β-galactosidase mutant and its application

Abstract

The present invention discloses a β-galactosidase mutant and its application, whose activity provides degradation and transglycosidic function in lactose degradation, and is a protein as follows a) or b): a) the histidine at position 331 of the amino acid sequence as shown in SEQ ID NO: 1 is substituted with valine or isoleucine, or, the glutamic acid at position 469 of the amino acid sequence as shown in SEQ ID NO: 1 is substituted with valine, or, the histidine at position 517 of the amino acid sequence as shown in SEQ ID NO: 1 is substituted with isoleucine; b) the amino acid sequence in a) is substituted, deleted or added with one or more amino acids and has a protein derived from (a) with β-galactosidase activity. The mutant of the present invention has the excellent properties of fast catalytic efficiency and high yield of oligosaccharides, which is conducive to reducing the cost of prebiotics and has large industrial production and application potential and economic value.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a beta-galactosidase mutant and an application thereof. Background Art

[0002] Galactooligosaccharides (GOS) are functional oligosaccharides with varying numbers (2 to 8) of galactose attached to the non-reducing end of lactose. They are widely used in the food industry and other industries as a health food ingredient and have attracted much attention for many years. In addition to their beneficial properties, GOS also has benefits such as preventing tooth decay and regulating lipid metabolism. The safety of GOS has been widely recognized. For example, the United States has designated it as a generally recognized safe food ingredient, and my country has also approved it as a nutritional enhancer and new food ingredient. GOS has certain structural and functional similarities with human milk oligosaccharides in human milk and can be specifically utilized by intestinal microorganisms in the human body. It is currently approved for use in infant formula.

[0003] Currently, the main method for industrially producing GOS is the use of β-galactosidase to catalyze the synthesis of GOS from lactose or whey. This method, characterized by mild reaction conditions, simple operation, and high efficiency, is considered effective and suitable for large-scale production. Currently, most β-galactosidases used in industrial GOS production are derived from yeasts such as Kluyveromyces lactis, Aspergillus oryzae, and Bacillus circulans. However, most of these enzymes produce low GOS yields, ranging from 12% to 50%, insufficient to meet the needs of large-scale and efficient industrial production of GOS. Summary of the Invention

[0004] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide β-galactosidase mutants and uses thereof.

[0006] To this end, the technical solution provided by the present invention is:

[0007] A β-galactosidase mutant, wherein the activity of the β-galactosidase mutant provides a degradation function in lactose degradation, and is a protein of the following a) or b):

[0008] a) the histidine at position 331 of the amino acid sequence set forth in SEQ ID NO: 1 is substituted with valine or isoleucine, and the amino acid sequences thereof are set forth in SEQ ID NOs: 2 and 3, respectively; or, the glutamic acid at position 469 of the amino acid sequence set forth in SEQ ID NO: 1 is substituted with valine, and the amino acid sequence thereof is set forth in SEQ ID NO: 4; or, the histidine at position 517 of the amino acid sequence set forth in SEQ ID NO: 1 is substituted with isoleucine, and the amino acid sequence thereof is set forth in SEQ ID NO: 5;

[0009] b) A protein derived from (a) having a substitution, deletion or addition of one or more amino acids in the amino acid sequence in (a) and having β-galactosidase activity.

[0010] A composition containing one or more of the β-galactosidase mutants.

[0011] A DNA molecule encoding the β-galactosidase mutant. For example, the base sequence of one DNA molecule encoding the H331V mutant enzyme set forth in SEQ ID NO: 2 is shown in SEQ ID NO: 6, the base sequence of one DNA molecule encoding the H331I mutant enzyme set forth in SEQ ID NO: 3 is shown in SEQ ID NO: 7, the base sequence of one DNA molecule encoding the E469V mutant enzyme set forth in SEQ ID NO: 4 is shown in SEQ ID NO: 8, and the base sequence of one DNA molecule encoding the H517I mutant enzyme set forth in SEQ ID NO: 5 is shown in SEQ ID NO: 9. Due to codon degeneracy, any DNA molecule capable of encoding the aforementioned amino acid sequence may be used.

[0012] Preferably, the base sequence of the DNA molecule is as shown in SEQ ID NO: 6.

[0013] A recombinant vector contains the DNA molecule and a regulatory sequence for expression that is operably linked to the DNA molecule.

[0014] The enzyme mutant genetically engineered bacteria, the host cell contains the DNA molecule or the recombinant vector.

