A beta-galactosidase mutant with high transglycosylation activity and thermal stability and application thereof

Abstract

This invention relates to a β-galactosidase mutant with high transglycosylation activity and thermostability, and its applications, belonging to the fields of enzyme engineering and genetic engineering. This invention involves molecular modification of β-galactosidase derived from *Kluyveromyces lactis* and screening for hydrolytic activity, transglycosylation activity, and half-life at 40°C, resulting in β-galactosidase mutants V842G and L764T / V842G with significantly improved catalytic activity and thermostability. Compared to the wild-type enzyme, the mutant enzymes exhibit catalytic efficiency increased by 1.5–4.85 times, specific enzyme activity increased by 88.9 U / mg–92.3 U / mg, transglycosylation efficiency increased by up to 79.7%, and half-life at 40°C increased by 54.7%–101.5%. The β-galactosidase mutants of this invention show excellent performance in lactose degradation and galactooligosaccharide (GOS) synthesis.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and genetic engineering, and in particular to a β-galactosidase mutant with high transglycosylation activity and thermostability and its applications. Background Technology

[0002] β-galactosidase (β-gal, EC 3.2.1.23) is a glycoside hydrolase that specifically catalyzes the hydrolysis of the glycosidic bond between the terminal non-reducing β-D-galactosidic unit and the glycosidic aglycone in lactose. Simultaneously, it catalyzes the synthesis of galacto-oligosaccharides (GOS) through kinetically controlled lactose galactosylation. It has wide applications in the food, pharmaceutical, environmental, and other industries. For example, β-galactosidase can be used not only to construct polysaccharide blocks of key glycoconjugates to elucidate their biological functions but also for the glycosylation of important molecules. GOS synthesized by β-galactosidase can further stimulate the growth of beneficial intestinal bacteria, contributing to the regulation of gut microbiota balance and the treatment of inflammation and cancer. Furthermore, β-galactosidase can be used to process the large quantities of lactose-containing byproducts generated during dairy production, such as whey and whey permeate. Compared to chemical methods, the β-galactosidase-catalyzed reactions exhibit high specificity, safety, health benefits, and environmental friendliness.

[0003] Compared to β-galactosidases from other microbial sources (such as Escherichia coli, Aspergillus, and Bacillus), β-galactosidases from Kluyveromyces lactis have several advantages in terms of enzymatic properties, safety, and industrial adaptability, making them more suitable for the food industry, especially for the production of lactose-free or low-lactose dairy products. Kluyveromyces lactis is a naturally occurring yeast found in traditional dairy products, and its enzymes have received GRAS (Generally Recognized As Safe) status from numerous international regulatory agencies, including the US FDA. Furthermore, the optimal pH for β-galactosidases from Kluyveromyces lactis is typically highly consistent with the natural pH of fresh milk, whey, and other dairy products, and the optimal operating temperature is usually around 35℃-40℃. This temperature is ideal for enzymatic hydrolysis reactions without destroying the nutritional components of milk. Therefore, in commercial applications, especially in the dairy industry, β-galactosidases from Kluyveromyces lactis are among the most widely used and have the largest market share, with representative products such as Maxilact. TM It has been widely used by companies such as DSM in lactose-free dairy products.

[0004] β-galactosidase from Kluyveromyces lactis has been successfully produced through microbial fermentation. Recombinant expression offers advantages such as high purity and specificity. However, the resulting β-galactosidase exhibits limited hydrolytic activity, thermal stability, and transglycosylation activity, making it unsuitable for many industrial applications. Therefore, this study aims to provide a method to improve the catalytic activity and thermal stability of β-galactosidase for low-cost industrial applications, which is of significant value for the production and application of β-galactosidase. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problems of poor catalytic activity and poor thermal stability of β-gal molecules derived from Kluyveromyces lactis in the prior art, and to provide a molecularly modified β-galactosidase mutant.

[0006] To solve the above-mentioned technical problems, the present invention provides a β-galactosidase mutant, the amino acid sequence of which is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0007] In one embodiment of the present invention, the β-galactosidase mutant is obtained by mutating wild-type β-gal from Kluyveromyces lactis. The mutation is based on the amino acid sequence shown in SEQ ID NO.1, replacing valine at position 842 with glycine, or replacing leucine at position 764 with threonine and replacing valine at position 842 with glycine.

[0008] A second objective of this invention is to provide a nucleic acid molecule encoding the β-galactosidase mutant.

[0009] A third objective of the present invention is to provide a gene expression cassette or recombinant plasmid carrying the nucleic acid molecule.

