阅读说明：本技术 一种源于双歧杆菌的β-半乳糖苷酶及其应用 (Beta-galactosidase derived from bifidobacterium and application thereof ) 是由 谢新强 杨双红 杜明珠 张菊梅 吴清平 蔡淑珍 蒋同 梁婷婷 李滢 柏建玲 陈玲 于 2021-09-07 设计创作，主要内容包括：本发明提供一种β-半乳糖苷酶,所述β-半乳糖苷酶的氨基酸序列如SEQID NO:1所示；编辑如SEQ ID NO:1所示的基酸序列的基因,其核苷酸序列如SEQ ID NO:2所示。本发明的β-半乳糖苷酶最适温度为45℃,最适pH＝5.5,在35～50℃都能具有良好的酶活性,良好的pH(4～9)耐受性。水解oNPG,比活力为3826.28±137.56U/mg,Km为1.75±0.2mmol/L,kcat为5127±183s-1,kcat/Km为2928mM·s-1。本发明的β-半乳糖苷酶B17-2在牛乳中降解乳糖的比活力为2.34±0.19U/mg,在酸性乳清的比活力为4.84±0.78U/mg。(The invention provides beta-galactosidase, wherein the amino acid sequence of the beta-galactosidase is shown as SEQ ID NO. 1; the gene of the amino acid sequence shown as SEQ ID NO. 1 is edited, and the nucleotide sequence is shown as SEQ ID NO. 2. The optimum temperature of the beta-galactosidase is 45 ℃, the optimum pH value is 5.5, the beta-galactosidase has good enzyme activity at 35-50 ℃, and the pH (4-9) tolerance is good. The hydrolyzed oNPG has the specific activity of 3826.28 +/-137.56U/mg, the Km of 1.75 +/-0.2 mmol/L, the kcat of 5127 +/-183 s-1 and the kcat/Km of 2928mM s-1. The specific activity of the beta-galactosidase B17_2 for degrading lactose in cow milk is 2.34 plus or minus 0.19U/mg, and the specific activity in acid whey is 4.84 plus or minus 0.78U/mg.)

1. The beta-galactosidase is characterized in that the amino acid sequence of the beta-galactosidase is shown as SEQ ID NO. 1.

2. The beta-galactosidase according to claim 1, wherein the gene having the amino acid sequence shown in SEQ ID NO. 1 is edited, and the nucleotide sequence thereof is shown in SEQ ID NO. 2.

3. An editing beta-galactosidase gene, characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

4. A recombinant expression vector is characterized in that the vector contains a nucleotide sequence shown as SEQ ID NO. 2.

5. A recombinant bacterium comprising the recombinant expression vector according to claim 4.

6. An enzyme preparation comprising the β -galactosidase of claim 1.

7. Use of a beta-galactosidase according to claim 1 for decomposing compounds having beta-1, 4-D-galactose residues in a dairy product or an acidic whey.

Technical Field

The invention belongs to the technical field of protease, and particularly relates to beta-galactosidase derived from bifidobacteria and application thereof.

Background

Beta-galactosidase is involved in catalyzing the hydrolysis of terminal beta-1, 4-D-galactose residues in beta-D-galactoside, such as lactose. Meanwhile, it can also participate in transglycosylation reaction to generate galacto-oligosaccharide and fructo-oligosaccharide. Carbohydrate active enzymes are classified into five different families according to their sequence, activity and structure in the database of carbohydrate active enzymes (GH1, GH2, GH35, GH42 and GH 59). Beta-galactosidase is widely used in the food industry to improve the flavour of dairy and baked products. It can also convert whey into useful products such as ethanol and sweet syrups, or solve the problem of whey processing by converting whey into lactic acid. In addition, its ability to produce colored products in chemical reactions makes it increasingly important in molecular biology.

For humans, β -galactosidase is an important glycoside hydrolase in the human body, which is responsible for the hydrolysis of dietary lactose to produce galactose and glucose. Some people normally produce beta-galactosidase in the intestine, so that they can normally digest lactose. While some particular populations, especially infants whose food is derived primarily from breast milk and lactose-enriched formula, not only do their intestinal tracts become lactose intolerant due to beta-galactosidase deficiency, but they also do not benefit the growth and development of the infant. Furthermore, lactose intolerant populations in europe and the united states are reported to be between 2% and 70%. The increasing world population suffering from lactose intolerance has led to an increasing market demand for lactose-free dairy products. In addition the market still needs beta-galactosidase with a wider range of thermal and ph stability to suit industrial down stream processes. Although the production of beta-galactosidase from microorganisms has been widely studied, many of them cannot be used directly in the food industry. However, beta-galactosidase derived from probiotic microorganisms is safe for human use, and therefore, the excavation of beta-galactosidase derived from probiotic bacteria is a technical problem to be solved by those skilled in the art.

