A kind of β-galactosidase and its application

Abstract

The present invention relates to a β-galactosidase and an application thereof, wherein the β-galactosidase is from Lactobacillus fermentum 9-4, wherein the gene of the β-galactosidase is composed of a gene encoding an α subunit and a gene encoding a β subunit, wherein the nucleotide sequence of the gene encoding the α subunit is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the β subunit is shown in SEQ ID NO.2. The β-galactosidase encoded by the β-galactosidase gene is composed of an α subunit and a β subunit, wherein the amino acid sequence of the α subunit of the enzyme is shown in SEQ ID NO.3, and the amino acid sequence of the β subunit is shown in SEQ ID NO.4. The β-galactosidase of the present invention has high transglycosidic activity and can be applied to the enzymatic production of galactosyl oligosaccharides, especially the application of β-galactosidase in the synthesis of galactosyl trehalose.

Description

Technical Field

[0001] The invention relates to a beta-galactosidase and an application thereof, in particular to a beta-galactosidase derived from lactobacillus fermentum 9-4 and an application thereof in synthesizing galacto-oligosaccharide and galactosyl trehalose. Background Art

[0002] β-galactosidase is a class of enzymes that have the property of hydrolyzing the β-D-(1-4) glycosidic bonds of β-D-galactoside compounds, and is commonly used to hydrolyze lactose to produce galactose and glucose. This enzyme is a functional enzyme widely found in microorganisms, plants and animal organs, and is commonly used in dairy processing. In addition, its transglycosidic properties are also commonly used in the industrial production of galactosyl oligosaccharides.

[0003] As the most common galactosyl oligosaccharide, galacto-oligosaccharide GOS is a functional oligosaccharide naturally present in animal whey and milk. It is the only oligosaccharide that can be used by eight beneficial bacteria in the human intestine, such as bifidobacteria and lactobacilli. The sweetness of galacto-oligosaccharide is low, only 20%-40% of sucrose, and the calorific value is low. It has good heat resistance and acid resistance, and can maintain high stability in the human digestive tract, and has high application value. The method of synthesizing galacto-oligosaccharide catalyzed by β-galactosidase has the advantages of simple reaction pathway, no need for other auxiliary factors, low cost of substrate lactose, and simple reaction by-products, so it has aroused widespread interest.

[0004] Galactosyl trehalose GTO is a trehalose derivative, an oligosaccharide formed by one or more galactoses connected to one trehalose, and the galactose and trehalose are mainly connected by β-(1-4) and β-(1-6) glycosidic bonds. As a derivative of trehalose, galactosyl trehalose has resistance to hydrolysis by trehalase due to the change of the chemical structure of trehalose. It is not easily decomposed into glucose in the human intestine. It has the characteristics of a low-calorie sweetener and retains the functional properties of trehalose. It is a new type of oligosaccharide with great development potential. Compared with trehalose, galactosyl trehalose shows significantly enhanced properties in terms of hygroscopicity, anti-caries, promotion of bifidobacterium growth and cryoprotection effect.

[0005] There are two methods for synthesizing galactosyl trehalose: chemical synthesis and enzymatic synthesis. In the chemical synthesis method, it is difficult to obtain C2 symmetry or achieve strict α-(1,1)-α isomer control, so it is difficult to achieve efficient chemical synthesis of trehalose derivatives. Although some pure chemical methods for desymmetrization of trehalose or for manipulating regioselective hydroxyl groups have been developed, these methods are usually time-consuming, inefficient and have low yields. In addition, the use of heavy metal catalysts and chemical reagents in chemical synthesis has toxic effects, which is not conducive to the development and production of food. Enzymatic synthesis of galactosyl trehalose, especially the use of enzymes derived from GRAS bacteria, is more environmentally friendly, efficient and has simple by-products.

[0006] Although enzymatic synthesis can efficiently produce galactosyl trehalose, there is little research on it, and the relevant properties of galactosyl trehalose produced by β-galactosidase from lactic acid bacteria have not been reported. Developing a method for enzymatic synthesis of galactosyl trehalose can effectively improve the production efficiency of galactosyl trehalose, so as to improve the application of galactosyl trehalose in food production. Summary of the invention

[0007] The purpose of the present invention is to provide a beta-galactosidase and apply the beta-galactosidase to produce galacto-oligosaccharides and galactosyl trehalose.

