WO2024021455A1 - β-GALACTOSIDASE GENE AND USE THEREOF IN ENCODING OF ENZYME - Google Patents

β-GALACTOSIDASE GENE AND USE THEREOF IN ENCODING OF ENZYME Download PDF

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WO2024021455A1
WO2024021455A1 PCT/CN2022/140522 CN2022140522W WO2024021455A1 WO 2024021455 A1 WO2024021455 A1 WO 2024021455A1 CN 2022140522 W CN2022140522 W CN 2022140522W WO 2024021455 A1 WO2024021455 A1 WO 2024021455A1
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galactosidase
seq
recombinant
lactose
enzyme
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李兆丰
张梓芊
李才明
顾正彪
班宵逢
程力
洪雁
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江南大学
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention relates to a ⁇ -galactosidase gene and the application of its encoding enzyme, specifically to a ⁇ -galactosidase gene that produces galacto-oligosaccharide and its application method, and belongs to the technical fields of genetic engineering and enzyme engineering.
  • ⁇ -galactooligosaccharides is a functional oligosaccharide with a degree of polymerization of 2 to 8, which uses galactose or glucose as the reducing end and is connected through ⁇ -glycosidic bonds 1 to 7
  • ⁇ -GOS has good taste, low sweetness, high solubility and strong moisturizing properties. It is an excellent food sweetener.
  • ⁇ -GOS has good anti-digestive properties and can resist degradation by digestive enzymes in the small intestine, maintaining a relatively intact structure and reaching the large intestine, thereby exerting many probiotic functions.
  • the specific performance is as follows: (1) It can 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 intestinal barrier function and relieve colitis.
  • Supplementing ⁇ -GOS in early life can help babies establish a healthy colon 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 ⁇ -GOS can also accelerate wound healing, which is beneficial to the treatment of colitis. Postoperative recovery. (3) Improve metabolism and delay aging. Synbiotics containing ⁇ -GOS can alleviate intestinal flora imbalance and significantly enhance the antioxidant capacity of the liver, exerting anti-aging effects through the liver-gut axis. (4) Improve diabetes symptoms. Due to its excellent antioxidant capacity and ability to balance intestinal flora, ⁇ -GOS has also been proven to reduce the levels of diabetes-related markers in the blood and delay the development of type II diabetes.
  • SCFAs short-chain fatty acids
  • ⁇ -GOS has no adverse effects on bodies of different ages or different organisms, and is safe and reliable. Therefore, ⁇ -GOS, as a prebiotic with rich nutritional value, can be used in infant food or dietary supplements for dietary treatment of special patients, and has broad application prospects in the fields of food, medicine and health. my country approved the use of ⁇ -GOS as a food additive in 2008. As its application market increases year by year, research on the functional development and production technology of ⁇ -GOS is of great significance.
  • ⁇ -GOS mainly relies on the enzymatic process, which uses ⁇ -galactosidase with transglycosidic activity to act on high-concentration lactose.
  • Japan prepared the first commercial product of ⁇ -GOS, and then its preparation process was introduced to Europe. The two monopolized the current production of high-purity ⁇ -GOS.
  • my country's ⁇ -GOS production started late and has not yet reached the industrial scale of 1,000 tons.
  • the main limiting factor is the lack of ⁇ -galactosidase with good properties.
  • ⁇ -galactosidase for commercial preparation of ⁇ -GOS: Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans.
  • the price of ⁇ -galactosidase derived from Aspergillus oryzae is relatively low, the conversion rate is about 30%, and the main product is galactooligosaccharide (about 18%); ⁇ -galactosidase derived from Kluyveromyces lactis
  • the highest conversion rate of glycosidase is also about 30%, and the disaccharide content in the product is the highest, but the probiotic activity of the disaccharide has not been confirmed; the conversion rate of ⁇ -galactosidase derived from Bacillus circulans is higher, up to 40% Around 26%, the product is mainly trisaccharide (about 26%).
  • the current enzymes used for ⁇ -GOS production have problems such as low conversion rate and low proportion of main products.
  • the composition of the transglycoside products of ⁇ -galactosidase is complex and contains different types of glycosidic bonds, which makes subsequent separation more difficult and is not conducive to functional research on ⁇ -GOS with different structures. Therefore, in order to reduce preparation and separation costs, it is an important research direction to find ⁇ -galactosidase with high transglycosylation efficiency and strong product specificity.
  • the purpose of the present invention is to make up for the shortcomings of the current enzymatic synthesis of ⁇ -GOS and provide a gene encoding ⁇ -galactosidase.
  • the gene is derived from Paenibacillus macquariensis, and the nucleotide sequence is as shown in SEQ ID NO.1 Show.
  • the present invention also provides a ⁇ -galactosidase encoded by the above nucleotide sequence, whose amino acid sequence is shown in SEQ ID NO. 2.
  • the present invention also provides a recombinant plasmid carrying the above-mentioned ⁇ -galactosidase gene.
  • the recombinant plasmid uses E. coli expression plasmid pET-20b(+) as a vector.