Disclosure of Invention

In order to solve the above technical problems, the present invention aims to provide a beta-galactosidase derived from bifidobacterium and having a high enzymatic activity.

The invention adopts the following technical scheme to realize the purpose of the invention:

the first purpose is to provide beta-galactosidase, wherein the amino acid sequence of the beta-galactosidase is shown as SEQ ID NO. 1.

The beta-galactosidase in the present invention is derived from bifidobacterium and its primary structure consists of 719 amino acids. Where E161 and E321 are critical catalytic sites, R122 and N160 may be binding sites, Y170 may be a metal binding site, and N332 may be a catalytic site. The beta-galactosidase of the invention has good pH tolerance, can keep 80% or more of activity within the range of pH 4.0-9.0, and has specific activity of 3826.28U/mg. The specific activity of the beta galactosidase for degrading lactose in cow milk is 2.34 plus or minus 0.19U/mg, and the specific activity in acid whey is 4.84 plus or minus 0.78U/mg. The beta-galactosidase of the invention is proved to have excellent enzymological properties.

Preferably, the gene editing the amino acid sequence shown as SEQ ID NO. 1 has the nucleotide sequence shown as SEQ ID NO. 2.

The second purpose of the invention is to provide a gene, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

The third purpose of the invention is to provide a recombinant expression vector, which contains a nucleotide sequence shown as SEQ ID NO. 2.

The fourth purpose of the invention is to provide a recombinant bacterium, wherein the recombinant bacterium contains the recombinant expression vector.

A fifth object of the invention is to provide an enzyme preparation comprising the beta-galactosidase of the invention. The beta-galactosidase of the invention can not only decompose beta-1, 4-D-galactose residue compounds in dairy products, but also decompose beta-1, 4-D-galactose residue compounds in acidic whey, and has certain acid resistance.

The sixth purpose of the invention is to provide the application of the beta-galactosidase in decomposing beta-1, 4-D-galactose residue compounds in dairy products or acidic whey.

The invention has the beneficial effects that: the beta-galactosidase of the invention has an optimum temperature of 45 ℃ and an optimum pH of 5.5, has good enzyme activity at 35-50 ℃, has good pH tolerance, and can almost maintain 80% or more of activity particularly in the pH range of 4.0-9.0. The specific activity of the hydrolyzed oNPG is 3826.28 +/-137.56U/mg, Km is 1.75 +/-0.2 mmol/L, kcat is 5127 +/-183 s-1, and kcat/Km is 2928mM s-1. The specific activity of the beta-galactosidase B17_2 for degrading lactose in cow milk is 2.34 plus or minus 0.19U/mg, and the specific activity in acid whey is 4.84 plus or minus 0.78U/mg.

Drawings

FIG. 1 shows the expression electrophoresis of recombinant protein B17_2, wherein M: protein molecular weight Marker

FIG. 2B17_2 protein sequence alignment in NCBI database

FIG. 3B17_2 is a graph showing the conserved amino acid residue positions in known alignments with the sequence of Bifidobacterium beta-galactosidase

FIG. 4B17_2 is a graph of key amino acid residue positions aligned with the beta-galactosidase sequence of Thermus thermophiles

FIG. 5 hydrolysis activity profile of recombinant beta-galactosidase B17_2 on oNPG

FIG. 6 enzyme kinetic fitting graph of recombinant beta galactosidase B17_2

Detailed Description

In order to show technical solutions, purposes and advantages of the present invention more concisely and clearly, the technical solutions of the present invention are described in detail below with reference to specific embodiments. Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1: obtaining beta galactosidase (referred to as: B17_2 in the invention)

1. Construction of recombinant expression vector for β -galactosidase B17_ 2:

according to the sequence characteristics of the gene and an expression vector pET28a, online software NEBcuter V2.0 and GenScript are adopted for primer design:

PrimerF：AAAAAACATATGACTACTCGTAGAACGTTCAGG

PrimerR：GTGGTGCTCGAGTTAGCAGGACGTTTTAGCG

using genome of Bifidobacterium strain B.longum020402 as templateHigh-Fidelity DNA polymers (New England Biolabs) were obtained by PCR amplificationB17_2, the nucleotide sequence of the fragment is shown as SEQ ID NO:2, and the PCR system is as follows:

TABLE 1

The PCR reaction conditions are pre-denaturation at 95 ℃ for 5min, melting at 95 ℃ for 30s, annealing at 72 ℃ for 2min, extension at 72 ℃ for 10min, and 32 cycles.

