Bifidobacterium bifidum-derived beta-galactosidase mutant and application thereof

Abstract

The invention discloses a beta-galactosidase mutant derived from bifidobacterium bifidum and application thereof, and belongs to the technical field of enzyme engineering. The invention aims to improve the GOS synthesis yield and shorten the treatment time. The invention provides a beta-galactosidase mutant derived from bifidobacterium, and the mutant is obtained by mutating a 531 site, a 682 site, a 747 site or a 710 site by taking SEQ ID No.1 as a starting sequence. The glucoside conversion rate of the mutant W710F after reacting in a 350g / L lactose system for 5 hours reaches 45.90%, and is increased by 53.87% compared with that of a wild type. The beta-galactosidase mutant has the excellent properties of high catalytic efficiency and high yield of galactooligosaccharide, the cost of prebiotics is reduced, the conversion efficiency of producing galactooligosaccharide by using the beta-galactosidase mutant is improved, and the beta-galactosidase mutant has great industrial production and application potential and economic value.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and particularly relates to a β-galactosidase mutant derived from Bifidobacterium bifidum and its application. Background Art

[0002] Galacto-oligosaccharides (GOS) are functional oligosaccharides formed by linking one or more galactose groups with glucose or galactose through β(1-3), β(1-4) or β(1-6) bonds. GOS cannot be directly digested and absorbed by the human gastrointestinal tract, can be utilized by intestinal probiotics, and has the effects of regulating the intestinal flora, promoting mineral absorption, enhancing immunity, improving constipation, etc. In addition, GOS has positive effects in regulating blood sugar and blood lipids, as well as skin care. There are mainly four methods for the preparation of GOS: separation method, hydrolysis method, chemical synthesis method and enzyme synthesis method. The products obtained by the separation method, hydrolysis method and chemical synthesis method have complex components, difficult separation, and are prone to residual harmful substances, making it difficult to be widely applied in the food industry. Currently, commercial GOS is mainly obtained by catalyzing lactose reaction with β-galactosidase with transglycosylation activity.

[0003] β-galactosidase (β-galactosidase, EC 3.2.1.23) is an important biocatalyst widely used in food processing, pharmaceuticals and other fields, and belongs to glycoside hydrolases (GH). β-galactosidase is classified according to protein sequence similarity and belongs to GH1, GH2, GH35, GH42, and GH59. β-galactosidase has a wide range of sources, and β-galactosidase is contained in plant seeds, animal viscera and microorganisms. Among them, β-galactosidase from microorganisms has large differences in properties and a large number, which is also the focus of current research. At present, commercial β-galactosidases with high transglycosylation activity mainly come from Aspergillus oryzae, Bacillus circulans, Kluyveromyces lactis, etc. The transglycosylation process catalyzed by β-galactosidase is affected by the properties of β-galactosidase and reaction conditions, and the generated GOS components are usually relatively complex, with diverse degrees of polymerization and glycosidic bond types. At the same time, in the process of synthesizing GOS with lactose as the substrate by β-galactosidase, the synthesis and decomposition reactions of GOS occur simultaneously, and the yield of GOS will change. Therefore, the reaction time of β-galactosidase should be strictly controlled to obtain the highest GOS yield. At present, the reported synthesis of GOS by β-galactosidase has problems such as long reaction time and low production efficiency. Therefore, screening β-galactosidases with safety, high efficiency and high specificity is of great significance for GOS synthesis. Bifidobacterium is a common probiotic and is considered a microorganism of "Generally Recognized As Safe (GRAS)", and has a long application history in the field of food fermentation and is also a high-quality source of β-galactosidase. Therefore, exploring β-galactosidase derived from Bifidobacterium is of great significance for providing new options for dairy production and GOS synthesis. Summary of the Invention

[0004] The object of the present invention is to improve the yield of GOS synthesis and shorten the processing time.

[0005] The present invention provides a mutant β-galactosidase derived from Bifidobacterium, which is obtained by mutating positions 531, 682, 747 or 710 with SEQ ID NO.1 as the starting sequence.