[0015] A method for producing galacto-oligosaccharides comprises the following steps: using lactose as a substrate, adding the β-galactosidase mutant according to claim 1 as a catalyst, reacting in a MOPS buffer at pH 7.5, and harvesting galacto-oligosaccharides from the reaction product after the reaction is completed.

[0016] Preferably, in the production method, the concentration of lactose is 300-400 g / L, and the dosage of the β-galactosidase mutant is 5 U / mL.

[0017] Preferably, the production method comprises the following steps: the β-galactosidase mutant is added in the form of a β-galactosidase mutant enzyme solution, and the preparation method of the β-galactosidase mutant enzyme solution comprises: culturing the enzyme mutant genetically engineered bacteria according to claim 6 in a liquid culture medium, and when the bacterial solution OD 600 When the value reaches 0.6-0.8, the temperature is lowered, isopropylthiogalactopyranoside is added to the bacterial solution for induction, and then the bacteria are disrupted and purified to obtain the β-galactosidase mutant enzyme solution.

[0018] The β-galactosidase mutant or the composition is used in the preparation of medical products, food or feed containing galactose.

[0019] The present invention has at least the following beneficial effects:

[0020] The present invention uses the β-galactosidase GH2-pBbGal2A (amino acid sequence shown in SEQ ID NO: 1) from Paenibacillus barrengarzi as a template to construct multiple single-point mutant enzymes, including H331V, H331I, E469V, and H517I. After optimizing reaction conditions, H331V achieved a maximum yield of 76.3% in producing galacto-oligosaccharides. Experimental results showed that the mutants significantly improved the reaction rate and galacto-oligosaccharide yield of the enzyme. These mutants exhibit high catalytic efficiency and high galacto-oligosaccharide yield, which helps reduce the cost of prebiotics and improves the conversion efficiency of galacto-oligosaccharide production using the enzyme. These β-galactosidase mutants have significant potential for industrial production and application, as well as economic value.

[0021] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram showing the results of SDS-PAGE electrophoresis analysis of protein purification of mutants such as β-galactosidase pBbGal2A H331V in the examples of the present invention.

[0023] Figure 2 This is a graph showing the HPLC analysis results of GOS synthesis by the wild type and different mutants of β-galactosidase pBbGal2A in the examples of the present invention. DETAILED DESCRIPTION

[0024] The present invention is described in further detail below so that those skilled in the art can implement the invention with reference to the description.

[0025] It should be understood that terms such as “having,” “including,” and “comprising” used herein do not prescribe the existence or addition of one or more other elements or combinations thereof.

[0026] The present invention uses Paenibacillus barrenguzzi β-galactosidase (pBbGal2A) to synthesize GOS with a yield of 47.9%. We rationally designed the wild-type strain of pBbGal2A and further obtained the H331V mutant with a GOS yield of up to 76.3%. Compared with the wild-type, the GOS yield was increased by 28.4%, which is the highest level reported so far. This provides a new and feasible solution for the industrial application of efficient enzymatic synthesis of GOS.

[0027] The present invention utilizes conventional techniques and methods used in the fields of genetic engineering and molecular biology. These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art may employ other conventional methods, experimental protocols, and reagents in the art based on the technical solutions described herein, without being limited to the specific embodiments of the present invention. For example, the present invention may utilize the following experimental materials and reagents:

[0028] Strains and vectors: Escherichia coli DH5α Trans1-T1 (Quanshijin, Beijing, China) was used for gene cloning; Escherichia coli T7 Express (Bomaide Gene, Beijing, China) was used for protein expression; and the vector pET-28a(+) with a T7 promoter (Novagen) was used for recombinant plasmid construction.

[0029] Enzymes and kits: DNA polymerase, ligase, and DpnI enzyme were purchased from Takara, restriction endonucleases were purchased from NEB, and plasmid extraction kits and gel purification and recovery kits were purchased from Tiangen. Lactose was purchased from Sigma-Aldrich, and glucose and galactose were purchased from Solarbio. Other reagents were domestically produced (all available from general biochemical reagent companies).

[0030] Medium formula: Liquid LB medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0; solid LB medium: 0.5% yeast extract, 1% peptone, 1% NaCl, 1.5% agar powder; all of the above media were sterilized by high-pressure steam sterilization at 103 kPa, 121°C for 20 min. The solid medium was cooled to approximately 50°C, 100 mg / mL kanamycin was added, and the culture medium was poured into a plate in a laminar flow hood for later use.

