Wide-temperature-range low-temperature beta-galactosidase OUC-JST derived from flavobacterium fargesii as well as coding gene and application of beta-galactosidase OUC-JST

Abstract

The invention discloses wide-temperature-range low-temperature beta-galactosidase OUC-JST derived from flavobacterium fusiformis as well as a coding gene and application of the wide-temperature-range low-temperature beta-galactosidase OUC-JST, and belongs to the technical field of functional enzymes. The beta-galactosidase OUC-JST has an amino acid sequence as shown in SEQ ID No.1, the optimum reaction temperature is 20 DEG C, the relative enzyme activity in a wide temperature range from 5 DEG C to 30 DEG C is kept at 50% or above, 20% of the enzyme activity is still kept at 0 DEG C, and the beta-galactosidase OUC-JST shows excellent wide-temperature-range low-temperature adaptability. The invention also provides a gene SEQ ID No.2 for coding the enzyme, a recombinant expression vector and recombinant engineering bacteria containing the gene, and a preparation method of the enzyme. The beta-galactosidase OUC-JST disclosed by the invention is suitable for hydrolyzing lactose at low temperature, and has a wide application prospect in the field of lactose-free dairy product production.

Description

Technical Field

[0001] This invention relates to a wide-temperature-range, low-temperature β-galactosidase OUC-JST derived from *Xanthomonas aeruginosa*, 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 enzyme 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 ) and other cold-adapted enzymes. However, the temperature-activity profiles of these enzymes differ, and their application under actual production conditions still has room for improvement. In addition, multiple predicted β-galactosidase genes may exist in the same strain, but not all predicted sequences can be successfully expressed as active enzymes, which increases the unpredictability of new enzyme development.

[0006] To address the aforementioned problems and existing technologies, this invention utilizes *Xanthomonas aeruginosa* (… Flavobacterium algicola A β-galactosidase OUC-JST with unique wide temperature range characteristics was screened out, and its hydrolytic effect on lactose was investigated, in order to provide a new enzyme preparation option for alleviating lactose intolerance. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method derived from *Xanthomonas auricula-judae* (…). Flavobacterium algicola) wide-temperature-range low-temperature β-galactosidase OUC-JST.

[0008] Another object of the present invention is to provide a gene encoding the β-galactosidase OUC-JST.

[0009] The present invention also aims to provide a recombinant expression vector containing the said gene and a recombinant engineered bacteria.

[0010] Another objective of this invention is to provide a method for preparing the β-galactosidase OUC-JST.

[0011] Another objective of this invention is to provide the application of the β-galactosidase OUC-JST in the hydrolysis of lactose at low temperatures.

[0012] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a β-galactosidase OUC-JST having the amino acid sequence shown in SEQ ID No. 1 and having the following temperature-activity characteristics: (a) The optimal reaction temperature is 20°C; (b) The relative enzyme activity is not less than 50% within a temperature range of 5°C to 30°C; (c) At 0°C, the relative enzyme activity is not less than 20%.

[0013] In one embodiment of the present invention, the β-galactosidase OUC-JST is derived from *Xanthomonas auricula-judae* (… Flavobacterium algicola ), and its relative molecular mass is 121.3 kDa.

[0014] In one embodiment of the present invention, the residual enzyme activity of the β-galactosidase OUC-JST is not less than 50% after being stored at 4°C for 200 h.

[0015] In one embodiment of the present invention, the β-galactosidase OUC-JST is added to cow's milk at 10°C and treated with an enzyme dosage of 1 U / mL for 12 hours, and the lactose hydrolysis rate is not less than 89%.

[0016] In a second aspect of the invention, a gene encoding the β-galactosidase OUC-JST described in the first aspect is provided.

[0017] In one embodiment of the present invention, the nucleotide sequence of the gene is shown in SEQ ID No. 2.

[0018] In a third aspect of the invention, a recombinant expression vector containing the gene described in the second aspect is provided.

[0019] Preferably, the recombinant expression vector is pET28a-OUC-JST, which is obtained by cloning the gene shown in SEQ ID No. 2 into the multiple cloning site of the pET-28a(+) vector.

[0020] In a fourth aspect of the invention, a recombinant engineered bacterium containing the recombinant expression vector described in the third aspect is provided.