  • the present invention also provides a microbial cell carrying the above-mentioned ⁇ -galactosidase gene or the recombinant plasmid.
  • the microbial cell is recombinant E. coli.
  • the recombinant Escherichia coli uses Escherichia coli BL21 (DE3) as the expression host.
  • the construction method of the recombinant E. coli is: using a seamless cloning method, the ⁇ -galactosidase gene whose nucleotide sequence is shown in SEQ ID NO.1 is spliced into the expression vector pET -20b(+), construct the recombinant plasmid pmgal/pET-20b(+) and transform it into E.coli BL21(DE3).
  • the present invention also provides a method for producing ⁇ -galactosidase.
  • the method uses lactose as a substrate and utilizes the ⁇ -galactosidase with an amino acid sequence as shown in SEQ ID NO.2 to catalyze the substrate to generate oligomers.
  • Galactose is a method for producing ⁇ -galactosidase.
  • the ⁇ -galactosidase is added in an amount of no less than 500 U/g substrate.
  • the ⁇ -galactosidase is added in an amount of 500 to 1000 U/g substrate.
  • the ⁇ -galactosidase is added in an amount of 1000 U/g substrate.
  • the substrate is lactose, and the lactose concentration is 200-400g/L.
  • the lactose concentration is 400g/L.
  • the reaction is carried out at 45-55°C and pH 5.0-7.0 for 48-72 hours.
  • the reaction is carried out at 50°C.
  • reaction is carried out at pH 6.5 and 50°C for 60 hours.
  • the nucleotide shown in SEQ ID NO. 1 is connected to an expression vector and transferred into E. coli to obtain recombinant E. coli.
  • the fermentation involves inserting a certain amount of recombinant cells or recombinant E. coli into an LB medium containing ampicillin, culturing it at 37°C to the logarithmic growth phase, and preparing a seed liquid.
  • the seed liquid is used Proceed with fermentation.
  • the seed liquid is inoculated into TB medium containing ampicillin and 0-15% (w/v) lactose at an inoculation amount of 2% to 5% (v/v), and the seed liquid is inoculated at 25 to 37 Incubate in a shake flask at °C for 24 to 72 hours, and centrifuge to obtain the supernatant, which is the ⁇ -galactosidase crude enzyme solution.
  • the present invention also provides the ⁇ -galactosidase gene, the recombinant plasmid containing the ⁇ -galactosidase gene, and the E. coli expressing the ⁇ -galactosidase in the production of ⁇ -galactooligosaccharide.
  • the application uses ⁇ -galactosidase or an enzyme preparation containing the enzyme as a catalyst and a lactose solution as a substrate to convert lactose into functional galactooligosaccharides.
  • the enzyme preparation is the ⁇ -galactosidase crude 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.
  • the concentration of the lactose solution is 200-400g/L.
  • the present invention screened out a ⁇ -galactosidase with a specific amino acid sequence, and successfully achieved heterologous expression in E. coli, which can be used in food and pharmaceutical production.
  • the expressed ⁇ -galactosidase The catalytic activity can reach 12378.6U/mg;
  • the ⁇ -galactosidase of the present invention has a specific enzyme activity higher than most of the currently reported similar enzymes, and can maintain a high level in a wide temperature range. Vitality, adaptable to different reaction temperature conditions;
  • the ⁇ -galactosidase of the present invention has obvious advantages in preparing galacto-oligosaccharides, such as higher substrate conversion rate, stronger product specificity, and can effectively improve The yield of galactooligosaccharide reduces its preparation difficulty and subsequent separation and purification costs.
  • the substrate conversion rate can reach 70.9%.
  • the galactooligosaccharide content in the product accounts for approximately 63.1% of the total sugar.
  • the conversion rate and galactooligosaccharide content All have reached the highest level in the existing technology and have high industrial application value.
  • Figure 1 shows the SDS-PAGE analysis of purified recombinant ⁇ -galactosidase.
  • Figure 2 shows the thermal stability of recombinant ⁇ -galactosidase.
  • Figure 3 shows the effect of pH on the thermal stability of recombinant ⁇ -galactosidase.
  • Figure 4 shows HPAEC-PAD analysis when using recombinant ⁇ -galactosidase to prepare galactooligosaccharides.
  • the ⁇ -galactosidase enzyme activity assay method involved in the following examples is as follows:
  • Enzyme activity is defined as: under certain conditions, the amount of enzyme that hydrolyzes oNPG and releases 1 ⁇ mol oNP per minute by ⁇ -galactosidase is one unit of enzyme activity (U).
  • galacto-oligosaccharide content (%) 100% ⁇ mass of galacto-oligosaccharide in the product/mass of all sugars in the product.
  • the mass of galactooligosaccharide is the sum of the masses of transfer disaccharide to pentasaccharide.
  • the homologous recombination reaction system is: 1 ⁇ L of purified ⁇ -galactosidase fragment (50 ng/ ⁇ L), 1 ⁇ L of purified pET-20b(+) vector PCR fragment (50 ng/ ⁇ L), 4 ⁇ L of 5 ⁇ CEIIBuffer, 2 ⁇ L of ExnaseII, ddH 2 O 12 ⁇ L.