[0010] Furthermore, the gene expression cassette or recombinant plasmid contains elements necessary for gene expression, such as promoters, terminators, RBS, etc.

[0011] A fourth objective of this invention is to provide recombinant cells expressing the β-galactosidase mutant.

[0012] Furthermore, the host of the recombinant cells includes bacteria, fungi, or animal cells.

[0013] Furthermore, the host of the recombinant cells is preferably Escherichia coli or Kluyveromyces lactis.

[0014] A fifth objective of this invention is to provide the application of the β-galactosidase mutant, nucleic acid molecule, gene expression cassette, recombinant plasmid, or recombinant cell in the preparation of lactose-degrading products.

[0015] A sixth object of the present invention is to provide the use of the β-galactosidase mutant, nucleic acid molecule, gene expression cassette, recombinant plasmid, or recombinant cell in the preparation of lactose-free or low-lactose dairy products.

[0016] A seventh object of the present invention is to provide the use of the β-galactosidase mutant, nucleic acid molecule, gene expression cassette, recombinant plasmid, or recombinant cell in the preparation of galactooligosaccharides.

[0017] In one embodiment of the present invention, galactooligosaccharides are synthesized using lactose as a substrate and a mutant enzyme is used as a catalyst to react at 30-50°C and pH 7.4.

[0018] In one embodiment of the invention, the galactooligosaccharide comprises two to eight sugar units, including galactose and glucose.

[0019] An eighth object of the present invention is to provide a method for improving β-galactosidase activity, transglycosylation activity, or thermostability, comprising the step of mutating amino acid position 842 of the β-galactosidase as shown in SEQ ID NO.1, or simultaneously mutating amino acids position 764 and 842.

[0020] Alternatively, it may include the step of mutating the site homologous to position 842 on the amino acid sequence shown in SEQ ID NO.1 in the starting sequence of the β-galactosidase to be modified, or simultaneously mutating the sites homologous to positions 764 and 842.

[0021] The amino acid resulting from a mutation at position 842 or its homologous site is glycine, and the amino acid resulting from a mutation at position 764 or its homologous site is threonine.

[0022] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0023] This invention involves molecularly modifying β-galactosidase from Kluyveromyces lactis by mutating its 762 and 842 sites, resulting in mutant V842G with improved hydrolytic activity (enzyme activity) and thermostability, as well as combined mutant L764T / V842G with improved catalytic activity (hydrolytic activity and transglycosylation activity) and stability. Compared to the catalytic efficiency of wild-type β-galactosidase, the β-galactosidase mutant V842G provided by this invention exhibits 1.5 times the catalytic efficiency of the wild type, with a specific enzyme activity increased to 88.9 U / mg. Its half-life at 40℃ reaches a maximum of 25.8 min, representing a 101.5% improvement over the wild type. The combined mutant L764T / V842G demonstrates 4.85 times the catalytic efficiency of the wild type, with a specific enzyme activity increased to 92.3 U / mg. Its transglycosylation efficiency is 79.7% higher than the wild type, and its half-life at 40℃ reaches a maximum of 19.8 min, representing a 54.7% improvement over the wild type. These improvements significantly enhance the catalytic activity and thermal stability of β-galactosidase, solving the problems of low catalytic efficiency and poor thermal stability, thus laying the foundation for its industrial application. Attached Figure Description

[0024] 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.

[0025] Figure 1 The specific enzyme activity of β-gal and its mutants.

[0026] Figure 2 This refers to the transglycosylation activity of β-gal and its mutants.

[0027] Figure 3 The temperature stability diagram of β-gal and its mutants; temperature stability of β-gal and mutants at 40°C, with the original activity of unincubated β-gal or mutants at 100%.

[0028] Figure 4 The image shows the high performance liquid chromatography (HPLC) results of β-gal and its mutants.

[0029] Figure 5 The diagram shows the optimal conditions for transglycosylation of the mutant. Detailed Implementation

[0030] 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.

[0031] The materials and methods involved in the embodiments of the present invention are as follows:

[0032] (1) Culture medium:

[0033] LB / seed medium (g / L): tryptone 10, yeast extract 5, NaCl 10.

[0034] Fermentation medium (g / L): yeast extract 24, tryptone 12, glycerol 5, K2HPO4 16.43, KH2PO4 2.31.