[0021] Preferably, the recombinant engineered bacteria is Escherichia coli BL21(DE3) / pET28a-OUC-JST, which is obtained by transforming pET28a-OUC-JST into Escherichia coli BL21(DE3) competent cells.

[0022] In a fifth aspect of the present invention, a method for preparing the β-galactosidase OUC-JST is provided, comprising the following steps: (1) Construct a recombinant expression vector containing the genes described in the second aspect; (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 β-galactosidase OUC-JST; (4) Purify the β-galactosidase OUC-JST expressed in step (3).

[0023] 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.

[0024] 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.

[0025] In a sixth aspect of the invention, the application of the β-galactosidase OUC-JST described in the first aspect in the hydrolysis of lactose at low temperature is provided.

[0026] Furthermore, the application involves adding β-galactosidase OUC-JST to the dairy product to be treated and carrying out a hydrolysis reaction under low temperature conditions.

[0027] Furthermore, the low temperature condition is 0-30℃, preferably 10-20℃, and most preferably 20℃.

[0028] Furthermore, the dairy product is cow's milk.

[0029] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) It provides a novel enzyme resource, and the enzyme has sequence non-substitutability. This invention is the first to obtain *Xanthomonas aeruginosa* ( Flavobacterium algicola β-galactosidase OUC-JST was isolated from the strain. More importantly, based on the genomic information of this strain, the inventors predicted several candidate genes. However, under the same expression and detection conditions, only the gene shown in SEQ ID No. 2 successfully expressed a soluble protein with significant enzymatic activity; the other candidate genes did not show any effective enzymatic activity. This result confirms that the amino acid sequence shown in SEQ ID No. 1 is a specific sequence with actual enzymatic activity that has been experimentally verified. Its activity is not a universal characteristic of this type of enzyme, but rather an unexpected discovery obtained by the inventors through experimental screening. This process of "screening out a unique active sequence from numerous candidates" itself embodies the inventiveness of this invention.

[0030] (2) Unique wide temperature range low temperature adaptability The OUC-JST provided by this invention has excellent low-temperature adaptability and a wide temperature activity range: The optimal reaction temperature is 20℃, making it a typical low-temperature enzyme, suitable for low-temperature processing of dairy products. Wide temperature activity range: The relative enzyme activity can be maintained at more than 50% of the maximum enzyme activity within a wide temperature range of 5℃ to 30℃. Ultra-low temperature activity: It can still retain 20% of the enzyme activity at 0℃.

[0031] This combination of low-temperature high activity and wide suitable temperature range allows it to better adapt to temperature fluctuations that may occur in industrial production (such as temperature changes during cold chain transportation and differences in production conditions across different seasons), giving it significant practical value. No existing technology teaches or implies that GH2 family refractory enzymes necessarily possess such a wide temperature range characteristic.

[0032] (3) Good storage stability OUC-JST retains more than 50% of its initial enzyme activity after being stored at 4°C for 200 hours, which is beneficial for the transportation and storage of enzyme preparations and reduces cold chain costs in industrial applications.

[0033] (4) Excellent practical application effect Using lactose as a substrate, the specific enzyme activity of OUC-JST was determined to be 2.59 U / mg. In milk at 10°C, treatment with an enzyme dosage of 1 U / mL resulted in: 89.64% lactose hydrolysis rate after 12 hours; and over 96% after 48 hours. This data demonstrates that the enzyme is not only temperature-adaptable but can also be directly applied in practical production scenarios, effectively addressing the needs of people with lactose intolerance. This complete "from laboratory to application" validation represents a substantial contribution of this application to the prior art.

[0034] (5) Provides an excellent framework for protein engineering This invention unexpectedly revealed that the protein backbone of OUC-JST possesses excellent structural plasticity, laying the foundation for further molecular modification. This characteristic not only gives it direct application value but also the potential to serve as a protein engineering platform. Attached Figure Description

[0035] 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.

[0036] Figure 1 Evolutionary kinship analysis diagram of OUC-JST.

[0037] Figure 2 SDS-PAGE protein electrophoresis image of OUC-JST. 1 is Marker, 2 is crude enzyme solution, 3 is permeation solution, 4 is pET28a(+) empty vector, 5 is 10 mM imidazole elution buffer, 6 is 20 mM imidazole elution buffer, and 7-9 are 120 mM imidazole elution buffer.

