Application of a β-galactosidase gene and an enzyme encoded thereby

Abstract

The present invention discloses the application of a β-galactosidase gene and the enzyme encoded thereby, belonging to the technical fields of genetic engineering and enzyme engineering. The present invention screened a β-galactosidase with a specific amino acid sequence and successfully carried out heterologous expression of the β-galactosidase in Escherichia coli. The method of the present invention is safe and efficient, and the catalytic activity of the crude enzyme solution of the expressed β-galactosidase can reach 12378.6 U / mg, which can be applied to the food and pharmaceutical fields. This β-galactosidase is used to convert lactose to produce galactooligosaccharides, wherein the conversion rate can reach 70.9%, and the proportion of galactooligosaccharides generated can reach 63.1%, which is beneficial to industrial production.

Description

Technical Field

[0001] The present invention relates to the application of a β-galactosidase gene and the enzyme encoded thereby, and particularly to a β-galactosidase gene for producing galactooligosaccharides and an application method thereof, belonging to the technical fields of genetic engineering and enzyme engineering. Background Art

[0002] β-Galactooligosaccharides (β-GOS) are functional oligosaccharides with a degree of polymerization of 2-8, that is, with galactose or glucose as the reducing end, and 1-7 galactose molecules are linked by β-glycosidic bonds, and the glycosidic bonds therein may be β-1,1, β-1,3, β-1,4 or β-1,6-glycosidic bonds. β-GOS has a good taste, low sweetness, high solubility and strong moisture retention, and is an excellent food sweetener.

[0003] Importantly, β-GOS has good anti-digestive properties, can resist the degradation of digestive enzymes in the small intestine, and maintain a relatively intact structure to reach the large intestine, thereby exerting many probiotic functions. Specifically, it can: (1) selectively promote the proliferation of beneficial intestinal bacteria, especially Bifidobacterium and Lactobacillus, and at the same time inhibit the growth of putrefactive bacteria (such as some Clostridium). (2) Improve the intestinal barrier function and relieve colitis. Supplementing β-GOS in the early stage of life can help infants establish a healthy colonic environment, increase the content of short-chain fatty acids (SCFAs) in the intestine, and reduce the risk of colitis; at the same time, dietary supplementation of β-GOS can also accelerate wound healing and is beneficial to the postoperative recovery of colitis. (3) Improve metabolism and delay aging. The synbiotic containing β-GOS can relieve intestinal flora imbalance and significantly enhance the antioxidant capacity of the liver, and play an anti-aging role through the liver-intestine axis. (4) Improve diabetes symptoms. Due to its excellent antioxidant capacity and the effect of balancing the intestinal flora, β-GOS has also been proven to be able to reduce the content of diabetes-related markers in the blood and delay the development of type II diabetes. In addition, toxicological studies have shown that β-GOS has no adverse effects on organisms of different ages or different organisms, and is safe and reliable. Therefore, as a prebiotic with rich nutritional value, β-GOS can be applied to dietary supplements for infant foods or special patient diet therapies, and has broad application prospects in the fields of food and medicine health. China approved the use of β-GOS as a food additive in 2008. With the increasing application market year by year, it is of great significance to study the development of the functionality of β-GOS and its production technology.