[0006] Further defined, the mutant is any one of the following:

[0007] (1) The histidine at position 531 of the amino acid sequence shown in SEQ ID NO.1 is replaced with valine;

[0008] (2) Substitute the histidine at position 531 of the amino acid sequence shown in SEQ ID NO.1 with tyrosine;

[0009] (3) Substitute the histidine at position 682 of the amino acid sequence shown in SEQ ID NO.1 with isoleucine;

[0010] (4) Substitute the histidine at position 682 of the amino acid sequence shown in SEQ ID NO.1 with glutamine;

[0011] (5) Substitute the aspartic acid at position 747 of the amino acid sequence shown in SEQ ID NO.1 with glutamic acid;

[0012] (6) Substitute the aspartic acid at position 747 of the amino acid sequence shown in SEQ ID NO.1 with proline;

[0013] (7) Substitute the tryptophan at position 710 of the amino acid sequence shown in SEQ ID NO.1 with phenylalanine.

[0014] The present invention provides a gene encoding the above mutant.

[0015] The present invention provides a recombinant vector containing the above gene.

[0016] Further defined, the starting vector of the recombinant vector is a PET series vector.

[0017] The present invention relates to a recombinant microbial cell containing the above gene or expressing the above mutant.

[0018] Further defined, the microbial cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

[0019] The present invention provides the use of the above mutant, the above recombinant vector or the above recombinant microbial cell in the preparation of galactooligosaccharides.

[0020] The present invention provides a method for preparing galactooligosaccharides, using lactose as a substrate, adding the above β-galactosidase mutant as a catalyst, reacting in a PBS buffer at pH 6.5 to obtain galactooligosaccharides, wherein the concentration of lactose is 350 g / L, and the dosage of the β-galactosidase mutant is 20 - 40 U / g lactose.

[0021] The present invention provides the use of the above mutant, the above recombinant vector or the above recombinant microbial cell in the preparation of pharmaceutical products, foods or feeds containing galactooligosaccharides.

[0022] Beneficial effects: The present invention relates to the β-galactosidase β-gal derived from Bifidobacterium ATCC29521(The amino acid sequence shown in SEQ ID NO.1) was subjected to homology modeling to determine the β-galactosidase protein structure, and molecular docking was performed with the substrate galactose to determine the active site where the substrate binds to the enzyme protein. Multiple single-point mutant enzymes were constructed by site-directed mutagenesis. The highest GOS yields of the mutants H531V, H682I, H531Y, H682Q, D747P, and D747E were 31.25%, 35.15%, 42.18%, 40.98%, 42.67%, and 32.59% respectively. Compared with the wild type (29.83%), they were increased by 4.76%, 15.14%, 41.40%, 37.38%, 43.04%, and 9.25% respectively. The GOS yield of the mutant W710F showed an increasing trend, and the highest GOS yield was 45.90%, which was increased by 53.87% compared with the wild type. The mutants of the present invention have the excellent properties of fast catalytic efficiency and high yield of galactooligosaccharides, which are beneficial to reducing the cost of prebiotics, improving the conversion efficiency of producing galactooligosaccharides using this enzyme, and the β-galactosidase mutants have great potential for industrial production and application as well as economic value. Description of the Drawings

[0023] Figure 1 It is a three-dimensional structure result diagram; A is β-gal ATCC29521 Three-dimensional structure result diagram of homology modeling; B is β-gal ATCC29521 Three-dimensional structure evaluation result diagram;

[0024] Figure 2 It is β-gal ATCC29521 Molecular docking result diagram with the substrate lactose;

[0025] Figure 3 It is the alanine scanning mutagenesis energy result diagram of the site;

[0026] Figure 4 It is β-gal ATCC29521 Wild type and mutant COS yield result diagram;

[0027] Figure 5 Active center surface display result diagram and active center hydrophobicity analysis result diagram; A is the active center surface display result diagram of β-gal ATCC29521; B is the active center surface display result diagram of the mutant W710F; C is β-gal ATCC29521 Active center hydrophobicity analysis result diagram of the interaction with lactose; D is the active center hydrophobicity analysis result diagram of the interaction of the mutant W710F (D) with lactose; E is β-gal ATCC29521 Active center hydrophobicity analysis result diagram of; F is the active center hydrophobicity analysis result diagram of the mutant W710F. Detailed Implementation Modes

[0028] The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0029] When passing, for the molecular biology experimental methods not specifically described in the following embodiments, they are all carried out with reference to the specific methods listed in "Molecular Cloning: A Laboratory Manual", or according to the kits and product instructions; the reagents and biological materials, unless otherwise specified, can be obtained from commercial channels.

