within twenty years from the date of this patent application.

[0045] Example 1: Prediction and Screening of Mutation Sites (1) Construction of molecular docking model Using wild-type β-galactosidase OUC-JST (amino acid sequence shown in SEQ ID No. 1) as a template, AlphaFold 3 was used for modeling and analysis. The protein structure was preprocessed using the Schrödinger Protein Preparation Workflow, and the energy of the preprocessed protein was minimized using an OPLS4 force field to ensure the structure reached the lowest potential energy state. A molecular docking model was obtained by docking the lactose substrate to the active site of OUC-JST. The molecular docking results are shown below. Figure 1 As shown.

[0046] (2) Virtual saturation mutation analysis Virtual saturation mutation analysis was re-performed on the amino acid residues within the lactose-binding pocket using Discovery Studio software, and the results are as follows: Figure 2 As shown.

[0047] Based on the results of virtual saturation mutagenesis and predictions from the HotSpot Wizard website, a mutant library of 18 mutants was constructed. Experimental results showed that the enzyme activities of R110I, E476Y, K522A, N1018K, N1018R, K1023V, K1023D, and K1023M were significantly improved. Among them, the enzyme activity of K1023D was increased by 3.07 times, and the enzyme activities of R110I, K522A, N1018R, and K1023V were all increased by more than 1.5 times.

[0048] Example 2: Construction and screening of single-point mutants (1) Design of mutant primers Specific site-directed mutagenesis primers were designed for the selected mutation sites. Using the pET28a-OUC-JST plasmid containing the gene shown in SEQ ID No. 2 as a template, the corresponding mutations were introduced by PCR amplification.

[0049] (2) Construction of mutant plasmids PCR reaction system: 1 μL template DNA, 1.5 μL each of forward and reverse primers, 10 μL dNTPs, 1 μL high-fidelity DNA polymerase, 25 μL KOD Buffer, and sterile water to a final volume of 50 μL.

[0050] PCR reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 20 sec, 55℃ annealing for 20 sec, 72℃ extension for 3.5 min, for a total of 30 cycles; 72℃ final extension for 10 min.

[0051] PCR products were digested with DpnI enzyme (37℃, 1 h) to remove methylated template plasmids. The digested products were transformed into E. coli DH5α competent cells, plated on LB agar plates containing 50 μg / mL kanamycin sulfate, and incubated overnight at 37℃. Single colonies were picked for sequencing verification to confirm the correct introduction of the mutation.

[0052] (3) Construction of mutant expression engineered bacteria The correctly sequenced mutant plasmids were transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin sulfate, and incubated overnight at 37°C to obtain recombinant engineered bacteria expressing each mutant.

[0053] (4) Expression and purification of mutants Single colonies of the recombinant engineered bacteria were picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate, and activated at 37°C and 220 rpm for 12 hours. A 1% inoculum was then added to 50 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate, and cultured at 37°C and 220 rpm until the OD600 value reached 0.8. IPTG was then added to a final concentration of 0.1 mM, and induction was performed at 37°C and 220 rpm for 20 hours.

[0054] Take the induced culture medium, centrifuge at 10,000 rpm for 15 minutes at 4℃, collect the cells, resuspend them in water (pH 7.0), sonicate for 15 minutes, centrifuge at 10,000 rpm for 10 minutes at 4℃ to remove cell debris, and the supernatant is the crude enzyme solution.

[0055] The crude enzyme solution was purified by affinity chromatography using a Ni-NTA column: First, the column was equilibrated with 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). After loading the sample, impurities were eluted with 20 mM imidazole solution, and then the target protein was eluted with 120 mM imidazole solution. The eluent was collected, which was the purified mutant protein.

[0056] The purified proteins were analyzed by SDS-PAGE. All mutants yielded high-purity proteins with a molecular weight of approximately 121.3 kDa, consistent with the wild type.

[0057] (5) Screening for enzyme activity of mutants Using oNPG as a substrate, the enzyme activity of each purified mutant was determined at 20℃. The enzyme activity assay method was as follows: 90 μL of 10 mM oNPG substrate was mixed with 400 μL of 50 mM pH 7.0 PBS buffer, incubated at 20℃ for 10 min, 10 μL of purified enzyme was added, and the reaction was allowed to proceed for 5 min. The reaction was then terminated by adding 500 μL of 200 mM Na2CO3, and the OD410 was measured. The relative enzyme activity of each mutant was calculated with the enzyme activity of wild-type OUC-JST as 100%.

[0058] The results are as follows Figure 3 As shown, in the constructed mutant library, the following mutant enzyme activities were significantly increased: K1023D: 307% (3.07x improvement), R110I: 187% (1.87x improvement), K1023V: 176% (1.76x improvement), N1018R: 168% (1.68x improvement), K522A: 159% (1.59x improvement), K1023M: 148% (1.48x improvement), N1018K: 135% (1.35x improvement), E476Y: 128% (1.28x improvement).

[0059] The enzyme activity at other mutation sites was not significantly increased or was lower than that at wild type, indicating that the mutation effect is site-specific.

[0060] Example 3: Determination of the optimal reaction temperature for highly active mutants Following the method in Example 2 (5), the optimal reaction temperature of five mutants (R110I, K522A, N1018R, K1023V, K1023D) with enzyme activity increased by more than 1.5 times was determined in the range of 0-60℃.

[0061] The results are as follows Figure 4 As shown: the optimal reaction temperature for the R110I mutant decreased from 20℃ for the wild type to 15℃; The optimal reaction temperature for K522A, N1018R, K1023V, and K1023D remains at 20℃, consistent with the wild type.