[0031] β-galactosidase activity assay and definition: 75 mL of o-nitrophenyl β-D-galactopyranoside (oNPG) solution (15 mM) was added to 150 mL of MOPS buffer (50 mM, pH 7.5). After preheating in a 40°C water bath for 3 minutes, 25 mL of appropriately diluted enzyme solution was added and the mixture was reacted in a 40°C water bath for 10 minutes. The reaction was terminated by adding 750 mL of Na2CO3 solution (2 M). The absorbance was measured at 410 nm. The same method was performed with inactivated enzyme solution as a blank control. One unit of enzyme activity (U) is defined as the amount of enzyme required to generate 1 mmol of o-nitrophenol per minute under the above conditions.

[0032] β-galactosidase reaction product determination method: High-performance liquid chromatography (HPLC) was used. Samples were filtered through a 0.22 mm pore size syringe filter before loading. Column A was BP-800 Pb++ (Benson Polymeric, Reno, NE, USA), with a column temperature of 80°C, a flow rate of 0.6 mL / min, and ultrapure water as the mobile phase. The injection volume was 20 μL. Column B was Sugar-D (250 × 4.6 mm) amino column, with a column temperature of 40°C, a flow rate of 0.6 mL / min, and 73% acetonitrile in water as the mobile phase. The injection volume was 20 μL. The eluted products were monitored using a refractive index (RI) detector. The amounts of glucose and galactose in the product were determined using the external standard method based on the retention time and peak area on the Pb column, and the amount of lactose in the product was determined based on the retention time and peak area on the Sugar-D column.

[0033] The GOS yield was calculated as follows: GOS (%) = (initial lactose - remaining lactose - glucose - galactose) / initial lactose x 100%.

[0034] SEQ ID NO: 1beta-galactosidase[Paenibacillus barengoltzii]

[0035]

[0036] SEQ ID NO: 2H331V protein sequence:

[0037]

[0038] SEQ ID NO: 3H331I protein sequence:

[0039]

[0040] SEQ ID NO: 4E469V protein sequence:

[0041]

[0042] SEQ ID NO: 5H517I protein sequence:

[0043]

[0044] SEQ ID NO.6:H331V DNA sequence

[0045] >KP714731.1Paenibacillusbarengoltzii strain CAU904 beta-galactosidase genes,completecds

[0046]

[0047] SEQ ID NO: 7H331I nucleic acid sequence:

[0048]

[0049] SEQ ID NO: 8E469V nucleic acid sequence:

[0050]

[0051] SEQ ID NO: 9H517I nucleic acid sequence:

[0052]

[0053] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the following embodiments are provided for illustration:

[0054] Example 1

[0055] 1) Construction of β-galactosidase mutants: Using the recombinant plasmid pET-28a(+) carrying the wild-type β-galactosidase pBbGal2A encoding gene as a template, the corresponding primers were designed:

[0056] H331V: F': 5′-CCGCCACGATTCCGTGCCGGAGCTGGGGCAGACCAT-3′ (SEQ ID NO: 10),

[0057] R': 5'-GCCCCAGCTCCGGATGGGAATCCACGCGGTTCACTC-3' (SEQ ID NO: 11),

[0058] H331I: F′: 5′-CCGCCACGATTCCATTCCGGAGCTGGGGCAGACCAT-3′ (SEQ ID NO: 12),

[0059] R': 5′-GCCCCAGCTCCGGATGGGAATCTAAGCGGTTCACTC-3′ (SEQ ID NO: 13),

[0060] E469V: F': 5′-TTTAGTTCATTACGTGGGAGCGGATCCTCGGTATTA-3′ (SEQ ID NO: 14),

[0061] R': 5′-GAGGATCCGCTCCCACGTAATGAACTAAACGTGAGG-3′ (SEQ ID NO: 15),

[0062] H517I: F': 5′-TTGTGAATATAGCATTGCGATGGGTAACGGACCAGG-3′ (SEQ ID NO: 16),

[0063] R': 5′-CGTTACCCATCGCAATGCTATATTCACAAAGGAATA-3′ (SEQ ID NO: 17),

[0064] In vitro site-directed mutagenesis was performed by the Quickchange PCR method. The PCR products were treated with Dpn I overnight and transformed into Escherichia coli DH5α Trans1-T1 competent cells. The resulting plasmids were confirmed by DNA sequencing.