[0030] Experimental materials and reagents:

[0031] The materials and reagents used in the embodiments are as follows:

[0032] pH 6.5 phosphate buffer: 8.8 g / L potassium dihydrogen phosphate, 8.0 g / L dipotassium hydrogen phosphate trihydrate, 0.25 g / L magnesium sulfate heptahydrate, 18.6 mg / L EDTA dihydrate.

[0033] Amino acid sequence source: Bifidobacterium bifidum ATCC 29521.

[0034] Culture medium:

[0035] LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, pH 7.0.

[0036] LB solid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, 20 g / L agar powder, pH 7.0. The main test methods used in the embodiments are as follows:

[0037] β-galactosidase activity assay (ONPG method): Prepare a 2.5 mg / mL ONPG solution (the solvent is 50 mM Na2HPO4-NaH2PO4 buffer, pH 7.5). Take 1 mL of appropriately diluted enzyme solution, add 5 mL of ONPG solution (both the enzyme solution and the ONPG solution are incubated at 40 °C for 2 min), and accurately react at 40 °C for 10 min, then add 2 mL of 10% Na2CO3 to terminate the reaction, and measure the absorbance at 420 nm. Each group has three parallels, and the inactivated enzyme solution is used as the blank. The definition of enzyme activity unit is: under the above conditions, 1 mL of enzyme solution converts ONPG to generate 1 μM o-nitrophenol (ONP) per unit time, which is 1 enzyme activity unit (U).

[0038] Determination of β-galactosidase reaction products: High performance liquid chromatography (HPLC) method. Before sample injection, the sample must be filtered through a filter with a pore size of 0.22 μm. The chromatographic column is NH2P-504E (250 mm × 4.6 mm, 5 μm), the mobile phase is 70% acetonitrile + 30% ultrapure water, the injection volume is 10 μL, the flow rate is 1.0 mL / min, the column temperature is 30 °C, and the detector is a refractive index detector (RID). The external standard method is used to determine the amounts of glucose and galactose in the product based on the column retention time and peak area.

[0039] The calculation formula for the GOS yield is: GOS (%) = (initial lactose - remaining lactose - glucose - galactose) / initial lactose × 100%.

[0040] Example 1. Obtaining β-galactosidase mutation sites

[0041] I. β-gal ATCC29521 Molecular homology modeling

[0042] Use the SWISS-MODEL (http: / / swissmodel.expasy.org / ) online server to perform homology modeling on the amino acid sequence of β-gal (NCBI ID: BAQ97366.1). Use the online analysis website ProCheck() to evaluate and optimize the structural model of the homology modeling to obtain a model with higher accuracy, as ATCC29521 shown. Figure 1 shown.

[0043] II. Docking of β-gal ATCC29521 molecule with the substrate lactose molecule

[0044] Use Discovery Studio 2019 to dock β-gal ATCC29521Perform molecular docking with the substrate lactose. The specific steps are as follows: (1) Lactose pretreatment: Use the Prepare method in the Small Molecules module of Discovery Studio software. The Prepare Ligands program modifies the lactose molecule, such as adding hydrogen and balancing charges. (2) Receptor protein pretreatment: Use the Prepare Protein method in the Macromolecules module of Discovery Studio software. The Prepare Protein program modifies the receptor protein, such as removing water, adding hydrogen, and balancing charges. (3) Docking: In the Receptor-Ligand Interactions module of Discovery Studio software, use the Define and Edit Binding Site method to select the protein as the receptor and choose the binding site between the ligand and the receptor. Use the Dock Ligands (CDOCKER) program in the Dock Ligands method for docking. Select the binding conformation with the minimum Binding Energy in the docking results to obtain the key sites of the interaction between the enzyme and the substrate and the amino acid sites that may affect the enzyme catalytic efficiency. As Figure 2 shown, there are 18 amino acids within the lactose molecule range. Amino acids N127, D225, H531, E529, E589, and C1266 form hydrogen bond interactions with the lactose molecule, stabilizing the lactose molecule within the active site through hydrogen bond interactions; the indole ring on the side chain of W710 and the galactose molecule are stabilized through Π-Π interactions. In addition, amino acids F128, H504, R535, N588, M637, E679, H682, F744, D747, S1265, and G1267 form the binding pocket for the reaction of β-gal ATCC29521 with lactose.