Plasmid pET28a was double digested simultaneously with the purified PCR product. The enzyme cutting system is as follows: PCR purified product/plasmid 20 u L, endonuclease NdeI 2 u L, endonuclease XhoI 2 u L, buffer 5 u L, sterile water 21 u L. The digestion conditions are 37 ℃/1.5 h. And purifying the digested plasmid and the PCR product, and then connecting the digested plasmid and the PCR product by using T4 ligase under the condition of room temperature for 2 hours. The ligation product is transformed into DH5 alpha plasmid by a heat shock method to construct a recombinant strain, the recombinant strain is coated on an LB (containing kanamycin) plate, after overnight culture, a positive colony is selected, and after PCR detection, sequencing verification (Shanghai Biotechnology Co., Ltd.) is sent to compare with a gene nucleotide sequence. And selecting a recombinant plasmid without mutation, and transforming the BL21 expression strain by a heat shock method.

2. Preparation of recombinant β -galactosidase B17_ 2:

inoculating a BL21 expression strain containing a recombinant plasmid into a SB liquid culture medium containing kanamycin, performing shake culture at 37 ℃ for 4-6 h, then reducing the temperature of a shaking table to 18 ℃, continuing to culture for 30min, adding 200 mu L of IPTG with the concentration of 1mol/L, and continuing to culture for 48-72 h. The cultured bacterial solution was centrifuged at low temperature and high speed (9000 Xg, 4 ℃, 20min) to collect the bacterial cells, which were then lysed with 35mL of lysis buffer (1mol/L NaCl,50mmol/L NaH)₂PO₄,70mmol/L Na₂HPO₄and 50mmol/L imidazole, pH 7.6-7.8) in a 50mL centrifuge tube, adding 0.2g lysozyme for splitting, and then placing the mixture in a refrigerator at-80 ℃ for freezing. After the bacterial suspension is taken out from a refrigerator at minus 80 ℃ after being frozen for 12h, the bacterial suspension is naturally thawed, and an ultrasonic crusher is used for carrying out intermittent crushing (600W, 40-50 min) until the bacterial solution is not turbid any more. The disrupted liquid was transferred to a high-speed centrifuge tube for low-temperature high-speed centrifugation (4 ℃, 24000 Xg, 30min), after which the supernatant was carefully aspirated andfiltration was performed with a 0.8 μm filter. The protein-purified nickel column, which had been stored refrigerated at 4 ℃ was removed and the column was first equilibrated with a lysis buffer (about 50 mL). And then loading the filtered protein solution onto the column at the speed of 3mL/min, and then feeding lysis solution at the speed of 5mL/min to remove the hybrid protein between the gaps of the column after the loading on the column is finished. The wash was then exchanged for the eluent (150mmol/L NaCl,150mmol/L NaH)₂PO₄,50mmol/L Na₂HPO₄and 200 mmol/limdazole, pH 7), the speed can be kept constant. Purified enzyme exchange solution (100mmol/L NaCl,100mmol/L NaH)₂PO₄ and 50mmol/L Na₂HPO₄pH 7.2). The molecular weight of the protein detected by SDS-PAGE is 75kDa, and the purity is more than 90% (figure 1).

Example 2: sequence analysis of recombinant beta-galactosidase B17_2

After analysis, the primary structure of the recombinant beta galactosidase B17_2 consists of 719 amino acids, and the specific amino acid sequence is shown in SEQ ID NO. 1. Comparison with the sequence in NCBIblastp showed that the sequence of the enzyme was already present in the database, but there was no relevant characterization. Comparison of the conserved domains of the proteins shows that the structure of the enzyme B17_2 consists of the domains of beta-galactosidase (amino acid sequence 25-398) belonging to the GH42 family and beta-galactosidase forming several polymers (amino acid sequence 411-622) (FIG. 2).