[0065] 2) Preparation of mutant enzymes: H331V-pET-28a(+), H331I-pET-28a(+), E469V-pET-28a(+), H517I-pET-28a(+) and other recombinant plasmids were heat-shocked and transformed into E. coli T7 Express competent cells to construct four genetically engineered bacteria expressing mutant enzymes. The above four engineered bacteria were respectively subjected to the following operations: inoculated into LB liquid medium at 37°C and 220rpm and cultured until OD 600 When the pH reaches 0.6-0.8, cool to 20°C, add 1 mM IPTG to the bacterial solution, induce for 2 hours, and collect the bacteria by centrifugation. Use a high-pressure homogenizer to disrupt the bacteria, centrifuge the broken bacterial solution at 12000 rpm and 4°C for 50 minutes, collect the supernatant, and elute it through Ni column affinity chromatography and gel filtration chromatography (Superdex 200) in sequence to obtain enzyme solution (5 mg / mL), which is quickly frozen in liquid nitrogen after aliquoting and stored at -80°C. Among them, the steps of Ni column affinity chromatography are: ① Column pretreatment: prepare a gravity column containing 2mL of column material for each liter of bacterial liquid, wash it with ultrapure water, and then balance the column with 10mL of lysis buffer; ② Loading: pour the supernatant of the broken bacteria after centrifugation into the affinity column. If the protein binding force is weak, it can be loaded twice; ③ Washing: use lysis buffer, washing buffer, and desalting buffer in sequence to wash away non-specifically bound proteins and high-concentration salt ions; ④ Elution: use an appropriate amount of elution buffer to elute all the proteins (using Bradford non-blueing as the standard). The steps of gel filtration chromatography are: ① Column equilibration: rinse 30mL with molecular sieve buffer; ② Loading: inject the concentrated protein into the sample loop; ③ Elution: the elution volume is 40mL, and the required target protein is collected according to the peak graph and SDS-PAGE identification. Figure 1 As shown, four mutant enzymes were obtained.

[0066] Example 2

[0067] 1) Transglycosylation reaction to synthesize GOS: A 350 g / L lactose solution was prepared in MOPS buffer (50 mM, pH 7.5), and 5 U / mL of the H331V mutant enzyme was added. The mixture was then stirred in a 40°C water bath for 24 hours. Samples were taken periodically and boiled for 10 minutes to terminate the reaction. The samples were diluted 40-fold and the product was analyzed by HPLC. The same method was performed with an inactivated enzyme solution as a blank control. GOS yield (%) = (initial lactose - residual lactose - galactose produced - glucose produced) / initial lactose × 100.

[0068] 2) Product analysis: HPLC analysis showed that the product contained 0.33 mg / ml lactose, 1.55 mg / ml glucose, and 0.51 mg / ml galactose. The calculated GOS yield was 76.3%.

[0069] Example 3

[0070] GOS were prepared using the same strategy as in Example 2, except that a different substrate concentration of 300 g / L lactose was used. HPLC analysis revealed that the product contained 0.90 mg / ml lactose, 2.51 mg / ml glucose, and 0.56 mg / ml galactose, resulting in a calculated GOS yield of 61.17%.

[0071] Example 4

[0072] GOS were prepared using the same strategy as in Example 2, except that a different substrate concentration of 400 g / L lactose was used. HPLC analysis revealed that the product contained 0.23 mg / ml lactose, 2.16 mg / ml glucose, and 1.21 mg / ml galactose, resulting in a calculated GOS yield of 64.69%.

[0073] Example 5

[0074] GOS were prepared using the same strategy as in Example 2, except that a different mutant, H331I, was used. HPLC analysis showed that the product contained 0.90 mg / ml lactose, 2.50 mg / ml glucose, and 0.56 mg / ml galactose. The calculated GOS yield was 61.17%.

[0075] Example 6

[0076] GOS were prepared using the same strategy as in Example 2, except that a different mutant, E469V, was used. HPLC analysis showed that the product contained 0.98 mg / ml lactose, 1.96 mg / ml glucose, and 0.71 mg / ml galactose. The calculated GOS yield was 64.17%.

[0077] Example 7

[0078] GOS were prepared using the same strategy as in Example 2, except that a different mutant, H517I, was used. HPLC analysis showed that the product contained 0.71 mg / ml lactose, 2.39 mg / ml glucose, and 0.79 mg / ml galactose. The calculated GOS yield was 61.75%.