  • Example 2 Expression, isolation and purification of recombinant ⁇ -galactosidase
  • Example 1 Inoculate the genetically engineered bacteria preservation solution prepared in Example 1 into LB liquid culture medium containing 100 ⁇ g/mL ampicillin, and culture it at 37°C for 8 to 10 hours to prepare a seed solution;
  • the crude enzyme solution is purified by a nickel ion affinity chromatography column.
  • the buffers are A (10mmol/L Tris-HCl, 500mmol/L NaCl, pH 7.5) and B (10mmol/L). Tris-HCl, 500mmol/L NaCl, 500mmol/L imidazole, pH 7.5), the flow rate is 2mL/min. Equilibrate the nickel column with 25 to 30 mL of buffer A until it is stable, load the sample, and then use buffer A to elute the unbound protein in the purification column.
  • the thermal stability determination method of recombinant ⁇ -galactosidase pure enzyme is as follows: dilute the pure enzyme in 10mmol/L K 2 HPO 4 -KH 2 PO 4 buffer (pH 6.0), and incubate at 50°C and 55°C , incubate at 60°C for 60 minutes, take samples at different time points, and measure the enzyme activity. Taking the activity without incubation as 100%, calculate the relative residual enzyme activity at different times. The results are shown in Figure 2. The half-life of the enzyme at 50°C is approximately 50 minutes.
  • Recombinant ⁇ -galactosidase is used to prepare galactooligosaccharides.
  • the reaction process is as follows: prepare 200-400g/L lactose as substrate, adjust the pH to 5.0-7.0, add 500-1000U/g of pure enzyme as substrate, and 48 to 72 hours at 50°C (react until the residual lactose in the solution no longer degrades). Take part of the reaction solution and put it in a boiling water bath for 10 minutes to terminate the reaction, centrifuge at 8000r/min for 5 minutes, take the supernatant and dilute it to an appropriate multiple, and pass it through a 0.22 ⁇ m water filter membrane for testing.
  • HPAEC-PAD was used to determine the content of each component in the enzymatic hydrolyzate.
  • a ternary gradient elution program was adopted, eluent A was 0.25M sodium hydroxide, eluent B was 1.0M 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 ⁇ L, and the sugar four-potential waveform was used for detection.
  • the chromatographic analysis results are shown in Figure 4.
  • the substrate conversion rate is about 58% to 70%, and the galacto-oligosaccharide content is about 46% to 63% (% total sugar).
  • the substrate conversion rate is approximately 70.9%, and the galactooligosaccharide content in the product accounts for approximately 63.1% of total sugars.
  • the specific implementation is the same as in Examples 1 to 2 and 4. The difference is that the gene derived from Paenibacillus macquariensis is replaced with the ⁇ -galactosidase gene from other sources reported in the past. According to the steps in Examples 1 to 2 Methods Genetically engineered bacteria were constructed and cultured to prepare pure enzyme liquid. The product was analyzed according to the method in Example 4. The comparison results are shown in Table 1. It can be seen from the data in the table that the ⁇ -galactosidase encoded by the gene shown in SEQ ID NO.1 is superior to most ⁇ -galactosidase in existing reports in terms of substrate conversion rate and galactooligosaccharide yield. , has very broad application prospects.

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Abstract

The present invention relates to the technical field of genetic engineering and enzyme engineering, and provides a β-galactosidase, an encoding gene thereof, and a method for preparing galactooligosaccharide. The amino acid sequence of the β-galactosidase is as shown in SEQ ID NO. 2, and the nucleotide sequence of the encoding gene of the β-galactosidase is as shown in SEQ ID NO. 1. Heterologous expression is performed on the β-galactosidase by using Escherichia coli, and the catalytic activity of an expressed β-galactosidase crude enzyme solution can reach 12,378.6 U/mg. The β-galactosidase can catalyze lactose to generate galactooligosaccharide.

Description

一种β-半乳糖苷酶基因及其编码酶的应用Application of a β-galactosidase gene and its encoding enzyme 技术领域Technical field
本发明涉及一种β-半乳糖苷酶基因及其编码酶的应用，具体涉及一种产低聚半乳糖的β-半乳糖苷酶基因及其应用方法，属于基因工程和酶工程技术领域。The invention relates to a β-galactosidase gene and the application of its encoding enzyme, specifically to a β-galactosidase gene that produces galacto-oligosaccharide and its application method, and belongs to the technical fields of genetic engineering and enzyme engineering.
背景技术Background technique
β-低聚半乳糖(β-galactooligosaccharides，β-GOS)是一种聚合度为2～8的功能性低聚糖，即以半乳糖或葡萄糖为还原端，通过β-糖苷键连接1～7个半乳糖分子，其中的糖苷键可能为β-1,1、β-1,3、β-1,4或β-1,6-糖苷键。β-GOS口感好、甜度低、溶解度高、保湿性强，是一种优良的食品甜味剂。β-galactooligosaccharides (β-GOS) is a functional oligosaccharide with a degree of polymerization of 2 to 8, which uses galactose or glucose as the reducing end and is connected through β-glycosidic bonds 1 to 7 A galactose molecule, in which the glycosidic bond may be β-1,1, β-1,3, β-1,4 or β-1,6-glycosidic bond. β-GOS has good taste, low sweetness, high solubility and strong moisturizing properties. It is an excellent food sweetener.