[0004] Industrially, the production of β-GOS mainly relies on enzymatic processes, that is, using β-galactosidase with transglycosylation activity to act on high-concentration lactose. As early as 1988, Japan obtained the first commercial product of β-GOS, and subsequently its preparation process was introduced into Europe, and the two monopolize the production of high-purity β-GOS at the present stage. In contrast, the production of β-GOS in China started relatively late, and the industrial scale has not reached the thousand-ton level. The main limiting factor is the lack of β-galactosidase with excellent properties. At present, the β-galactosidase for commercial preparation of β-GOS mainly has the following three sources: Aspergillus oryzae, Kluyveromyces lactis, and Bacillus circulans. Among them, the β-galactosidase from Aspergillus oryzae has a relatively low price, with a conversion rate of about 30%, and the main product is galactooligosaccharide trisaccharide (about 18%); the β-galactosidase from Kluyveromyces lactis also has a maximum conversion rate of about 30%, and the content of disaccharide in the product is the highest, but the probiotic activity of the disaccharide has not been confirmed; the β-galactosidase from Bacillus circulans has a higher conversion rate, up to about 40%, and the product is mainly trisaccharide (about 26%). It can be seen that the enzymes used in the current production of β-GOS have problems such as low conversion rate and low proportion of main products. At the same time, the transglycosylation products of β-galactosidase have a complex composition, containing different types of glycosidic bonds, which increases the difficulty of subsequent separation and is not conducive to the functional research of β-GOS with different structures. Therefore, in order to reduce the preparation and separation costs, it is an important research direction to find β-galactosidase with high transglycosylation efficiency and strong product specificity. Summary of the Invention

[0005] The purpose of the present invention is to make up for the deficiencies of the current enzymatic synthesis of β-GOS, and provides a gene encoding β-galactosidase, which is derived from Paenibacillus macquariensis, and the nucleotide sequence is as shown in SEQ ID NO.1.

[0006] The present invention also provides a β-galactosidase encoded by the above nucleotide sequence, and its amino acid sequence is as shown in SEQ ID NO.2.

[0007] The present invention also provides a recombinant plasmid carrying the above β-galactosidase gene.

[0008] In one embodiment, the recombinant plasmid uses the Escherichia coli expression plasmid pET-20b(+) as a vector.

[0009] The present invention also provides a microbial cell carrying the above β-galactosidase gene or the above recombinant plasmid.

[0010] In one embodiment, the microbial cell is recombinant Escherichia coli.

[0011] Preferably, the recombinant Escherichia coli uses Escherichia coli BL21(DE3) as the expression host.

[0012] In one embodiment, the method for constructing the recombinant Escherichia coli is as follows: Using the seamless cloning method, splice the β-galactosidase gene with the nucleotide sequence shown in SEQ ID NO.1 onto the expression vector pET-20b(+), construct the recombinant plasmid pmgal / pET-20b(+), and transform it into E. coli BL21(DE3).

[0013] The present invention also provides a method for producing β-galactosidase. The method uses lactose as a substrate and uses β-galactosidase with the amino acid sequence shown in SEQ ID NO.2 to catalyze the substrate to generate galactooligosaccharides.

[0014] In one embodiment, the β-galactosidase is added in an amount of not less than 500 U / g of the substrate.

[0015] Preferably, the β-galactosidase is added in an amount of 500 - 1000 U / g of the substrate.

[0016] More preferably, the β-galactosidase is added in an amount of 1000 U / g of the substrate.

[0017] In one embodiment, the substrate is lactose, and the lactose concentration is 200 - 400 g / L.

[0018] Preferably, the lactose concentration is 400 g / L.

[0019] In one embodiment, the reaction is carried out at 45 - 55 °C and pH 5.0 - 7.0 for 48 - 72 h.

[0020] Preferably, the reaction is carried out at 50 °C.

[0021] More preferably, the reaction is carried out at pH 6.5 and 50 °C for 60 h.

[0022] In one embodiment, the nucleotide shown in SEQ ID NO.1 is ligated to an expression vector and transferred into Escherichia coli to obtain recombinant Escherichia coli.

[0023] In one embodiment, the fermentation is to inoculate a certain amount of recombinant cells or recombinant Escherichia coli into an LB medium containing ampicillin, culture at 37 °C until the logarithmic growth phase to prepare a seed solution, and use the seed solution for fermentation.

[0024] In one embodiment, the seed liquid is inoculated into TB medium containing ampicillin and 0 - 15% (w / v) lactose at an inoculum size of 2% - 5% (v / v), and cultured in a shaking flask at 25 - 37°C for 24 - 72 h. The supernatant obtained by centrifugation is the crude β-galactosidase enzyme solution.