[0038] Figure 3 : OUC-JST optimal reaction temperature diagram (showing wide temperature range characteristics from 5-30℃).

[0039] Figure 4 OUC-JST storage stability diagram.

[0040] Figure 5 : Optimal reaction pH diagram of OUC-JST.

[0041] Figure 6 pH stability graph of OUC-JST.

[0042] Figure 7 : Effect of chemical reagents on the activity of OUC-JST enzyme.

[0043] Figure 8 Analysis of the effect of OUC-JST on lactose hydrolysis in cow's milk.

[0044] Figure 9 Image showing the results of OUC-JST molecular docking. Detailed Implementation

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The *Xanthomonas auricula-judae* used 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, Seundo 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.

[0049] Example 1: Screening and sequence analysis of β-galactosidase OUC-JST 1. Screening and activity verification of candidate genes Based on seaweed flavobacterium ( Flavobacterium algicola Based on genomic information, the inventors predicted and selected several potential β-galactosidase encoding genes as candidates. Through PCR amplification, construction of expression vectors, transformation of *E. coli* BL21(DE3), and induction of expression, recombinant expression and enzyme activity assays were performed on three representative candidate genes.

[0050] The experimental results unexpectedly showed that, although multiple candidate genes were predicted to encode β-galactosidase, under the same expression and detection conditions, only the gene shown in SEQ ID No. 2 was able to successfully express a soluble protein with significant β-galactosidase activity. The recombinant expression products of the other two candidate genes did not show any effective enzyme activity.

[0051] This result confirms that the amino acid sequence shown in SEQ ID No. 1 (encoded by SEQ ID No. 2) is a specific sequence with actual enzyme activity that has been experimentally verified. Its activity is not a general characteristic of GH2 family sequences, but an unexpected discovery obtained by the inventors through experimental screening.

[0052] 2. Sequence Analysis Analysis of the gene sequence shown in SEQ ID No. 2 revealed a length of 3135 bp, encoding 1045 amino acids (SEQ ID No. 1). OUC-JST was predicted to belong to the GH2 family, with an isoelectric point of 6.17 and a relative molecular mass of 121.3 kDa. Evolutionary phylogenetic analysis is as follows: Figure 1 As shown.

[0053] Example 2: Expression and purification of β-galactosidase OUC-JST β-galactosidase OUC-JST was heterologously expressed using conventional methods, followed by extraction and purification. The steps are as follows: (1) Construction of recombinant expression vector by F. marinum Using the genome as a template, specific primers were designed to amplify the β-galactosidase OUC-JST encoding gene by PCR. The PCR reaction system consisted of: 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.

[0054] 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; final extension at 72℃ for 10 min. The PCR product was recovered by agarose gel electrophoresis to obtain the gene fragment, which was ligated with the PET-28a(+) cloning vector using seamless cloning technology to obtain the pET28a plasmid containing the gene encoding β-galactosidase OUC-JST.

[0055] The plasmids obtained above were transformed into *E. coli* DH5α competent cells. Positive transformants were screened using LB agar plates containing kanamycin sulfate. After verification by PCR and DNA sequencing, recombinant plasmids were obtained.

[0056] (2) Construction of recombinant engineered bacteria The correctly sequenced recombinant plasmid was extracted and transformed into host E. coli BL21 competent cells. The constructed engineered bacteria grew on kanamycin sulfate resistant plates.

[0057] (3) Expression of β-galactosidase OUC-JST Recombinant engineered bacterial strains grown on kanamycin sulfate-resistant plates were selected and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate. The culture was activated at 37°C and 220 rpm for 12 hours. 1% of the culture was then inoculated into 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. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM to induce the expression of β-galactosidase OUC-JST for 20 hours.

[0058] (4) Extraction and purification of β-galactosidase OUC-JST After the above-mentioned induction of expression, the culture medium was taken, centrifuged at 10,000 rpm for 15 minutes at 4°C, the bacterial cells were collected, resuspended in water (pH 7.0), sonicated for 15 minutes, and centrifuged at 10,000 rpm for 10 minutes at 4°C to remove cell debris. The supernatant was the crude protein.

[0059] The crude protein 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, weakly binding contaminating proteins were eluted with 20 mM imidazole solution (20 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). Then, the target protein was eluted with 120 mM imidazole solution (120 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). The eluent was collected, which was the purified protein.