重要的是，β-GOS具有良好的抗消化特性，能够抵抗小肠中消化酶的降解，保持相对完整的结构到达大肠，从而发挥诸多益生功能。具体表现为：(1)能够选择性地促进肠道有益菌的增殖，特别是双歧杆菌与乳酸杆菌，同时能够抑制腐败菌(如部分梭状芽孢杆菌)的生长。(2)改善肠道屏障功能，缓解结肠炎。在生命早期补充β-GOS能够帮助婴儿建立健康的结肠环境，增加肠道中短链脂肪酸(SCFAs)含量，降低结肠炎风险；同时β-GOS的膳食补充也可加速伤口愈合，有利于结肠炎的术后恢复。(3)提高新陈代谢，延缓衰老。含有β-GOS的合生元能够缓解肠道菌群失调，并显著增强肝脏的抗氧化能力，通过肝-肠轴发挥抗衰老的作用。(4)改善糖尿病症状。由于出色的抗氧化能力与平衡肠道菌群的功效，β-GOS也被证实能够降低血液中糖尿病相关标志物含量，延缓II型糖尿病的发展。此外，毒理学研究表明β-GOS对于不同年龄段机体或不同生物均无任何不良影响，安全可靠。因此，β-GOS作为富有营养价值的益生元，可应用于婴幼儿食品或特殊病人饮食治疗的膳食补充剂中，在食品及医药健康领域具有广阔的应用前景。我国在2008年批准将β-GOS作为食品添加剂使用，随着其应用市场的逐年增加，对β-GOS功能性的开发与生产技术的研究具有重要意义。Importantly, β-GOS has good anti-digestive properties and can resist degradation by digestive enzymes in the small intestine, maintaining a relatively intact structure and reaching the large intestine, thereby exerting many probiotic functions. The specific performance is as follows: (1) It can 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 intestinal barrier function and relieve colitis. Supplementing β-GOS in early life can help babies establish a healthy colon 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, which is beneficial to the treatment of colitis. Postoperative recovery. (3) Improve metabolism and delay aging. Synbiotics containing β-GOS can alleviate intestinal flora imbalance and significantly enhance the antioxidant capacity of the liver, exerting anti-aging effects through the liver-gut axis. (4) Improve diabetes symptoms. Due to its excellent antioxidant capacity and ability to balance intestinal flora, β-GOS has also been proven to reduce the levels 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 bodies of different ages or different organisms, and is safe and reliable. Therefore, β-GOS, as a prebiotic with rich nutritional value, can be used in infant food or dietary supplements for dietary treatment of special patients, and has broad application prospects in the fields of food, medicine and health. my country approved the use of β-GOS as a food additive in 2008. As its application market increases year by year, research on the functional development and production technology of β-GOS is of great significance.
工业上，β-GOS的生产主要依靠酶法工艺，即利用具有转糖苷活性的β-半乳糖苷酶作用于高浓度乳糖而成。早在1988年，日本制备得到了第一个β-GOS商业化产品，随后其制备工艺引入欧洲，二者垄断了现阶段高纯度β-GOS的生产。与此相比，我国的β-GOS生产起步较晚，工业上尚未达到千吨规模。主要限制因素在于缺少性质优良的β-半乳糖苷酶。目前，商业化制备β-GOS的β-半乳糖苷酶主要有以下3个来源：米曲霉(Aspergillus oryzae)、乳酸克鲁维酵母(Kluyveromyces lactis)和环状芽孢杆菌(Bacillus circulans)。其中，米曲霉来源的β-半乳糖苷酶价格相对较低，转化率约为30％，主产物为低聚半乳三糖(约18％)；乳酸克鲁维酵 母来源的β-半乳糖苷酶最高转化率同样约为30％，产物中二糖含量最高，但二糖的益生活性尚未被证实；环状芽孢杆菌来源的β-半乳糖苷酶转化率较高，可达40％左右，产物以三糖为主(约26％)。可见，目前的β-GOS生产用酶存在转化率低、且主产物比例低的问题。同时，β-半乳糖苷酶的转苷产物组成复杂，包含不同类型的糖苷键，导致后续分离难度增加，且不利于不同结构β-GOS的功能性研究。因此，为降低制备及分离成本，寻找转苷效率高、产物专一性强的β-半乳糖苷酶是重要的研究方向。Industrially, the production of β-GOS mainly relies on the enzymatic process, which uses β-galactosidase with transglycosidic activity to act on high-concentration lactose. As early as 1988, Japan prepared the first commercial product of β-GOS, and then its preparation process was introduced to Europe. The two monopolized the current production of high-purity β-GOS. In comparison, my country's β-GOS production started late and has not yet reached the industrial scale of 1,000 tons. The main limiting factor is the lack of β-galactosidase with good properties. At present, there are three main sources of β-galactosidase for commercial preparation of β-GOS: Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans. Among them, the price of β-galactosidase derived from Aspergillus oryzae is relatively low, the conversion rate is about 30%, and the main product is galactooligosaccharide (about 18%); β-galactosidase derived from Kluyveromyces lactis The highest conversion rate of glycosidase is also about 30%, and the disaccharide content in the product is the highest, but the probiotic activity of the disaccharide has not been confirmed; the conversion rate of β-galactosidase derived from Bacillus circulans is higher, up to 40% Around 26%, the product is mainly trisaccharide (about 26%). It can be seen that the current enzymes used for β-GOS production have problems such as low conversion rate and low proportion of main products. At the same time, the composition of the transglycoside products of β-galactosidase is complex and contains different types of glycosidic bonds, which makes subsequent separation more difficult and is not conducive to functional research on β-GOS with different structures. Therefore, in order to reduce preparation and separation costs, it is an important research direction to find β-galactosidase with high transglycosylation efficiency and strong product specificity.