[0008] To achieve the above object, the technical solution of the present invention is: a gene of β-galactosidase capable of producing galactosyltrehalose is derived from Lactobacillus fermentum Lactobacillus fermentum 9-4, the β-galactosidase gene consists of a gene encoding an α subunit and a gene encoding a β subunit, the nucleotide sequence of the gene encoding the α subunit is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the β subunit is shown in SEQ ID NO.2.

[0009] The β-galactosidase encoded by the gene consists of an α subunit and a β subunit. The amino acid sequence of the α subunit of the enzyme is shown in SEQ ID NO.3, and the amino acid sequence of the β subunit is shown in SEQ ID NO.4.

[0010] The preparation method of the β-galactosidase is to introduce the nucleic acid molecule into Escherichia coli BL21 (DE3) for inducing expression to obtain the β-galactosidase.

[0011] The optimal temperature of the β-galactosidase is 45° C., the optimal pH is 7.0, the specific enzyme activity for hydrolyzing oNPG is 197.42 U / mg, and the enzyme has transglycosidic activity.

[0012] The method for producing galactosyl trehalose and galacto-oligosaccharides using the β-galactosidase comprises the following steps:

[0013] a. providing a mixture comprising: a galactose donor comprising a galactosyl group bound to a leaving group, wherein the galactose donor is lactose, and a galactose acceptor, wherein the galactosyl acceptor is trehalose or lactose;

[0014] b. the β-galactosidase having transglycosidic activity, contacting the enzyme with the mixture;

[0015] c. Incubate the mixture and the enzyme, add the enzyme in step b to the donor and acceptor mixture in step a to catalyze the transglycosylation reaction, the reaction conditions are: enzyme addition amount: 10 U / mL, reaction time: 12 h, reaction temperature: 30°C, to prepare a mixture of the galactosyl trehalose or galacto-oligosaccharide and the substrate.

[0016] d. If the reaction acceptor is trehalose, 20 U / mL trehalase is added to the product mixture and reacted at 40 °C for 12 h to remove the remaining trehalose in the substrate;

[0017] e. Pass through a 200-mesh silica gel chromatography column 30×400 mm, use n-butanol: isopropanol: acetic acid: water = 7:5:2:4 as the eluent, at a flow rate of 1 mL / min, collect the eluted samples every 10 min and number them in sequence;

[0018] f. The sample eluted in step e was subjected to TLC detection. 10 μL of the sample was spotted with an activated silica gel aluminum plate using a capillary tube. The developing solvent was n-butanol: isopropanol: acetic acid: water = 7:5:2:4. The sample was developed in a chromatography cylinder and chromatographed to 1 cm from the top of the silica gel plate. The sample was taken out and air-dried. 20% concentrated sulfuric acid was evenly sprayed on the sample. After air-drying, the sample was baked in an oven at 105°C for 10 min for color development. The glycoside products with the same migration distance were combined and concentrated and dried.

[0019] g. Place the sample in a rotary evaporator at 60°C and 60 rpm for rotary evaporation and concentrate to 10 mL to remove the organic reagent. After concentration, the sample is freeze-dried into a solid powder, which is the obtained pure galactosyl trehalose or galacto-oligosaccharide.

[0020] The leaving group of the galactosyl donor of step a is a glycosyl group.

[0021] The content of the galactosyl donor in step a is not less than 25% (W / V), and the content of the galactosyl acceptor is not less than 25% (W / V).

[0022] The beta-galactosidase is used for synthesizing galactosyl trehalose.

[0023] The beta-galactosidase is used in the enzymatic production of galactosyl oligosaccharides.

[0024] Compared with the prior art, the present invention has the following advantages and effects:

[0025] (1) For the first time, β-galactosidase from Lactobacillus fermentum 9-4 was used to produce galactosyl trehalose.

[0026] (2) An enzymatic method for synthesizing galactosyl trehalose was established using β-galactosidase from Lactobacillus fermentum 9-4, which effectively improved the production efficiency of galactosyl trehalose and was suitable for the demand for galactosyl trehalose in food production.