[0025] The present invention also provides the application of the β-galactosidase gene, the recombinant plasmid containing the β-galactosidase gene, and the Escherichia coli expressing the β-galactosidase in the production of β-galactooligosaccharides. The application uses β-galactosidase or an enzyme preparation containing this enzyme as a catalyst, and lactose solution as a substrate to convert lactose into functional galactooligosaccharides.

[0026] In one embodiment, the enzyme preparation is the crude β-galactosidase enzyme solution or the pure enzyme obtained after separation and purification, and is added to the reaction system in the form of a solution or dry powder.

[0027] In one embodiment, the concentration of the lactose solution is 200 - 400 g / L.

[0028] The beneficial effects of the present invention are as follows:

[0029] (1) A β-galactosidase with a specific amino acid sequence was screened in the present invention, and its heterologous expression in Escherichia coli was successfully achieved. It can be applied in food and drug production, and the catalytic activity of the expressed β-galactosidase can reach 12378.6 U / mg;

[0030] (2) Compared with the similar enzymes reported currently, the specific enzyme activity of the β-galactosidase of the present invention is higher than that of most of the reported similar enzymes, and it can maintain high activity within a relatively wide temperature range, and can adapt to different reaction temperature conditions;

[0031] (3) Compared with most of the reported similar enzymes, the β-galactosidase of the present invention has obvious advantages in preparing galactooligosaccharides, with a higher substrate conversion rate, stronger product specificity, can effectively improve the yield of galactooligosaccharides, reduce the preparation difficulty and subsequent separation and purification cost. The substrate conversion rate can reach 70.9%, and the content of galactooligosaccharides in the product accounts for about 63.1% of the total sugar. Both the conversion rate and the content of galactooligosaccharides reach the highest level in the existing technology, and it has high industrial application value. Description of the Drawings

[0032] Figure 1 SDS-PAGE analysis for purifying recombinant β-galactosidase.

[0033] Figure 2 Thermal stability of recombinant β-galactosidase.

[0034] Figure 3Effect of pH on the thermal stability of recombinant β-galactosidase.

[0035] Figure 4 HPAEC-PAD analysis for the preparation of galactooligosaccharides using recombinant β-galactosidase. Detailed implementation manners

[0036] The method for determining the enzyme activity of β-galactosidase involved in the following examples is as follows:

[0037] Using 2-nitrophenyl-β-D-galactopyranoside (oNPG) as the substrate to evaluate the hydrolysis activity of β-galactosidase:

[0038] Prepare a 10 mmol / L oNPG solution as the substrate with a K2HPO4-KH2PO4 buffer solution (20 mmol / L, pH 5.5). Add 0.1 mL of the enzyme solution to 0.9 mL of the substrate, react at 55 °C for 15 min, then add 1 mL of 1 M Na2CO3 solution to terminate the reaction, and measure the absorbance at 420 nm. Calculate the content of oNP in the reaction system according to the o-nitrophenol (oNP) standard curve.

[0039] The enzyme activity is defined as: under certain conditions, the amount of enzyme that hydrolyzes oNPG to release 1 μmol of oNP per minute by β-galactosidase is one enzyme activity unit (U).

[0040] Calculation method of substrate conversion rate: Using a lactose solution with a concentration of 10 - 50 μg / mL as the standard product, analyze the lactose content in the system before and after enzymatic hydrolysis by HPAEC-PAD. Lactose conversion rate (%) = 100% × (lactose mass before reaction - lactose mass after reaction) / lactose mass before reaction.

[0041] Calculation method of the percentage content of galactooligosaccharides: Galactooligosaccharide content (%) = 100% × mass of galactooligosaccharides in the product / mass of all sugars in the product. Among them, the mass of galactooligosaccharides is the total mass of transfer disaccharides to pentasaccharides.