[0035] (2) Amino acid sequence:

[0036] β-gal: SEQ ID NO.1

[0037]

[0038] V842G: SEQ ID NO.2

[0039]

[0040] L764T / V842G: SEQ ID NO.3

[0041]

[0042] (3) Determination of GOS content:

[0043] The reaction was carried out in the following manner: 2-4 U / mL of wild-type β-galactosidase (β-Gal) or its mutant was added to a PBS buffer (pH 7.5) solution containing 10-30% (w / v) lactose. The mixture was incubated at 30-50°C for 4 hours, followed by termination by boiling in a water bath for 10 minutes. After centrifugation, the supernatant was collected, filtered through a 0.22 μm filter, and analyzed by high-performance liquid chromatography (HPLC) equipped with a refractive index detector (RID). The GOS yield was calculated using the following formula:

[0044] GOS yield (%) = (GOS content in product / initial lactose content) × 100%

[0045] Example 1: Construction of mutant libraries

[0046] Based on computer-aided rational design, target mutants were obtained by amplification of the whole plasmid using site-directed mutagenesis primers. Mutagenesis was induced at the corresponding mutation sites using primers containing the bases corresponding to the target amino acids (Table 1, starting sequences are shown in SEQ ID NO. 1). The PET-gal series of plasmids were constructed using the pET28a(+) plasmid as the backbone. PCR was performed using the following amplification program: initial denaturation at 98°C for 3 min before 20 cycles (including 10 s at 98°C, 5 s at 55°C, and 3 min at 72°C). The PCR reaction mixture was digested with Dpn I to remove the template, and then the PCR reaction mixture was purified and recovered. Positive clones were obtained by transformation into *E. coli* JM109. Specific primer sequences are shown in Table 1.

[0047] Table 1 Primers used for mutations

[0048]

[0049] Example 2: Mutant Screening

[0050] Plasmids were extracted from the positive clones constructed in Example 1, and the plasmids were transformed into Escherichia coli BL21 to express β-gal protein.

[0051] Specifically:

[0052] (1) E. coli seed culture: positive single colonies were inoculated into LB liquid medium (containing 100 μg / ml kanamycin) and cultured at 37°C and 220 rpm for 12 h to obtain seed culture.

[0053] (2) Fermentation culture conditions of E. coli: The seed culture was inoculated into a 250 ml shake flask containing 1% fermentation medium (containing 100 μg / ml kanamycin) at a volume of 20%, and cultured at 37°C and 220 rpm until the cell density (OD600) reached 0.4-0.6. IPTG was added to a final concentration of 0.5 mM and cultured at 30°C for 18 h.

[0054] (3) Protein separation and purification: β-gal protein was separated and purified using nickel affinity chromatography. The specific process is as follows: Cell extracts from different recombinant bacteria were centrifuged at 4°C and 12000 rpm for 8 min to collect the supernatant, which was then filtered through a 0.22 μm mixed cellulose membrane to remove impurities. The sample was loaded into a nickel affinity chromatography column (Histrap 5 ml FF) pre-equilibrated with buffer A. After reequilibration, impurities were eluted with 20% buffer B. The target β-gal was eluted with 100% buffer B, followed by ultrafiltration for subsequent experimental analysis.

[0055] (4) The mutant screening steps are as follows:

[0056] (a) Determination of specific enzyme activity

[0057] Method for β-galactosidase activity assay: Dilute β-gal with pH 7.4 phosphate buffer. Take 100 μL of the appropriately diluted enzyme solution and add 500 μL of preheated o-nitrobenzene-β-D-galactopyranoside (oNPG) solution. After reacting in a 30℃ water bath for 10 min, add 200 μL of Na2CO3-EDTA solution to terminate the reaction. Measure the absorbance of the product at 420 nm using a microplate reader. In all experiments, inactivated β-gal was used as a blank control. One unit of β-gal activity (U) is defined as the amount of enzyme that releases 1 μmol of nitrophenol in 1 min. The protein concentration of the purified enzyme was determined using a kit method for calculating specific enzyme activity. Experimental data are the average of three experimental results, with the error being the standard deviation. Results are as follows: Figure 1 As shown, compared to the wild type (42.5 U / mg), the specific enzyme activity of mutant V842G increased to 88.9 U / mg; the specific enzyme activity of the combined mutant L764T / V842G increased to 92.3 U / mg.

[0058] (b) Glycoside activity

[0059] The purified enzyme solution was added to a lactose substrate solution with a final concentration of 100 mg / mL at a concentration of 0.2 mg / mL. The mixture was incubated at 40°C with shaking for 4 hours. Then, it was incubated in a 100°C water bath for 10 minutes to inactivate the enzyme protein. After cooling to room temperature, the mixture was centrifuged at 13000 rpm for 10 minutes. The supernatant was filtered through an aqueous membrane (0.22 μm) and used for high-performance liquid chromatography (HPLC) to determine the GOS yield. The results are as follows: Figure 2 As shown, the transglycosylation efficiency of mutant V842G is comparable to that of wild type; the transglycosylation efficiency of the combined mutant L764T / V842G is 79.7% higher than that of wild type.