The primary sequence of B17_2 was compared with the primary sequences of other β -galactosidases of the GH42 family (fig. 3), the latter sequences belonging to the GH42 family of carbohydrate databases and to bifidobacteria. Alignment results show the presence of a series of conserved amino acid residues, P28, D39, a46, G47, N49, W58, D75, D78, T93, P97, W99, P106, G114, G120, R122, P130, N160, E161, N193, a195, W201, P214, F235, D238, T262, D288, Y290, W329, G339, a350, G352, D354, E369, G393, W424, D457, P460, Y468, P474, G494, G495, D507, P518, G530, a601, G610, L619, and G658, which are 100% conserved. Of these absolutely conserved amino acid residues, E161 is a key catalytic site, and E321 is also a key catalytic site. Furthermore, comparison with ABI35985.1(β -galactosidase from Thermus thermophilus) showed that R122 and N160 could be binding sites, Y170 could be a metal binding site, N332 could be a catalytic site (fig. 4), while ABE95118.1 was 86.90% similar to B17 — 2.

Example 3: hydrolytic activity of recombinant beta galactosidase B17_2

The amount of enzyme that releases 1. mu. mol ONP (o-nitrophenol) within 1min upon hydrolysis of ONPG (o-nitrophenyl beta-D-galactopyranoside, as substrate for beta-galactosidase) was taken as an activity unit of 1U. Group beta-galactosidase B17_2 and commercial beta-galactosidase (Diamond, China), the enzyme concentrations of which are 0.87 mug/mL and 0.044mg/mL respectively, were added into a PBS buffer solution reaction system containing 2mmol/L ONPG, and reacted at different temperatures, and the influence of temperature on the enzyme activity was determined. The recombinant beta-galactosidase B17_2 and the commercial beta-galactosidase are added into a buffer solution reaction system containing oNPG with different pH values of 2mmol/L to react at the optimal temperature, and the influence of the pH value on the enzyme activity is measured. Under the condition of optimum pH, the recombinant beta-galactosidase B17_2 and the commercial beta-galactosidase are incubated at different temperatures for different times, and then added into a reaction system containing 2mmol/L of oNPG solution, and the temperature stability of the enzymes is measured at the optimum temperature. Under the condition of optimal temperature, recombinant beta-galactosidase B17_2 and commercial beta-galactosidase are added into buffer solutions with different pH values and are kept warm for 30min, and then are added into reaction systems of respective optimal pH buffer solutions containing 2mmol/L oNPG, and the acid-base tolerance of the enzyme is measured. A standard curve was prepared by using the absorbance at 410nm of oNP in PBS buffer.

The results showed that β -galactosidase B17 — 2 had an optimum temperature of 45 ℃ and an optimum pH of 5.5 (fig. 5A, B), had good pH tolerance (residual enzyme activity was measured after incubating the zymogen solution with buffer solutions of different pH values at 37 ℃ for 30min), and was able to maintain almost 80% or more activity particularly in the pH range of 4.0 to 9.0 (fig. 5E). Beta-galactosidase B17_2 hydrolyzed ONPG (FIG. 6A), with specific activity of 3826.28 + -137.56U/mg, Km of 1.75 + -0.2 mmol/L, kcat of 5127 + -183 s-1, kcat/Km of 2928 mM. multidot.s-1. Whereas commercial β -galactosidase had an optimum temperature of 45 ℃, an optimum pH of 7.0 (fig. 5A, B), was sensitive to the temperature of the enzyme reaction (fig. 5C) and was not pH tolerant. Commercial β -galactosidase hydrolyzed ONPG (FIG. 6B), specific activity 57.02 + -1.50U/mg, Km 0.34 + -0.04 mmol/L.

Example 4: degradation rate of recombinant beta-galactosidase B17_2 to lactose in cow milk and acidic whey

Recombinant beta galactosidase B17_2, added into cow's milk or acidic whey, reacted at 45 deg.c for 10 min. The enzyme activity is determined by a3, 5-dinitrosalicylic acid DNS method. The amount of enzyme that hydrolyzes lactose at 1min to yield 1. mu. mol of reducing sugars was taken as one activity unit of 1U.

The results show that the specific activity of recombinant beta galactosidase B17_2 for degrading lactose in cow's milk is 2.34 plus or minus 0.19U/mg, and the specific activity in acid whey (pH 3.6) is 4.84 plus or minus 0.78U/mg.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

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