[0042] Example 1: Construction of an Escherichia coli secretion expression system

[0043] Synthesize the nucleotide sequence as shown in SEQ ID NO.1, and ligate the above sequence to the pET-20b(+) vector by homologous recombination. The PCR amplification program involved is as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 5 min, repeating 35 cycles; finally, incubation at 72°C for 10 min. The homologous recombination reaction system is: 1 μL of purified β-galactosidase fragment (50 ng / μL), 1 μL of purified pET-20b(+) vector PCR fragment (50 ng / μL), 4 μL of 5×CEⅡ Buffer, 2 μL of ExnaseⅡ, and 12 μL of ddH2O. After reacting the above homologous recombination system at 37°C for 30 min, transform it into E. coli JM109, spread it on an LB plate containing ampicillin, pick a single colony for activation, sequencing, and obtain the recombinant plasmid pmgal / pET-20b(+); transform the above recombinant plasmid into E. coli BL21(DE3) to obtain the genetically engineered bacterium pmgal / pET-20b(+) / E. coli BL21(DE3).

[0044] Example 2: Expression, isolation and purification of recombinant β-galactosidase

[0045] The specific steps are as follows:

[0046] (1) Inoculate the preservation solution of the genetically engineered bacterium prepared in Example 1 into an LB liquid medium containing 100 μg / mL ampicillin, and culture it at 37°C for 8 - 10 h to prepare a seed solution;

[0047] Transfer the above seed solution to a TB medium containing 100 μg / mL ampicillin and 0 - 15% (w / v) lactose at an inoculation amount of 2% - 5% (v / v), and culture it at 25 - 37°C and 200 r / min for 24 - 72 h. Collect the supernatant, which is the crude enzyme solution of β-galactosidase, and its enzyme activity is 11397.1 U / mL.

[0048] (2) After passing the crude enzyme solution through a 0.45 μm aqueous membrane, purify it through a nickel ion affinity chromatography column. The buffers are A (10 mmol / L Tris-HCl, 500 mmol / L NaCl, pH 7.5) and B (10 mmol / L Tris-HCl, 500 mmol / L NaCl, 500 mmol / L imidazole, pH 7.5), and the flow rate is 2 mL / min. After equilibrating the nickel column with 25 - 30 mL of buffer A until stable, load the sample, and then elute the unbound proteins in the purification column with buffer A. After the elution curve is balanced, perform gradient elution with 35% (v / v) buffer B, collect the eluate for identification, as Figure 1As shown, the protein concentration of the obtained pure β-galactosidase solution was measured, and the specific enzyme activity was calculated to be 12378.6 U / mg.

[0049] Example 3: Stability of recombinant β-galactosidase

[0050] The thermal stability of the recombinant β-galactosidase prepared in Example 2 and the effect of pH on its thermal stability were measured respectively, and the specific steps were as follows:

[0051] (1) The method for measuring the thermal stability of the recombinant β-galactosidase pure enzyme was as follows: The pure enzyme was diluted in 10 mmol / L K2HPO4-KH2PO4 buffer (pH 6.0), incubated at 50 °C, 55 °C, and 60 °C for 60 min, samples were taken at different time points to measure the enzyme activity, and the activity without incubation was taken as 100%, and the relative residual enzyme activity at different times was calculated. The results were as Figure 2 shown, and the half-life of the enzyme at 50 °C was about 50 min.

[0052] (2) To analyze the effect of pH on the thermal stability of the recombinant β-galactosidase pure enzyme, the measurement method was as follows: 10 mmol / L oNPG was prepared as a substrate with 10 mmol / L CH3COOK-CH3COOH buffer (pH 3.0 - 5.0), K2HPO4-KH2PO4 buffer (pH 5.0 - 8.0), and NaOH-Gly buffer (pH 8.0 - 10.0) respectively. The pure enzyme was incubated at 50 °C for 1 h, samples were taken at different time points to measure the residual enzyme activity, and the enzyme activity without incubation was taken as 100%, and the relative enzyme activity at different pH values was calculated. The results showed that the enzyme could maintain more than 95% activity at pH 5.0 - 6.0, as Figure 3 shown.