发明内容Contents of the invention
本发明的目的在于弥补目前酶法合成β-GOS的不足之处，提供了一种编码β-半乳糖苷酶的基因，该基因来源于Paenibacillus macquariensis，核苷酸序列如SEQ ID NO.1所示。The purpose of the present invention is to make up for the shortcomings of the current enzymatic synthesis of β-GOS and provide a gene encoding β-galactosidase. The gene is derived from Paenibacillus macquariensis, and the nucleotide sequence is as shown in SEQ ID NO.1 Show.
本发明还提供了一种上述核苷酸序列编码的β-半乳糖苷酶，其氨基酸序列如SEQ ID NO.2所示。The present invention also provides a β-galactosidase encoded by the above nucleotide sequence, whose amino acid sequence is shown in SEQ ID NO. 2.
本发明还提供了一种携带上述β-半乳糖苷酶基因的重组质粒。The present invention also provides a recombinant plasmid carrying the above-mentioned β-galactosidase gene.
在一种实施方式中，所述重组质粒以大肠杆菌表达质粒pET-20b(+)为载体。In one embodiment, the recombinant plasmid uses E. coli expression plasmid pET-20b(+) as a vector.
本发明还提供了一种携带上述β-半乳糖苷酶的基因，或所述重组质粒的微生物细胞。The present invention also provides a microbial cell carrying the above-mentioned β-galactosidase gene or the recombinant plasmid.
在一种实施方式中，所述微生物细胞是重组大肠杆菌。In one embodiment, the microbial cell is recombinant E. coli.
优选地，所述重组大肠杆菌以Escherichia coli BL21(DE3)为表达宿主。Preferably, the recombinant Escherichia coli uses Escherichia coli BL21 (DE3) as the expression host.
在一种实施方式中，所述重组大肠杆菌的构建方法为：利用无缝克隆的方法，将核苷酸序列为SEQ ID NO.1所示的β-半乳糖苷酶基因拼接到表达载体pET-20b(+)上，构建重组质粒pmgal/pET-20b(+)，并将其转化至E.coli BL21(DE3)中。In one embodiment, the construction method of the recombinant E. coli is: using a seamless cloning method, the β-galactosidase gene whose nucleotide sequence is shown in SEQ ID NO.1 is spliced into the expression vector pET -20b(+), construct the recombinant plasmid pmgal/pET-20b(+) and transform it into E.coli BL21(DE3).
本发明还提供一种生产β-半乳糖苷酶的方法，所述方法是以乳糖为底物，利用氨基酸序列如SEQ ID NO.2所示的β-半乳糖苷酶催化底物生成低聚半乳糖。The present invention also provides a method for producing β-galactosidase. The method uses lactose as a substrate and utilizes the β-galactosidase with an amino acid sequence as shown in SEQ ID NO.2 to catalyze the substrate to generate oligomers. Galactose.
在一种实施方式中，按照不少于500U/g底物的量添加所述β-半乳糖苷酶。In one embodiment, the β-galactosidase is added in an amount of no less than 500 U/g substrate.
优选地，按照500～1000U/g底物的量添加所述β-半乳糖苷酶。Preferably, the β-galactosidase is added in an amount of 500 to 1000 U/g substrate.
更优选地，按照1000U/g底物的量添加所述β-半乳糖苷酶。More preferably, the β-galactosidase is added in an amount of 1000 U/g substrate.
在一种实施方式中，所述底物为乳糖，乳糖浓度为200～400g/L。In one embodiment, the substrate is lactose, and the lactose concentration is 200-400g/L.
优选地，所述乳糖浓度为400g/L。Preferably, the lactose concentration is 400g/L.
在一种实施方式中，在45～55℃、pH 5.0～7.0下反应48～72h。In one embodiment, the reaction is carried out at 45-55°C and pH 5.0-7.0 for 48-72 hours.
优选地，在50℃下反应。Preferably, the reaction is carried out at 50°C.