[0045] III. Alanine scanning and virtual amino acid mutation

[0046] Alanine scanning: Using the Design Protein method in the Macromolecules module of Discovery Studio software, the Calculate Mutation Energy (Binding) program was used to perform virtual amino acid mutations based on intermolecular forces on the enzyme-substrate complex. Set Input Typed Molecule to complex:complex; set LigandChain to complex:Ligand; set Mutation Site to Single Mutations; in Mutation, all amino acids at the active center were mutated to alanine. The remaining parameters were set to their default values. The results of alanine scanning are as Figure 3 shown. The key amino acids for the binding of the enzyme and the substrate are F128, D225, H531, R535, H682, W710, and D747.

[0047] Virtual mutation: Using the Design Protein method in the Macromolecules module of Discovery Studio software, the Calculate Mutation Energy (Binding) program, set Input Typed Molecule to complex:complex; set Ligand Chain to complex:Ligand; set Mutation Site to Single Mutations; in Mutation, all the key amino acids obtained from alanine scanning were mutated to the other 19 amino acids. The remaining parameters were set to their default values. If the mutation energy is greater than 0.5 kcal / mol and the Effect is destabilizing, it indicates that the affinity decreases after mutation; if the mutation energy is less than -0.5 kcal / mol and the Effect is stabilizing, it indicates that the affinity increases and the interaction relationship is enhanced after mutation. If the mutation energy is between -0.5 and +0.5, then the Effect is neutral, that is, this mutation has no effect on the affinity. Through the analysis of the mutation energy, 9 mutants with increased affinity (shown in Table 1) were finally screened out, namely H531V, H531Y, H682I, H682Q, W710F, D747E, D747P, D747M, D747Q, that is, the histidine at position 531 was replaced by valine or tyrosine; the histidine at position 682 was replaced by isoleucine or glutamine; the aspartic acid at position 747 was replaced by glutamic acid, proline, methionine or glutamine; the tryptophan at position 710 was replaced by phenylalanine.

[0048] Table 1 Mutation energies of saturated mutations of key amino acids

[0049]

[0050] Example 2. β-gal ATCC29521 Mutant strain construction

[0051] Design the primers for site-directed mutagenesis of the whole plasmid (Table 2) and use pET28a-β-gal ATCC29521 The recombinant plasmid (pET28a connected to the gene sequence of SEQ ID NO.2) was used as a template for whole plasmid PCR. The PCR amplification system was: 1 μL of pET28a-β-gal with a concentration of 50 ng / uL ATCC29521 Recombinant plasmid, 1 μL of primer F and primer R at a concentration of 10 μM, 25 μL of 2× PrimeSTAR MAXDNA Polymerase high-fidelity DNA polymerase, and 22 μL of ultrapure water. PCR amplification conditions are: pre-denaturation at 98°C for 3 minutes, followed by temperature cycling at 98°C for 10 seconds; 60°C for 10 seconds; 72°C for 2 minutes; a total of 30 cycles, with a termination temperature of 4°C.

[0052] The whole plasmid site-directed mutagenesis PCR product was digested with DpnI for 90 minutes and purified using a common DNA product purification kit. The purified PCR product was transformed into E. coli Top10 competent cells by heat shock method and spread on LB plates containing 50μg / ml kanamycin, cultured at 37℃ for 12h, and the transformants were picked for colony PCR, restriction enzyme verification and sequencing. The correctly sequenced mutant plasmid was transformed into E. coli BL21 (DE3) competent cells, cultured on LB plates containing 50μg / ml kanamycin at 37℃ for 12h, and the transformants were picked and verified. The verified correct strains were added with glycerol and stored at -80℃.

[0053] Table 2 Site-directed mutagenesis primers

[0054]

[0055] Note: Lowercase bold fonts indicate mutant bases, which are optimized bases for E. coli.

[0056] Example 3. β-gal ATCC29521 Expression of mutant proteins

[0057] Take 10 μL of glycerol bacteria and add it to 10 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture it at 37°C and 200 rpm for 12 hours to obtain seed solution. Inoculate the seed solution into 200 mL of LB liquid medium containing 50 μg / mL kanamycin at a 1% (v / v) inoculum, and culture it at 37°C and 200 rpm until OD 600When it is at 0.6 - 0.8, add isopropyl β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.05 mmol / L and cool down to 27°C, and induce culture for 16 h under the condition of 200 rpm. After cell disruption, collect the supernatant, and perform desalting treatment with 50 mmol / L HEPES solution at pH 7.0 after nickel column affinity chromatography. Measure the enzyme activity and protein content of the enzyme solution after dialysis.