[0027] (3) For the first time, it was proposed to use specific trehalase to hydrolyze the mixed trehalose substrate in the galactosyltrehalose product, thereby improving the purification efficiency and helping to reduce the cost of industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the SDS-PAGE image of β-galactosidase. Figure 1 Lane 1 is the crude enzyme; Lane 2 is the purified enzyme.

[0029] Figure 2 The effect of temperature on β-galactosidase. Figure 2 A is the optimum temperature diagram of LacLM; B is the temperature tolerance diagram of LacLM.

[0030] Figure 3 The effect of pH on β-galactosidase. Figure 3 A is the optimal pH diagram of LacLM; B is the pH tolerance diagram of LacLM.

[0031] Figure 4 The generated product galacto-oligosaccharide was analyzed by TLC and HPLC. Figure 4 A in the figure is a TLC chart, lane 1 is the substrate lactose, lane 2 is the product; B is a HPLC chart.

[0032] Figure 5 TLC analysis of the galactosyltrehalose produced by the reaction. Lane 1 is the substrate lactose and trehalose; Lane 2 is the product.

[0033] Figure 6 The purified galactosyltrehalose was analyzed by TLC. Lane 1 is the product after trehalase hydrolysis; Lanes 2 and 3 are the products purified by silica gel column.

[0034] Figure 7 This is the one-dimensional NMR spectrum of galactosyltrehalose. Figure 7A and B in 1 H spectrum; C and D are 1 C spectrum.

[0035] Figure 8 This is the two-dimensional NMR spectrum of galactosyltrehalose. Figure 8 A in the figure is the HH COSY spectrum; B is the HSQC spectrum; C is the HMBC spectrum. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0037] This example provides a method for preparing β-galactosidase and producing galactosyl trehalose, using β-galactosidase with transglycosylation activity to react with a mixture of lactose and trehalose, with lactose acting as a donor to provide galactosyl, and trehalose acting as an acceptor to bind to the galactosyl.

[0038] Example 1

[0039] Preparation and purification of β-galactosidase

[0040] (1) Using the codon preference of the Escherichia coli genome, the Lactobacillus fermentum The β-galactosidase gene of 9-4 was codon optimized, and the gene sequences encoding the large subunit LacL and the small subunit LacM of β-galactosidase after optimization were shown in sequence 3 and sequence 4 in the sequence list, respectively; the optimized gene was entrusted to Shanghai Sangon Biotechnology Co., Ltd. for chemical synthesis;

[0041] (2) Integrate the optimized synthetic gene into the E. coli expression vector pET-22b (+), and transform it into competent cells BL21 (DE3). Spread the bacteria onto LB plates with a final concentration of 100 µg / mL ampicillin resistance and culture at 37 °C overnight;

[0042] (3) Select a single colony and inoculate it into a finger-shaped bottle containing LBA liquid culture medium. Incubate at 37°C and 220 rpm for 4 h until the OD 600 = 1.0; transfer all the liquid as seed liquid into 800 mL of LBA liquid medium and culture at 37°C and 220 rpm for 4 h until OD 600 =0.4-0.6, add IPTG inducer with a final concentration of 0.3 mM, and culture at 37 °C, 220 rpm for 4 h;

[0043] (4) The bacterial solution induced for 4 h was centrifuged at 4 °C and 5000 rpm for 30 min, the culture medium was discarded, and the bacterial precipitate was collected. The bacterial cells were resuspended in 50 mM phosphate buffer with pH = 7.4; 1% of 10% TritionX-100 was added to the resuspended solution, and then the cells were ultrasonically disrupted. The working parameters of the ultrasonic cell disruptor were set as follows: ultrasonic power was 40%, ultrasonic working time was 2 s, intermittent time was 2 s, ultrasonic disruption was 45 min, and the disrupted liquid was centrifuged at 4 °C and 12000 rpm for 30 min. The cell fragments were discarded, and the supernatant was the crude β-galactosidase enzyme solution. After filtering through a 0.22 µm filter membrane, it was stored in a -20 °C refrigerator.