[0060] The components from the purification process were analyzed by SDS-PAGE protein electrophoresis, and the results are as follows: Figure 2 As shown, the purified protein exhibited a single, clear band in lanes 7-9, with a molecular weight of approximately 121.3 kDa, consistent with the theoretical prediction, indicating that high-purity OUC-JST enzyme was successfully obtained.

[0061] Example 3: Study on the enzymatic properties of β-galactosidase OUC-JST (1) Optimal reaction temperature and storage stability of OUC-JST 400 μL of 50 mM pH 7.0 PBS buffer was mixed with 90 μL of 10 mM ONPG substrate and incubated at 0–60 °C for 10 min each. Then, 10 μL of OUC-JST purified enzyme was added, and the reaction was carried out at the corresponding temperature for 5 min. The reaction was terminated by adding 500 μL of 200 mM Na2CO3 solution, and OD410 was measured. All experiments were performed in triplicate.

[0062] To determine the temperature stability of OUC-JST, 10 μL of pure OUC-JST enzyme was mixed with 400 μL of 50 mM pH 7.0 PBS buffer and stored at 4℃ and 25℃ for different times. Then, 90 μL of 10 mM ONPG substrate was added and reacted at the optimal temperature for 5 min. The reaction was terminated by adding 500 μL of 200 mM Na2CO3 solution, and the residual enzyme activity was measured.

[0063] The results are as follows Figure 3 As shown, the optimal reaction temperature for OUC-JST is 20℃. Within the temperature ranges of 5℃ and 15–30℃, the relative enzyme activity remains above 50% of the maximum enzyme activity, and even at 0℃, it retains 20% of its activity. This result demonstrates that OUC-JST not only possesses high activity at low temperatures but also exhibits a unique characteristic of a wide temperature activity range, enabling it to adapt to temperature fluctuations that may occur in industrial production.

[0064] The storage stability of OUC-JST was determined at 4℃ and 25℃, and its residual enzyme activity was obtained. Figure 4 As shown, OUC-JST retained more than 50% of its initial enzyme activity after being stored at 4℃ for 200 h, indicating that the enzyme has good stability at 4℃. However, after being stored at 25℃ for 48 h, OUC-JST retained 40% of its enzyme activity, and after 200 h, it almost completely lost its activity.

[0065] (2) Optimal reaction pH and pH stability of OUC-JST 400 μL of buffer solutions with different pH values ​​were mixed with 90 μL of 10 mM ONPG substrate and incubated at the optimal temperature for 10 min. Then, 10 μL of OUC-JST purified enzyme was added, and the reaction was continued at the optimal temperature for 5 min. Finally, 500 μL of 200 mM Na₂CO₃ solution was added to stop the reaction. OD₄¹⁰ was then measured.

[0066] To determine the pH stability of OUC-JST, 10 μL of pure OUC-JST enzyme was mixed evenly with buffers of different pH values ​​and incubated at 4°C for 1 h. Then, 90 μL of 10 mM ONPG substrate was added and reacted at the optimal temperature for 5 min. Finally, 500 μL of 200 mM Na2CO3 solution was added to stop the reaction, and the residual enzyme activity was measured.

[0067] The optimal pH of OUC-JST is as follows: Figure 5As shown, its optimal reaction pH is PBS buffer at pH 7.0. More than 60% of the enzyme activity is retained in PBS buffer at pH 6.0, while the enzyme activity is lower in strongly acidic or alkaline environments, reaching 0% at pH 4.0, pH 9.0, and pH 10.0. This indicates that OUC-JST is a neutral β-galactosidase, suitable for catalytic activity under neutral conditions.

[0068] The OUC-JST purified enzyme was added to buffer solutions of different pH values ​​(pH 3.0~10.0) and incubated at 4℃ for 1 h, followed by enzyme activity measurement. Figure 6 As shown, only at pH 7.0 did OUC-JST retain nearly 50% of its enzyme activity, and at PBS buffer pH 6.0 and 8.0, it retained more than 20% of its enzyme activity, indicating that the pH stability of β-galactosidase OUC-JST is poor.