[0058] The enzyme activity was measured by the ONPG method. The definition of enzyme activity unit is: under the above conditions, 1 μmol of o-nitrophenol (ONP) generated by the conversion of ONPG by 1 mL of enzyme solution per unit time is defined as 1 enzyme activity unit (U). The process of enzyme activity measurement: Prepare 2.5 mg / mL ONPG solution (the solvent is 50 mM Na2HPO4 - NaH2PO4 buffer, pH 7.5). Take 1 mL of appropriately diluted enzyme solution, add 5 mL of ONPG solution (both the enzyme solution and the ONPG solution are incubated at 40°C for 2 min), and react precisely at 40°C for 10 min, then add 2 mL of 10% Na2CO3 to terminate the reaction. Measure the absorbance of the sample at 420 nm using a microplate reader, calculate the content of the hydrolysis product o-nitrophenol (ONP) and calculate the enzyme activity. Each group has three parallels, and the inactivated enzyme solution is used as the blank. The protein content was measured using a BCA kit.

[0059] The enzyme expression levels of the mutants are shown in Table 3. Among the 9 mutants constructed, the enzyme activities of mutant W710F and mutant D747E increased by 158.29% and 46.15% respectively; the enzyme activities of mutants H531V, H682I, H531Y, H682Q, and D747P decreased to varying degrees, and their relative enzyme activities were all greater than 10%; the enzyme activities of mutants D747M and D747Q decreased significantly, only retaining 3.37% and 4.85% of the wild-type enzyme activity. The aspartic acid at position 747 mutated to methionine or glutamine, resulting in a significant decrease in enzyme activity, which is not conducive to the actual production needs and will not be considered for subsequent use in the synthesis of GOS. When β-galactosidase uses lactose as a substrate, it can catalyze hydrolysis and transglycosylation reactions, and the two reactions coexist and are in a competitive relationship. Previous studies have shown that β-galactosidase can be directionally modified to change the transglycosylation / hydrolysis ratio and increase the yield of transglycosylation reaction products. The changes in the enzyme activities of the 9 mutants indicate that the changes in key amino acids will affect the enzyme's participation in the hydrolysis reaction of the substrate ONPG, and it is speculated that it may also change the transglycosylation reaction participated by the enzyme.

[0060] Table 3 Enzyme expression levels of mutant strains

[0061]

[0062] Example 4. Method for synthesizing GOS

[0063] In β-gal ATCC29521The optimal conditions for the synthesis of GOS by β-gal and mutants are shown in Table 4. The lactose solution was prepared using 50 mmol / L Na2HPO4-NaH2PO4 buffer at pH 7.5, and the reaction was carried out at a constant temperature in a water bath shaker for 5 h. Samples were taken every 1 h, boiled for 10 min to terminate the reaction, and after diluting the samples 10-fold, the product amount was analyzed by HPLC. The inactivated enzyme solution was used as a blank control by performing the same method.

[0064] Table 4 β-gal ATCC29521 and the conditions for the synthesis of GOS by mutants

[0065]

[0066]

[0067] β-gal ATCC29521 and the HPLC results of the enzymatic reaction products of mutants are as Figure 4 shown. The highest GOS yield of the wild type is 29.83%. Among the mutants used for the synthesis of GOS, except for mutant W710F, the GOS yields of mutants all showed a trend of first increasing and then decreasing. The highest GOS yields of mutants H531V, H682I, H531Y, H682Q, D747P, and D747E are 31.25%, 35.15%, 42.18%, 40.98%, 42.67%, and 32.59% respectively. Compared with the wild type (29.83%), they are increased by 4.76%, 17.83%, 41.40%, 37.38%, 43.04%, and 9.25% respectively. The GOS yield of mutant W710F showed an increasing trend, and the highest GOS yield was 45.90%, which was increased by 53.87% compared with the wild type.