更优选地，在pH 6.5，50℃下反应60h。More preferably, the reaction is carried out at pH 6.5 and 50°C for 60 hours.
在一种实施方式中，将SEQ ID NO.1所示的核苷酸连接至表达载体上，转入大肠杆菌中获得重组大肠杆菌。In one embodiment, the nucleotide shown in SEQ ID NO. 1 is connected to an expression vector and transferred into E. coli to obtain recombinant E. coli.
在一种实施方式中，所述发酵是将一定量的重组细胞或重组大肠杆菌接入含有氨苄青霉素的LB培养基中，于37℃培养至对数生长期，制备得种子液，利用种子液进行发酵。In one embodiment, the fermentation involves inserting a certain amount of recombinant cells or recombinant E. coli into an LB medium containing ampicillin, culturing it at 37°C to the logarithmic growth phase, and preparing a seed liquid. The seed liquid is used Proceed with fermentation.
在一种实施方式中，将种子液按照2％～5％(v/v)的接种量接种至含有氨苄青霉素及0～15％(w/v)乳糖的TB培养基中，于25～37℃摇瓶培养24～72h，离心得上清液即为β-半乳糖苷酶粗酶液。In one embodiment, the seed liquid is inoculated into TB medium containing ampicillin and 0-15% (w/v) lactose at an inoculation amount of 2% to 5% (v/v), and the seed liquid is inoculated at 25 to 37 Incubate in a shake flask at ℃ for 24 to 72 hours, and centrifuge to obtain the supernatant, which is the β-galactosidase crude enzyme solution.
本发明还提供所述β-半乳糖苷酶基因、含有所述β-半乳糖苷酶基因的重组质粒、表达所述β-半乳糖苷酶的大肠杆菌在生产β-低聚半乳糖中的应用，所述应用是以β-半乳糖苷酶或含有该酶的酶制剂为催化剂，以乳糖溶液为底物，将乳糖转化为功能性低聚半乳糖。The present invention also provides the β-galactosidase gene, the recombinant plasmid containing the β-galactosidase gene, and the E. coli expressing the β-galactosidase in the production of β-galactooligosaccharide. The application uses β-galactosidase or an enzyme preparation containing the enzyme as a catalyst and a lactose solution as a substrate to convert lactose into functional galactooligosaccharides.
在一种实施方式中，所述酶制剂是所述β-半乳糖苷酶粗酶液或经分离纯化后得到的纯酶，以溶液或干粉的形式添加至反应体系中。In one embodiment, the enzyme preparation is the β-galactosidase crude 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.
在一种实施方式中，所述乳糖溶液的浓度为200～400g/L。In one embodiment, the concentration of the lactose solution is 200-400g/L.
本发明的有益效果在于：The beneficial effects of the present invention are:
(1)本发明筛选到了一种特定氨基酸序列的β-半乳糖苷酶，并且成功实现了在大肠杆菌中的异源表达，可应用于食品及药品生产中，所表达β-半乳糖苷酶催化活力可达12378.6U/mg；(1) The present invention screened out a β-galactosidase with a specific amino acid sequence, and successfully achieved heterologous expression in E. coli, which can be used in food and pharmaceutical production. The expressed β-galactosidase The catalytic activity can reach 12378.6U/mg;
(2)本发明的β-半乳糖苷酶与目前报道的同类酶相比，该酶比酶活高于现有报道的大多数同类酶，且在较宽的温度范围内均能保持较高活力，可适应不同的反应温度条件；(2) Compared with the currently reported similar enzymes, the β-galactosidase of the present invention has a specific enzyme activity higher than most of the currently reported similar enzymes, and can maintain a high level in a wide temperature range. Vitality, adaptable to different reaction temperature conditions;
(3)本发明的β-半乳糖苷酶与现有报道的大多数同类酶相比，制备低聚半乳糖的优势明显，底物转化率较高，产物专一性较强，可有效提高低聚半乳糖的产率，降低其制备难度以及后续分离纯化成本，底物转化率可达70.9％，产物中低聚半乳糖含量约占总糖的63.1％，转化率和低聚半乳糖含量均达到现有技术中的最高水平，具有较高的工业应用价值。(3) Compared with most similar enzymes currently reported, the β-galactosidase of the present invention has obvious advantages in preparing galacto-oligosaccharides, such as higher substrate conversion rate, stronger product specificity, and can effectively improve The yield of galactooligosaccharide reduces its preparation difficulty and subsequent separation and purification costs. The substrate conversion rate can reach 70.9%. The galactooligosaccharide content in the product accounts for approximately 63.1% of the total sugar. The conversion rate and galactooligosaccharide content All have reached the highest level in the existing technology and have high industrial application value.
附图说明Description of drawings
图1为纯化重组β-半乳糖苷酶的SDS-PAGE分析。Figure 1 shows the SDS-PAGE analysis of purified recombinant β-galactosidase.
图2为重组β-半乳糖苷酶的热稳定性。Figure 2 shows the thermal stability of recombinant β-galactosidase.