[0079] Our analysis suggests that H331 is a highly conserved transition-state stabilizer in β-galactosidase, with a consistent swing compared to homologous structures. Mutating it to hydrophobic amino acids such as V and I increases the hydrophobicity of the substrate-binding pocket, reducing the likelihood of water molecules participating in the reaction and thus minimizing hydrolysis.

[0080] The presence of a flexible loop in β-galactosidase causes residue Glu469 to swing outward from its homologous protein, resulting in a more acidic substrate-binding pocket. Mutating this residue to a hydrophobic amino acid further reduces water binding to the substrate pocket and reduces the occurrence of hydrolysis.

[0081] Residue H517 is located at the entrance of the catalytic pocket, and its swing direction is 90° rotated compared with the homologous structure, which is critical for the binding of galactose residues. Because the recognition pattern of galactose at subsite-1 is a common feature of all GH2β-galactosidases, the residues located in the galactosyl binding site (-1 subsite) play a vital role in regulating the transglycosylation synthesis ability of β-galactosidase. Therefore, mutation to hydrophobic amino acids increases the hydrophobicity of the active site and also increases the production of GOS. Figure 2 shown.

[0082] The number of modules and processing scales described herein are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.

[0083] The present invention discloses a β-galactosidase mutant that produces high-glucose oligosaccharides and its application, belonging to the field of enzyme genetic engineering technology. Using the β-galactosidase GH2-pBbGal2A from Paenibacillus barrengarzi as a template, the present invention constructed multiple single-point mutants, including H331V, H331I, and H517I. After optimizing reaction conditions, H331V achieved a maximum galactose-oligosaccharide yield of 76.3%. The production conditions were MOPS buffer at pH 7.5, 40°C, 350 g / L lactose, 5 U / mL enzyme, and a reaction time of 24 hours. Experimental results showed that the enzyme's reaction rate and galactose-oligosaccharide yield were significantly improved after the mutation. The mutants described herein exhibit excellent properties, including high catalytic efficiency and high galactose-oligosaccharide yield, which can help reduce the cost of prebiotics and improve the conversion efficiency of galactose-oligosaccharide production using the enzyme. These β-galactosidase mutants have significant potential for industrial production and economic value.

[0084] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A β-galactosidase mutant, characterized in that The activity of the β-galactosidase mutant provides degradation and transglycosidation functions in lactose degradation, and is the protein of a) as follows: a) the histidine at position 331 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with valine or isoleucine, or the glutamic acid at position 469 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with valine, or the histidine at position 517 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with isoleucine.

2. A composition comprising one or more β-galactosidase mutants according to claim 1.

3. A DNA molecule encoding the β-galactosidase mutant according to claim 1 or the combination of β-galactosidase mutants according to claim 2. The DNA molecule according to claim 3 , wherein the base sequence of the DNA molecule is shown in SEQ ID NO:

6.

5. A recombinant vector comprising the DNA molecule according to claim 3 and a regulatory sequence for expression operably linked to the DNA molecule.

6. An enzyme mutant genetically engineered bacterium, wherein the genetically engineered bacterium contains the DNA molecule according to claim 3 or the recombinant vector according to claim 5.

7. A method for producing galacto-oligosaccharide, characterized in that: The method comprises the following steps: using lactose as a substrate, adding the beta-galactosidase mutant according to claim 1 as a catalyst, reacting in a MOPS buffer at pH 7.5, and harvesting oligogalactose from the reaction product after the reaction is completed.

8. The production method according to claim 7, characterized in that The concentration of the lactose is 300-400 g / L, and the dosage of the β-galactosidase mutant is 5 U / mL.

9. The production method according to claim 7, characterized in that The method comprises the following steps: adding the β-galactosidase mutant in the form of a β-galactosidase mutant enzyme solution, and the preparation method of the β-galactosidase mutant enzyme solution comprises: culturing the enzyme mutant genetically engineered bacteria according to claim 6 in a liquid culture medium, and when the bacterial solution OD 600 When the value reaches 0.6-0.8, the temperature is lowered, isopropylthiogalactopyranoside is added to the bacterial solution for induction, and then the bacteria are disrupted and purified to obtain the β-galactosidase mutant enzyme solution.

10. Use of the β-galactosidase mutant according to claim 1 or the composition according to claim 2 in the preparation of medical products, food or feed containing galactose.

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