[0069] (3) Effect of chemical reagents on OUC-JST enzyme activity 10 μL of different metal ions were mixed with chemical reagents SDS and Na2EDTA and 10 μL of OUC-JST purified enzyme, respectively, to achieve final metal ion concentrations of 10 μmol / L and 1 μmol / L. Simultaneously, 390 μL of pH 7.0 PBS buffer was mixed with 90 μL of 10 mM ONPG substrate. The two solutions were incubated at the optimal temperature for 20 min, then mixed thoroughly and reacted at the optimal reaction temperature for 5 min. 500 μL of 200 mM Na2CO3 was added to stop the reaction, and the residual enzyme activity was measured.

[0070] The effects of chemical reagents on the enzyme activity of OUC-JST are as follows: Figure 7 As shown, Na + K + Ba 2+ Ni 2+ Cu 2+ Fe 3+ SDS and Na2EDTA have significant inhibitory effects on OUC-JST, among which Ni 2+ Cu 2+ Fe 3+ SDS and Na2EDTA showed strong inhibitory effects on OUC-JST enzyme activity, with high concentrations of Na... + K + Ba 2+ The inhibition of enzyme activity is more pronounced. And Ca... 2+ Mn 2+ Mg 2+ Co 2+ It has a certain promoting effect on the enzyme activity of OUC-JST, and Ca 2+Mg 2+ The promoting effect becomes more significant with increasing concentration within a certain range.

[0071] (4) Enzyme activity assay of OUC-JST As a synthetically produced substrate, oNPG can be hydrolyzed by β-galactosidase into galactose and ortho-nitrophenol (oNP). oNP exhibits a yellow color under alkaline conditions and has a specific absorption peak at 410 nm. Furthermore, the oNP content shows a linear relationship with OD410 within a certain range. Therefore, the enzyme activity of β-galactosidase OUC-JST can be calculated by measuring the oNP content produced from the hydrolysis of oNPG.

[0072] ① Determination of oNPG hydrolase activity by OUC-JST Using 10 mM oNPG as the reaction substrate, the hydrolytic activity of OUC-JST on oNPG was calculated by measuring the absorbance of the hydrolysate oNP at 410 nm. For the reaction, 90 μL of 10 mM oNPG substrate was thoroughly mixed with 400 μL of 50 mM pH 7.0 PBS buffer and incubated at the optimal reaction temperature for 10 min. Then, 10 μL of purified OUC-JST enzyme was added, and the reaction was continued at the optimal temperature for 5 min. Finally, 500 μL of 200 mM Na2CO3 was added to terminate the reaction. 200 μL of the reaction solution was pipetted into a 96-well plate, and OD410 was measured. The amount of oNP generated in the reaction system was calculated using a standard curve.

[0073] Enzyme activity units (U) are defined as the amount of enzyme required to produce 1 μmol oNP or galactose per minute. Specific enzyme activity is defined as the enzyme activity units per milligram of OUC-JST.

[0074] ② The hydrolytic effect of β-galactosidase OUC-JST on lactose Using 20 mM lactose as the reaction substrate, the change in galactose concentration before and after the reaction was determined by high-performance liquid chromatography (HPLC), and the lactose hydrolytic activity of OUC-JST was calculated. For the reaction, 90 μL of 20 mM lactose substrate was thoroughly mixed with 400 μL of 50 mM pH 7.0 PBS buffer and incubated at the optimal reaction temperature for 10 min. Then, 10 μL of purified OUC-JST enzyme was added, and the reaction was carried out at the optimal temperature for 5 min, followed by enzyme inactivation in a boiling water bath for 10 min. A 200 μL sample was passed through a 0.22 μm filter to remove impurities before HPLC analysis.

[0075] The HPLC detection conditions were as follows: 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℃, sample loading volume 20 μL, and detection time 15 min.

[0076] Before starting the analysis, equilibrate the column with 50 mg / L EDTA-CaNa2 solution. Before using the mobile phase, heat it to boiling for 15 min to remove dissolved gases such as CO2. Prepare a fresh mobile phase every 24 h. For every 5 L of mobile phase used, backwash the column with at least 100 mL of 500 mg / mL EDTA-CaNa2 solution at 90 °C and a flow rate of 0.5 mL / min to extend the column's lifespan.

[0077] After purification, the protein concentration in the pure enzyme solution was 0.2000 mg / mL. Calculations showed that the specific enzyme activity of the OUC-JST pure enzyme for hydrolyzing oNPG was 41.25 U / mg, which is higher than that of previously studied enzymes derived from ONC-JST. Alkalilactibacillus ikkense, Halorubrum lacusprofundi The cold-tolerant enzyme purified from strains, OUC-JST, exhibits better catalytic activity at low temperatures.