[0060] Example 3: Analysis of the kinetic properties of mutants

[0061] (1) Thermal stability

[0062] The thermostability of recombinant β-gal is measured by the half-life (t) of the enzyme activity at 40°C. 1 / 2 The residual enzyme activity was expressed as (t0.05, min). The purified enzyme was diluted with buffer A to a protein concentration of 100 μg / ml and incubated at different temperatures. Samples were taken at 5-min intervals to determine residual enzyme activity. The residual enzyme activity was fitted and the half-life (t0.05) was calculated according to the method described in the literature (Rico-Díaz, A., Álvarez-Cao, ME., Escuder-Rodríguez, JJ. et al. Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications. Sci Rep 7, 45535 (2017). https: / / doi.org / 10.1038 / srep45535). 1 / 2 (min). The results are as follows Figure 3 As shown in Table 2, the mutant V842G has the longest half-life of 25.8 min at 40℃, which is 101.5% longer than the wild type (12.8 min); the combined mutant L764T / V842G has the longest half-life of 19.8 min at 40℃, which is 54.7% longer than the wild type.

[0063] (2) Determination of the kinetic constant of β-gal

[0064] Solutions of oNPG at concentrations of 0.5–5 mg / mL were prepared using buffer A. The catalytic rates of different oNPGs by β-gal were measured at 30°C. K was calculated using nonlinear regression fitting.M and k cat With β-gal (k cat / K M =130.21 L·g -1 s -1 Compared to the wild type, the catalytic efficiency of mutant V842G is 1.5 times that of wild type, and the catalytic efficiency of combined mutant L764T / V842G is 4.85 times that of wild type (see Table 2).

[0065] Table 2 Reaction kinetic parameters of β-gal and mutants

[0066]

[0067] Example 4: Transglycoside level analysis of mutants

[0068] The appropriately diluted enzyme solutions were incubated at 30-50°C and 150-300 mg / mL lactose substrate concentration for 4 hours, followed by high-performance liquid chromatography (HPLC) analysis to calculate their transglycosylation activity. The results are as follows: Figure 4-5 As shown, the optimal reaction temperature for all mutants was approximately 40°C, and the optimal substrate concentration was 300 mg / mL. Compared to the wild type (35.2%), the highest transglycosylation level of the hybrid mutant L764T / V842G was increased to 47.9%.

[0069] 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. A β-galactosidase mutant, characterized in that: The amino acid sequence of the β-galactosidase mutant is shown in SEQ ID NO.2 or SEQ ID NO.

3.

2. A nucleic acid molecule encoding the β-galactosidase mutant of claim 1.

3. A gene expression cassette or recombinant plasmid carrying the nucleic acid molecule of claim 2.

4. Recombinant cells expressing the β-galactosidase mutant of claim 1.

5. The recombinant cell according to claim 4, characterized in that: The host of the recombinant cells is bacteria, fungi, or animal cells; wherein the bacteria include Escherichia coli, and the fungi include Kluyveromyces lactis.

6. The use of the β-galactosidase mutant of claim 1, the nucleic acid molecule of claim 2, the gene expression cassette or recombinant plasmid of claim 3, or the recombinant cell of claim 4 or 5 in the preparation of lactose degradation products.

7. The use of the β-galactosidase mutant of claim 1, the nucleic acid molecule of claim 2, the gene expression cassette or recombinant plasmid of claim 3, or the recombinant cell of claim 4 or 5 in the preparation of lactose-free or low-lactose dairy products.

8. The use of the β-galactosidase mutant of claim 1, the nucleic acid molecule of claim 2, the gene expression cassette or recombinant plasmid of claim 3, or the recombinant cell of claim 4 or 5 in the preparation of galactooligosaccharides.

9. The application according to claim 8, characterized in that: Galacto-oligosaccharides were synthesized by reacting lactose as a substrate and a β-galactosidase mutant as a catalyst at 30-50℃.

10. A method for improving β-galactosidase activity, transglycoside activity, or thermal stability, characterized in that: The method includes the steps of mutating valine at position 842 of the β-galactosidase, as shown in SEQ ID NO.1, to glycine, or mutating leucine at position 764 to threonine and simultaneously mutating valine at position 842 to glycine.

Images