[0044] (5) Using an AKTA protein purifier, the crude β-galactosidase enzyme solution was purified by nickel ion metal chelate chromatography. The crude enzyme was first filtered through a 0.22 μm filter and then mixed with the binding buffer for loading. The mixture was then gradient eluted with an elution buffer containing 20-500 mM imidazole. The eluate corresponding to the peak was collected and verified by SDS-PAGE.

[0045] The purification results are as follows Figure 1 :M: Marker; 1: LacLM crude enzyme; 2: LacLM pure enzyme. It can be seen that the purified LacLM has two bands at 35-48 kDa and 63-75 kDa. Since LacLM has two subunits, LacL and LacM, the theoretical molecular weights are 72.26 kDa and 35.25 kDa, respectively, and its apparent molecular weight is consistent with the theoretical molecular weight. Example 2

[0046] Determination of enzymatic properties of β-galactosidase

[0047] (1) Optimal temperature and temperature tolerance of β-galactosidase

[0048] Optimum temperature: Take 10 μL of appropriately diluted enzyme solution and mix it with 90 μL of 0.02 M oNPG with pH=7.4, and then add 100 μL of ice-cold 1 M NaCO3 solution immediately after 10 min of reaction at 30-65 ℃, with each 5 ℃ as a gradient, and terminate the reaction. Determine the absorbance at 420 nm to calculate the enzyme activity. The highest enzyme activity in the determination result is 100%, and calculate the relative enzyme activity of each determination group. Set 3 parallels for each temperature. The results are shown in Figure 2 As shown in A, the optimal temperature of LacLM is 45 ℃.

[0049] Temperature tolerance: Take 10 µL of appropriately diluted enzyme solution and keep it at 20-60 ℃ for 1 h, set a gradient every 5 ℃, mix with 90 µL 0.02M oNPG with pH=7.4, react at 45 ℃ for 10 min, and measure the enzyme activity. The enzyme activity of the untreated enzyme is 100%, and the relative enzyme activity of each group is calculated. 3 parallels are set for each temperature. The results are shown in Figure 2 As shown in B, the enzyme activity of LacLM can be stably maintained at more than 98% at 20-30 ℃, and the enzyme activity decreases rapidly at 30-50 ℃ until LacLM is completely inactivated at 55 ℃.

[0050] (2) Optimal pH and pH tolerance of β-galactosidase

[0051] Optimum pH: Prepare 0.02 M oNPG solutions with pH values ​​of 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, and 11, respectively. Take 10 µL of the appropriately diluted enzyme solution and mix thoroughly with 90 µL of oNPG solutions with different pH values, then react at 45°C for 10 min and measure the enzyme activity. The highest enzyme activity in the measurement result was taken as 100%, and the relative enzyme activity of each measurement group was calculated. Three parallels were set for each pH value. The results are shown in Figure 2. Figure 3 As shown in A, the optimum pH of LacLM is 7.0, and the enzyme activity can be maintained at more than 80% in the pH range of 6.0-7.5.

[0052] pH tolerance: Prepare buffer solutions of different pH values, dilute the enzyme solution with the buffer solutions, store at 4 °C for 1 h, then take 10 µL of the diluted enzyme solution and react at 45 °C for 10 min to measure the enzyme activity. The relative enzyme activity of each test group was calculated with the enzyme activity of the untreated enzyme as 100%, and 3 parallels were set for each pH value. The results are shown in Table 1. Figure 3 As shown in B, LacLM can maintain more than 50% activity in the range of pH 5.5-9.5, with a wide pH tolerance range. It can maintain more than 80% activity in the range of pH 6.0-9.0. Example 3

[0053] Method for producing galacto-oligosaccharides by β-galactosidase

[0054] β-galactosidase was added to 50% (W / V) lactose for catalytic reaction. The reaction conditions were as follows: enzyme amount: 10 U / mL, reaction temperature: 30°C, reaction time: 12 h, and a reaction mixture containing galacto-oligosaccharides was obtained.