[0062] This result unexpectedly showed that the R110I mutation not only increased enzyme activity but also shifted the enzyme's optimal temperature to a lower temperature.

[0063] Example 4: Construction and performance evaluation of combined mutants (1) Construction of the combinatorial mutant OUC-JST-N The optimal reaction temperature-lowering mutation site R110I and the site with the highest enzyme activity-enhancing site K1023D were selected for combined mutation. Using the plasmid containing the R110I mutation as a template, K1023D mutation primers were designed, and the second mutation was introduced through a second round of site-directed PCR to construct a double mutant containing both R110I and K1023D, named OUC-JST-N, whose amino acid sequence is shown in SEQ ID No. 3.

[0064] (2) Expression and purification of OUC-JST-N The OUC-JST-N was induced to express and purified according to the method in Example 2 (4) to obtain high-purity protein.

[0065] (3) Enzyme activity assay of OUC-JST-N Using oNPG as a substrate, the enzyme activity of OUC-JST-N was measured at 20℃ and compared with that of wild-type OUC-JST.

[0066] The results are as follows Figure 5 As shown, the highest enzyme activity of OUC-JST-N is 2.80 times higher than that of wild-type OUC-JST.

[0067] (4) Determination of the optimal reaction temperature of OUC-JST-N The optimal reaction temperature of OUC-JST-N was determined according to the method in Example 3.

[0068] The results are as follows Figure 6 As shown, the optimal reaction temperature for OUC-JST-N remains at 15℃, consistent with the R110I single-point mutant. Enzyme activity is retained at over 50% within the temperature range of 15-30℃, and even at 0℃, it retains 20% of its maximum activity, maintaining the wide temperature range characteristic of the wild type.

[0069] Example 5: Hydrolytic effect of OUC-JST-N on lactose in milk (1) Experimental methods Purified OUC-JST and OUC-JST-N were added to fresh milk at 10°C to a final concentration of 1 U / mL. Samples were taken at 1, 2, 3, 6, 12, 24, and 48 hours, and the enzymes were inactivated by boiling in a water bath for 10 min. The samples were then filtered through a 0.22 μm filter membrane for HPLC analysis.

[0070] HPLC detection conditions: Sugar Pak I column (6.5 mm × 300 mm), RID-20A differential detector, 50 mg / mL EDTA-CaNa2 mobile phase, flow rate 0.5 mL / min, column temperature 75℃, injection volume 20 μL, detection time 15 min.

[0071] (2) Experimental results The results are as follows Figure 7 As shown, OUC-JST-N achieved a lactose hydrolysis rate of 90.86% within 6 hours, while wild-type OUC-JST required more than 12 hours to achieve the same hydrolysis rate, more than doubling the hydrolysis efficiency. After 48 hours, the OUC-JST-N treatment group achieved a lactose hydrolysis rate of 98.37%, with residual lactose at only 812.25 mg / L, exceeding the requirements of Chinese and EU lactose-free product standards (≤1000 mg / L). This result demonstrates that OUC-JST-N possesses rapid and efficient lactose hydrolysis capabilities in actual milk samples at low temperatures, exhibiting significant application potential in the industrial production of lactose-free dairy products.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A β-galactosidase mutant, characterized in that, The mutant is a protein with increased enzyme activity obtained by performing any of the following mutations on the amino acid sequence shown in SEQ ID No. 1: (a) Arginine at position 110 is mutated to isoleucine (R110I); (b) The glutamic acid at position 476 is mutated to tyrosine (E476Y); (c) The lysine at position 522 is mutated to alanine (K522A); (d) The asparagine at position 1018 is mutated to lysine (N1018K). (e) The asparagine at position 1018 is mutated to arginine (N1018R); (f) The lysine at position 1023 is mutated to valine (K1023V); (g) The lysine at position 1023 is mutated to aspartic acid (K1023D); (h) The lysine at position 1023 is mutated to methionine (K1023M).

2. The β-galactosidase mutant according to claim 1, characterized in that, The mutation is a combination mutation of R110I and K1023D.

3. The β-galactosidase mutant according to claim 2, characterized in that, The mutant has the amino acid sequence shown in SEQ ID No.

3.

4. The β-galactosidase mutant according to claim 1, characterized in that, The mutant possesses one or more of the following enzymatic properties: The enzyme activity is more than 1.5 times higher than that of the wild type; The optimal reaction temperature is 15-20℃; Within a temperature range of 15℃ to 30℃, the relative enzyme activity is not less than 50%; At 0°C, the relative enzyme activity is not less than 20%.

5. A gene encoding the β-galactosidase mutant according to any one of claims 1-4.

6. A recombinant expression vector containing the gene of claim 5.

7. Recombinant engineered bacteria containing the recombinant expression vector of claim 6.

8. A method for preparing the β-galactosidase mutant according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Construct a recombinant expression vector containing the gene described in claim 6; (2) The recombinant expression vector from step (1) was transformed into host cells to obtain recombinant engineered bacteria; (3) Cultivate the recombinant engineered bacteria and induce the expression of the β-galactosidase mutant; (4) Purify the β-galactosidase mutant expressed in step (3).

9. The method according to claim 8, characterized in that, The induction conditions described in step (3) are: in OD 600 When the value is 0.8, add IPTG to a final concentration of 0.1 mM and induce for 20 hours at 37℃ and 220 rpm; The purification described in step (4) includes: ultrasonically disrupting the bacterial cells, centrifuging to collect the supernatant, and then using a Ni-NTA column for affinity chromatography purification.

10. The use of the β-galactosidase mutant according to any one of claims 1-4 in the hydrolysis of lactose at low temperature.

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