[0078] Calculations show that the specific enzyme activity of OUC-JST for lactose hydrolysis is 2.59 U / mg, indicating that the enzyme has good hydrolytic activity for lactose at relatively low temperatures.

[0079] ③ The hydrolytic effect of β-galactosidase OUC-JST on lactose in cow's milk β-galactosidase OUC-JST was added to fresh milk at 10°C at final concentrations of 1 U / mL and 2 U / mL. Samples were taken at different time points (1, 2, 3, 6, 12, 24, and 48 hours), and galactose concentration was subsequently determined by high-performance liquid chromatography (HPLC) to evaluate the hydrolytic activity of OUC-JST. HPLC parameters included: Sugar Pak I column (6.5 mm × 300 mm), RID-20A detector, 50 mg / mL EDTA-CaNa2 mobile phase, and a flow rate of 0.5 mL / min. The column temperature was maintained at 75°C, the sampling volume was 20 μL, and the detection time was set to 15 min.

[0080] The results are as follows Figure 8As shown, when 1 U / mL of OUC-JST was added, the lactose hydrolysis rate reached 89.64% after 12 hours of hydrolysis, and exceeded 96% after 48 hours. OUC-JST showed significantly higher efficiency in hydrolyzing lactose in milk than other reported β-galactosidases, and also exhibited a lower optimum temperature, indicating its outstanding application potential in the industrial production of lactose-free dairy products.

[0081] (5) OUC-JST molecular docking simulation OUC-JST was modeled and analyzed using Alphfold 3. The protein structure was preprocessed using the Schrödinger Protein Preparation Workflow, and the energy of the preprocessed protein was minimized using the OPLS4 force field to ensure the structure reached the lowest potential energy state, providing an optimized structure for subsequent computational analysis. Molecular docking results are shown below. Figure 9 Show.

[0082] 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 wide-temperature-range low-temperature β-galactosidase OUC-JST derived from *Xanthomonas auricula-judae*, characterized in that, The β-galactosidase OUC-JST has the amino acid sequence shown in SEQ ID No. 1 and has the following temperature-activity characteristics: (a) The optimal reaction temperature is 20°C; (b) The relative enzyme activity is not less than 50% within a temperature range of 5°C to 30°C; (c) At 0°C, the relative enzyme activity is not less than 20%.

2. The wide-temperature-range low-temperature β-galactosidase OUC-JST derived from *Xanthomonas auricula-judae* according to claim 1, characterized in that, The β-galactosidase OUC-JST is derived from *Xanthomonas auricula-judae* (…). Flavobacterium algicola The relative molecular mass of the β-galactosidase OUC-JST is 121.3 kDa; the residual enzyme activity of the β-galactosidase OUC-JST is not less than 50% after being stored at 4℃ for 200 h.

3. The wide-temperature-range low-temperature β-galactosidase OUC-JST derived from *Xanthomonas auricula-judae* according to claim 1, characterized in that, The β-galactosidase OUC-JST, when treated in milk at 10°C with an enzyme dosage of 1 U / mL for 12 hours, achieves a lactose hydrolysis rate of not less than 89%.

4. The gene encoding the wide-temperature-range low-temperature β-galactosidase OUC-JST derived from *Xanthomonas juvenilee* as described in any one of claims 1-3.

5. The gene according to claim 4, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID No.

2.

6. A recombinant expression vector containing the gene of claim 4 or 5.

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

8. A method for preparing the wide-temperature-range low-temperature β-galactosidase OUC-JST derived from *Xanthomonas juvenilee* as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Construct a recombinant expression vector containing the gene described in claim 4 or 5; (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 β-galactosidase OUC-JST; (4) Purify the β-galactosidase OUC-JST expressed in step (3).

9. The application of the wide-temperature-range low-temperature β-galactosidase OUC-JST derived from *Xanthomonas juvenilee* as described in any one of claims 1-3 in the low-temperature hydrolysis of lactose.

10. The application as described in claim 9, characterized in that, The application involves adding β-galactosidase OUC-JST to the dairy product to be treated and carrying out a hydrolysis reaction under low-temperature conditions; the low-temperature conditions are 0-30℃; and the dairy product is cow's milk.

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