[0053] Example 4: Application of recombinant β-galactosidase

[0054] Galactooligosaccharides were prepared using the recombinant β-galactosidase, and the reaction process was as follows: Lactose with a concentration of 200 - 400 g / L was prepared as a substrate, the pH was adjusted to 5.0 - 7.0, and pure enzyme with a dosage of 500 - 1000 U / g substrate was added, and the reaction was carried out at 50 °C for 48 - 72 h (until the residual lactose in the solution no longer continued to degrade). After taking part of the reaction solution and terminating the reaction in a boiling water bath for 10 min, it was centrifuged at 8000 r / min for 5 min, the supernatant was taken and diluted by an appropriate multiple, and then passed through a 0.22 μm aqueous filter membrane for testing.

[0055] The contents of each component in the enzymatic hydrolysate were determined by HPAEC-PAD. A ternary gradient elution program was adopted, eluent A was 0.25 M sodium hydroxide, eluent B was 1.0 M sodium acetate, and eluent C was ultrapure water. The flow rate was 0.5 mL / min, the column temperature was 35 °C, the injection volume was 10 μL, and the sugar four-potential waveform was used for detection.

[0056] The chromatographic analysis results are as Figure 4 shown. The substrate conversion rate is about 58% - 70%, and the content of galactooligosaccharides is about 46% - 63% (% total sugar). In particular, under the optimal conditions (pH 6.5, 50 °C, 400 g / L lactose, enzyme dosage of 1000 U / g substrate, reaction for 60 h), the substrate conversion rate is about 70.9%, and the content of galactooligosaccharides in the product accounts for about 63.1% of the total sugar.

[0057] Comparative example

[0058] The specific implementation manners are the same as those in Examples 1 - 2 and Example 4, except that the gene derived from Paenibacillus macquariensis is replaced with a β-galactosidase gene from other sources reported in the literature. A genetically engineered bacterium is constructed according to the methods in Examples 1 - 2 and cultured to prepare a pure enzyme solution. The product is analyzed according to the method in Example 4. The comparison results are shown in Table 1. From the data in the table, it can be seen that the β-galactosidase encoded by the gene shown in SEQ ID NO.1 is superior to most of the β-galactosidases reported in the literature in terms of substrate conversion rate and galactooligosaccharide yield, and has very broad application prospects.

[0059] Table 1 Product situations of β-galactosidases from different sources

[0060]

[0061] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for preparing galactooligosaccharides, characterized in that, The method is to use lactose as a substrate, and β-galactosidase with an amino acid sequence as shown in SEQ ID NO.2 catalyzes the substrate to generate galactooligosaccharides. The nucleotide shown in SEQ ID NO.1 is ligated to an expression vector and transferred into Escherichia coli to obtain recombinant Escherichia coli, and the recombinant Escherichia coli expresses β-galactosidase with an amino acid sequence as shown in SEQ ID NO.

2.

2. The method according to claim 1, wherein The β-galactosidase is added in an amount of not less than 500 U / g of the substrate.

3. The method according to claim 1 or 2, characterized in that, The substrate is lactose, and the lactose concentration is 200 - 400 g / L.

4. The method according to claim 3, characterized in that, The reaction is carried out at 45 - 55 °C and pH 5.0 - 7.0 until lactose is no longer degraded.

5. The method according to claim 4, characterized in that, The recombinant Escherichia coli is fermented in TB medium at 25 - 37 °C for 24 - 72 h to obtain a culture solution, and the supernatant is taken after centrifugation of the culture solution as the crude enzyme solution of β-galactosidase.

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