[0068] The transglycosylation ability of mutant W710F was greatly improved, and the mechanism for the improvement of its transglycosylation ability was analyzed. First, the tertiary structure of mutant W710F was constructed. After mutating the tryptophan at position 710 to phenylalanine, the benzene ring of the side chain group of phenylalanine replaced the side chain group of tryptophan (benzene ring + indole ring), resulting in a smaller steric hindrance. The reduction of the steric hindrance formed a cavity at the bottom of the (α / β)8 barrel-shaped platform ( Figure 5 as shown by A in Figure 5 and B in ATCC29521 ), which was more conducive to the binding of large receptor molecules to the enzyme in a more flexible conformation, thus promoting the reaction and increasing the enzyme activity of mutant W710F. The above results indicate that the transglycosylation ability of the mutants obtained by semi-rational design of β-gal ATCC29521 was enhanced, and the GOS yield could be increased within a shorter reaction time. The docking results of β-gal Figure 5 and mutant W710F with lactose are as Figure 5As shown in D of [reference], after the tryptophan was mutated to phenylalanine, phenylalanine did not participate in lactose binding, resulting in the disappearance of π-π interaction. In the mutant W710F, although phenylalanine did not participate in substrate binding, its proximity to the active center made it possible to participate in the synthesis of GOS. In addition, aromatic amino acid residues could be well stacked in the hydrophobic cavity, generating hydrophobic and hydrogen bond interactions, making the structure more stable. As shown by Figure 5 D of [reference], phenylalanine changed the spatial arrangement of the amino acids in the active center, making the positions of the amino acid residues in the active center more compact, resulting in an increase in the number of amino acids in W710F that interacted with lactose. Compared with β-gal ATCC29521 , in the mutant W710F, H504 and Y638 interacted with lactose molecules through hydrogen bonds, improving the stability of the mutant W710F. At the same time, the hydrogen bonds formed by amino acids H504 and Y638 with lactose replaced the π-π interaction, thereby enhancing the binding stability between the enzyme and lactose molecules, increasing the stability of the galactose-W710F intermediate complex, which was consistent with the higher transglycosylation rate of the mutant W710F at high temperatures. In addition, as shown by Figure 5 E of [reference] and Figure 5 F of [reference], the hydrophobicity of the active center of the mutant W710F increased significantly. This was because the side chain of tryptophan contained an indole ring, which had a certain polarity. After mutating it to phenylalanine with stronger hydrophobicity, the hydrophobic side chains tended to aggregate inside the protein, forming a stronger hydrophobic core and increasing the hydrophobicity of the active center. The increase in the hydrophobicity of the active center reduced the possibility of water as a receptor substrate and also increased the competitiveness of sugar groups as substrates, facilitating the transglycosylation reaction and improving the transglycosylation ability of the mutant W710F.

[0069] In summary, the mutant of the present invention increased the GOS yield relative to the wild-type enzyme. Among them, the mutant W710F had the highest GOS production rate, reaching 45.90%. This might be related to the improvement of the transglycosylation ability of the mutant W710F and the reduction of its ability to degrade the transglycosylation product GOS. In addition, compared with other β-galactosidases, the mutant enzyme of the present invention took a shorter time to obtain the highest GOS production rate.

[0070] β-galactosidase β-gal ATCC29521 Amino acid sequence, SEQ ID NO.1:

[0071] MFIPRYYESLGHLHVGTQPNRAYYVPASTPMDTVGENRVNSDRFMLLNGDWDFKYYASI

[0072] YDLDAEVSRLRAAGRPVFYDVDFGGDDAPENADPRTAATGALAADGFTTTPVPSVWQNH

[0073] GFDRHQYTNFDYPFPFDPPFVPQDNPCGVYLCDFMHTSDPDAPCTYLNFEGVDSAFYAWV

[0074] NGEFVGYSQVSHSTSEFDVTDVLEDGVNTLAVLVLKWCDGSYQEDQDKFRMSGIFRDVY

[0075] LLDRPEYAIRDMFVHTSIWRNVDSALVEAGISDDEYDASPVDHATVDVDFAFFDDADVPV

[0076] KVQLFDEDGELVAETAAEPIDDPMAGDEDDVTVRTQPDASEADDATGTGSDDTAADDEEE

[0077] GVIESIDDVAAIDDDSTDAQAALRIASVTGTLAGGANGTGFTGDSAFAPTAHASLAVDDPH

[0078] LWTAETPYLYTIVYTTANEVITALVGIREVSVDGNVVKVNGKPIKLHGVNRHDSDPVTGPV

[0079] ISEEQLMRDLTLMKEHNVNAIRTSHYPNAPHFYDLYDRLGFYVVAEADNESHGAMRGVH

[0080] PDESDAAVNKRWNRPIADNPAWIAPTVDRAQRSVERDKNHASIIFWSMGNECAYGCTFEA

[0081] ALLWTQTFDPSRLTHYESARYVDEGQECDYSYLDVHSRMYPSVEEIDQYFSEEGPRTPDGL

[0082] RDGSNGDDGDNGVKPYVLCEFCHAMGNGPGDLEDYFTRIQRYDGLVGGFIWEWCDHAI

[0083] DRGTNAAGEREYAYGGDSGEYPHFGNFCMDGLVYPDRTPHTGLLEFKNVYRPVRVTGFD

[0084] AAAGTVTLHNYLDFLDAADAVFMTFELLVDGAETAWAAWESDPAAGATFREEHPGNYTP

[0085] SMPSIAPHGDAVVDIPAEILDAIPEAGNVTMLVKYYQATDTETLPIGFELGFDEVAVPTADPR

[0086] NQTVVAALADIADGIGSDDDVDDVADDGTDSVEGADDGANDAADAGYDDVVLADPLTV

[0087] MQTDASITVEGSTFRYVLDRRTGLFSSMSFANRSLLNRPMELNVWRAPTDNDQYIKADWI

[0088] RAQYDRAQARAYEVGVLVDEDDVIAKPESIELRADDEDAPVASFGDDAILDDGNVNAGD

[0089] VSAMATSEDGSVVVDGQVTIHATMALVAPIVQRIADIDADWTIAPDGSVALRMHVMRDTD

[0090] FPFLPRFGLRLFVPKPMRQIAYCGLGPNESYIDKRRSSYHGVFSGTPESLFEPYIKPQENGN

[0091] HHDCDWASIASDDAELLVLRAGDHAFDFQALPYTQEELTAKAHNSELKPADSTVVCVDYMQSGIGSNSCGPKLHEKYRLDDAEFDFDLVLRPQAL;

[0092] β-galactosidase β-gal ATCC29521 Gene sequence, SEQ ID NO.2:

[0093]

Claims

1. A β-galactosidase mutant derived from Bifidobacterium, characterized in that, The mutant is obtained by mutating positions 531, 682, 747 or 710 with SEQ ID NO.1 as the starting sequence.

2. The mutant according to claim 1, wherein The mutant is any one of the following: (1) The histidine at position 531 of the amino acid sequence shown in SEQ ID NO.1 is replaced with valine; (2) The histidine at position 531 of the amino acid sequence shown in SEQ ID NO.1 is replaced with tyrosine; (3) The histidine at position 682 of the amino acid sequence shown in SEQ ID NO.1 is replaced with isoleucine; (4) The histidine at position 682 of the amino acid sequence shown in SEQ ID NO.1 is replaced with glutamine; (5) The aspartic acid at position 747 of the amino acid sequence shown in SEQ ID NO.1 is replaced with glutamic acid; (6) The aspartic acid at position 747 of the amino acid sequence shown in SEQ ID NO.1 is replaced with proline; (7) The tryptophan at position 710 of the amino acid sequence shown in SEQ ID NO.1 is replaced with phenylalanine.

3. A gene encoding the mutant according to claim 2.

4. A recombinant vector containing the gene according to claim 3.

5. The recombinant vector according to claim 4, characterized in that, The starting vector of the recombinant vector is a PET series vector.

6. A recombinant microbial cell containing the gene according to claim 3 or expressing the mutant according to claim 2.

7. The recombinant microbial cell according to claim 6, wherein The microbial cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

8. Use of the mutant according to claim 1 or 2, the gene according to claim 3, the recombinant vector according to claim 4, or the recombinant microbial cell according to claim 6 or 7 in the preparation of galactooligosaccharides.

9. A method for preparing galactooligosaccharides, characterized in that, Using lactose as a substrate, adding the β-galactosidase mutant according to claim 1 or 2 as a catalyst, reacting in a PBS buffer at pH 6.5 to obtain galactooligosaccharides, wherein the concentration of the lactose is 350 g / L, and the dosage of the β-galactosidase mutant is 20 - 40 U / g lactose.

10. Use of the mutant according to claim 1 or 2, the gene according to claim 3, the recombinant vector according to claim 4, or the recombinant microbial cell according to claim 6 or 7 in the preparation of pharmaceutical products, foods or feeds containing galactooligosaccharides.

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