[0055] The reaction product was appropriately diluted and then tested by TLC. 10 μL of the sample was spotted with a capillary tube on an activated silica gel aluminum plate. The developing agent was n-butanol: isopropanol: acetic acid: water = 7:5:2:4. The chromatography was performed in a chromatography cylinder until 1 cm from the top of the silica gel plate. The plate was taken out and dried. 20% concentrated sulfuric acid was evenly sprayed on the plate. After drying, the plate was baked in an oven at 105 °C for 10 min for color development. The results are shown in the figure. Figure 4 As shown in A, lane 1 is the substrate lactose, and lane 2 is the product. Three galacto-oligosaccharide spots can be clearly seen, namely GOS1, GOS2, and GOS3.

[0056] The reaction product was appropriately diluted and filtered through a 0.22 μm filter membrane before HPLC detection. The chromatographic column was AminexHPX-87C, Bio-Rad, 300 mm×7.8 mm; mobile phase was ultrapure water; column temperature was 75 °C; detector was a differential refractive index display; flow rate was 0.6 mL / min; injection volume was 10 μL. Figure 4 As shown in B, two galacto-oligosaccharide peaks were observed, with the elution times being 7.848 min and 9.009 min, respectively, which were not detected due to the low content of GOS3. Example 4

[0057] Production, separation and identification of galactosyltrehalose

[0058] (1) Production of galactosyltrehalose by β-galactosidase

[0059] 25% (W / V) lactose and 25% (W / V) trehalose were prepared with 0.05 M phosphate buffer with pH=7.4 as reaction substrates, and the enzyme amount was 10 U / mL. After reacting at 30 ℃ for 12 h, the mixture was boiled and inactivated, and centrifuged at 13000 rpm for 10 min at room temperature. The supernatant was the product mixture. The product mixture obtained in the above steps was appropriately diluted and then subjected to TLC detection. 10 μL of the sample was spotted with an activated silica gel aluminum plate using a capillary tube, and developed with n-butanol: isopropanol: acetic acid: water = 7:5:2:4 as the developing agent and the chromatography cylinder. The chromatography was performed to 1 cm from the top of the silica gel plate, and then taken out and dried. 20% concentrated sulfuric acid was evenly sprayed, and after drying, it was baked in a 105 ℃ oven for 10 min for color development. The results are as follows Figure 5 As shown, lane 1 is the substrate lactose and trehalose, and lane 2 is the product. Two spots of transglycosidic products can be clearly seen, namely transglycosidic product 1 and transglycosidic product 2.

[0060] (2) Separation and purification of galactosyltrehalose

[0061] A method for separating galactosyl trehalose from a product mixture comprises the following steps:

[0062] a. Trehalase hydrolysis: Take the reaction supernatant and add commercial trehalase to adjust to 20 U / mL, react at 40 ℃ for 12 h to completely hydrolyze trehalose into glucose.

[0063] b. Silica gel column chromatography: Fill 200 mesh silica gel into a 30×400 mm chromatography column and use n-butanol: isopropanol: acetic acid: water = 7:5:2:4 as the eluent. Add 20 mL of the sample obtained in step a to the chromatography column for chromatography at a flow rate of 1 mL / min. Collect the eluted samples every 10 min and number them in sequence.

[0064] c. TLC: The sample eluted from step b was subjected to TLC analysis. The results were as follows Figure 6 As shown, a single spot can be seen in lanes 2 and 3, which is the transglycosylation product 2.

[0065] d. Concentration and drying: Place the sample in a rotary evaporator at 60 °C and 60 rpm and perform rotary evaporation to concentrate it to 10 mL to remove the organic reagent. After concentration, the sample is freeze-dried into a solid powder and stored in a drying dish.