图3为pH对重组β-半乳糖苷酶热稳定性的影响。Figure 3 shows the effect of pH on the thermal stability of recombinant β-galactosidase.
图4为应用重组β-半乳糖苷酶制备低聚半乳糖时的HPAEC-PAD分析。Figure 4 shows HPAEC-PAD analysis when using recombinant β-galactosidase to prepare galactooligosaccharides.
具体实施方式Detailed ways
下述实例中所涉及的β-半乳糖苷酶酶活测定方法如下：The β-galactosidase enzyme activity assay method involved in the following examples is as follows:
以2-硝基苯基-β-D-吡喃半乳糖苷(oNPG)为底物评价β-半乳糖苷酶的水解活力：Use 2-nitrophenyl-β-D-galactopyranoside (oNPG) as substrate to evaluate the hydrolysis activity of β-galactosidase:
以K 2HPO 4-KH 2PO 4缓冲液(20mmol/L，pH 5.5)配制10mmol/L oNPG溶液作为底物，在0.9mL底物中加入0.1mL酶液，于55℃下反应15min后，加入1mL 1M Na 2CO 3溶液终止反应，并于420nm下测定吸光值。根据邻硝基苯酚(oNP)标准曲线计算反应体系中oNP含量。 Use K 2 HPO 4 -KH 2 PO 4 buffer (20mmol/L, pH 5.5) to prepare a 10mmol/L oNPG solution as the substrate, add 0.1mL enzyme solution to 0.9mL substrate, and react at 55°C for 15 minutes. Add 1 mL of 1M Na 2 CO 3 solution to terminate the reaction, and measure the absorbance value at 420 nm. Calculate the oNP content in the reaction system according to the o-nitrophenol (oNP) standard curve.
酶活定义为：在一定条件下，以β-半乳糖苷酶每分钟水解oNPG释放1μmol oNP的酶量为一个酶活单位(U)。Enzyme activity is defined as: under certain conditions, the amount of enzyme that hydrolyzes oNPG and releases 1 μmol oNP per minute by β-galactosidase is one unit of enzyme activity (U).
底物转化率计算方法：以10～50μg/mL的乳糖溶液为标品，利用HPAEC-PAD分析酶解前后体系中的乳糖含量。乳糖转化率(％)＝100％×(反应前乳糖质量-反应后乳糖质量)/反应前乳糖质量。Calculation method of substrate conversion rate: Using 10-50 μg/mL lactose solution as the standard, use HPAEC-PAD to analyze the lactose content in the system before and after enzymatic hydrolysis. Lactose conversion rate (%) = 100% × (mass of lactose before reaction - mass of lactose after reaction)/mass of lactose before reaction.
低聚半乳糖百分含量计算方法：低聚半乳糖含量(％)＝100％×产物中低聚半乳糖的质量/产物中所有糖的质量。其中，低聚半乳糖质量为转移二糖～五糖的质量总和。Calculation method for the percentage content of galacto-oligosaccharide: galacto-oligosaccharide content (%) = 100% × mass of galacto-oligosaccharide in the product/mass of all sugars in the product. Among them, the mass of galactooligosaccharide is the sum of the masses of transfer disaccharide to pentasaccharide.
实施例1：大肠杆菌分泌表达系统的构建Example 1: Construction of E. coli secretion expression system
基因合成如SEQ ID NO.1所示的核苷酸序列，采用同源重组的方法将上述序列连接至pET-20b(+)载体。其中涉及的PCR扩增程序为：94℃预变性3min；94℃变性30s，55℃退火30s，72℃延伸5min，重复35个循环；最后72℃保温10min。同源重组反应体系为：纯化的β-半乳糖苷酶片段(50ng/μL)1μL，纯化的pET-20b(+)载体PCR片段(50ng/μL)1μL，5×CEⅡBuffer 4μL，ExnaseⅡ2μL，ddH 2O 12μL。将上述同源重组体系在37℃下反应30min后，转化E.coli JM109，涂布含有氨苄青霉素的LB平板，挑取单菌落活化、测序、获得重组质粒pmgal/pET-20b(+)；将上述重组质粒转化E.coli BL21(DE3)，得到基因工程菌pmgal/pET-20b(+)/E.coli BL21(DE3)。 Gene synthesize the nucleotide sequence shown in SEQ ID NO. 1, and use homologous recombination to connect the above sequence to the pET-20b(+) vector. The PCR amplification procedure involved is: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 5 minutes, repeated for 35 cycles; and finally incubation at 72°C for 10 minutes. 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×CEⅡBuffer, 2 μL of ExnaseⅡ, ddH 2 O 12μL. After reacting the above homologous recombination system at 37°C for 30 minutes, transform E.coli JM109, spread it on an LB plate containing ampicillin, pick a single colony, activate it, sequence it, and obtain the recombinant plasmid pmgal/pET-20b(+); The above recombinant plasmid was used to transform E.coli BL21(DE3) to obtain the genetically engineered bacterium pmgal/pET-20b(+)/E.coli BL21(DE3).