[0066] (3) Structural identification of galactosyltrehalose

[0067] The dried samples were dissolved in D2O for nuclear magnetic resonance spectroscopy analysis (NMR) using a Bruker AVANCE NEO500 NMR detector ( 1 H 500 MHz, 1 C 125 MHz), detection temperature was 27 °C, and one-dimensional NMR was performed 1 H spectrum, 1 C spectrum, two-dimensional nuclear magnetic NOESY (HH), COSY (HH), HSQC (HC), HMBC (HC) detection. One-dimensional nuclear magnetic spectrum such as Figure 7 (A, B: 1 H spectrum, C, D: 1 C spectrum), which was integrated, peak-marked and multi-analyzed. The substance is mainly composed of three monosaccharides (two α-type sugars and one β-type sugar). It is preliminarily determined that the two α-type sugars are trehalose and the β-type sugar is β-galactose. Figure 8 As shown, Figure 4 A in the figure is the HH COSY spectrum; B is the HSQC spectrum; C is the HMBC spectrum. Combined with the one-dimensional NMR spectrum, it is inferred that the structure of the substance is β-Gal(1-6)-α-Glc-(1-1)-α-Glc, that is, galactosyltrehalose connected by β, 1-6 glycosidic bonds.

[0068] The above examples demonstrate that the method can be used to produce galacto-oligosaccharides and galactosyl trehalose using the β-galactosidase, and to separate pure galactosyl trehalose.

[0069] The present invention is not limited to the above technical solution, and any improvement of the present invention, including making a variety of simple modifications to the technical solution, falls within the protection scope of the present invention.

[0070] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0071] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A β-galactosidase, characterized in that: It is composed of an α subunit and a β subunit, wherein the α subunit is shown in SEQ ID NO.1; and the β subunit is shown in SEQ ID NO.

2.

2. The gene encoding the β-galactosidase according to claim 1, characterized in that: It consists of a gene encoding an α subunit and a gene encoding a β subunit. The nucleotide sequence of the gene encoding the α subunit is shown in SEQ ID NO.3, and the nucleotide sequence of the gene encoding the β subunit is shown in SEQ ID NO.

4.

3. A method for preparing the β-galactosidase according to claim 1, characterized in that: The β-galactosidase is obtained by introducing a gene encoding an α subunit and a gene encoding a β subunit into Escherichia coli for inducing expression.

4. A method for producing galactosyl oligosaccharides using the β-galactosidase according to claim 1, characterized in that: The following steps are involved: a. providing a mixture comprising: a galactose donor comprising a galactosyl group bound to a leaving group, wherein the galactose donor is lactose, and a galactose acceptor, wherein the galactosyl acceptor is trehalose or lactose; b. the β-galactosidase having transglycosidic activity, contacting the enzyme with the mixture; c. Incubate the mixture and the enzyme, add the enzyme in step b to the donor and acceptor mixture in step a to catalyze the transglycosylation reaction, the reaction conditions are: enzyme amount: 10 U / mL, reaction time: 12 h, reaction temperature: 30 ° C, to prepare a mixture of galactosyl trehalose or galacto-oligosaccharide and substrate; d. If the reaction acceptor is trehalose, 20 U / mL trehalase is added to the product mixture and reacted at 40°C for 12 h to remove the remaining trehalose in the substrate; e. Pass through a 200-mesh silica gel chromatography column 30×400 mm, use n-butanol: isopropanol: acetic acid: water = 7:5:2:4 as the eluent, at a flow rate of 1 mL / min, collect the eluted samples every 10 min and number them in sequence; f. The sample eluted in step e was subjected to TLC detection. 10 μL of the sample was spotted with an activated silica gel aluminum plate using a capillary tube. The developing solvent was n-butanol: isopropanol: acetic acid: water = 7:5:2:

4. The sample was developed in a chromatography cylinder and chromatographed to 1 cm from the top of the silica gel plate. The sample was taken out and air-dried. 20% concentrated sulfuric acid was sprayed evenly. After air-drying, the sample was baked in an oven at 105°C for 10 min for color development. The glycoside products with the same migration distance were combined and concentrated and dried. g. Place the sample in a rotary evaporator at 60°C and 60 rpm for rotary evaporation and concentrate to 10 mL to remove the organic reagent. After concentration, the sample is freeze-dried into a solid powder, which is the obtained pure galactosyl trehalose or galacto-oligosaccharide.

5. The method according to claim 4, characterized in that The content of the galactosyl donor in step a is not less than 25% (W / V), and the content of the galactosyl acceptor is not less than 25% (W / V).

6. Use of the β-galactosidase according to claim 1 in the synthesis of galactosyltrehalose.

7. Use of the β-galactosidase according to claim 1 in the enzymatic production of galacto-oligosaccharides.

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