实施例2：重组β-半乳糖苷酶的表达与分离纯化Example 2: Expression, isolation and purification of recombinant β-galactosidase
具体步骤如下：Specific steps are as follows:
(1)将实施例1中制备得到的基因工程菌保存液接种于含有100μg/mL氨苄青霉素的LB液体培养基中，与37℃下培养8～10h，制备得种子液；(1) Inoculate the genetically engineered bacteria preservation solution prepared in Example 1 into LB liquid culture medium containing 100 μg/mL ampicillin, and culture it at 37°C for 8 to 10 hours to prepare a seed solution;
将上述种子液按2％～5％(v/v)的接种量转移至含有100μg/mL氨苄青霉素及0～15％(w/v)乳糖的TB培养基中，在25～37℃、200r/min条件下培养24～72h，收集上清液即为β-半乳糖苷酶粗酶液，其酶活为11397.1U/mL。Transfer the above seed liquid to the TB medium containing 100 μg/mL ampicillin and 0 to 15% (w/v) lactose at an inoculation amount of 2% to 5% (v/v), and incubate at 25 to 37°C, 200r /min conditions for 24 to 72 hours, collect the supernatant to become β-galactosidase crude enzyme solution, and its enzyme activity is 11397.1U/mL.
(2)粗酶液过0.45μm水系膜后，经镍离子亲和层析柱纯化，缓冲液分别为A(10mmol/L Tris-HCl、500mmol/L NaCl，pH 7.5)和B(10mmol/L Tris-HCl、500mmol/L NaCl、500mmol/L咪唑，pH 7.5)，流速为2mL/min。用25～30mL缓冲液A平衡镍柱至稳定后，上样，再以缓冲液A洗脱纯化柱中的未结合蛋白。待洗脱曲线平衡后，以35％(v/v)缓冲液B进行梯度洗 脱，收集洗脱液进行鉴定，如图1所示。得到的β-半乳糖苷酶纯酶液测定蛋白浓度，并计算得比酶活为12378.6U/mg。(2) After passing through a 0.45μm water-based membrane, the crude enzyme solution is purified by a nickel ion affinity chromatography column. The buffers are A (10mmol/L Tris-HCl, 500mmol/L NaCl, pH 7.5) and B (10mmol/L). Tris-HCl, 500mmol/L NaCl, 500mmol/L imidazole, pH 7.5), the flow rate is 2mL/min. Equilibrate the nickel column with 25 to 30 mL of buffer A until it is stable, load the sample, and then use buffer A to elute the unbound protein in the purification column. After the elution curve is balanced, gradient elution is performed with 35% (v/v) buffer B, and the eluate is collected for identification, as shown in Figure 1. The protein concentration of the obtained β-galactosidase pure enzyme solution was measured, and the specific enzyme activity was calculated to be 12378.6U/mg.
实施例3：重组β-半乳糖苷酶的稳定性Example 3: Stability of recombinant β-galactosidase
分别测定实施例2中制备得重组β-半乳糖苷酶纯酶的热稳定性及pH对热稳定性的影响，具体步骤如下：The thermal stability of the recombinant β-galactosidase pure enzyme prepared in Example 2 and the effect of pH on the thermal stability were measured respectively. The specific steps are as follows:
(1)重组β-半乳糖苷酶纯酶的热稳定性测定方法如下：将纯酶稀释于10mmol/L K 2HPO 4-KH 2PO 4缓冲液(pH 6.0)中，于50℃、55℃、60℃保温60min，在不同时间点取样，测定酶活力，以未保温时的活力为100％，计算不同时间的相对残余酶活。结果如图2所示，该酶在50℃下的半衰期约为50min。 (1) The thermal stability determination method of recombinant β-galactosidase pure enzyme is as follows: dilute the pure enzyme in 10mmol/L K 2 HPO 4 -KH 2 PO 4 buffer (pH 6.0), and incubate at 50°C and 55°C , incubate at 60°C for 60 minutes, take samples at different time points, and measure the enzyme activity. Taking the activity without incubation as 100%, calculate the relative residual enzyme activity at different times. The results are shown in Figure 2. The half-life of the enzyme at 50°C is approximately 50 minutes.
(2)分析pH对重组β-半乳糖苷酶纯酶的热稳定性的影响，测定方法如下：分别用10mmol/L的CH 3COOK-CH 3COOH缓冲液(pH 3.0～5.0)、K 2HPO 4-KH 2PO 4缓冲液(pH 5.0～8.0)和NaOH-Gly缓冲液(pH 8.0～10.0)配制10mmol/L oNPG作为底物，将纯酶在50℃下保温1h，于不同时间点取样测定残余酶活力，以未保温时的酶活为100％，计算不同pH下的相对酶活力。结果显示，该酶在pH 5.0～6.0均可保持95％以上的活性，如图3所示。 (2) Analyze the effect of pH on the thermal stability of recombinant β-galactosidase pure enzyme. The determination method is as follows: use 10mmol/L CH 3 COOK-CH 3 COOH buffer (pH 3.0~5.0) and K 2 respectively. HPO 4 -KH 2 PO 4 buffer (pH 5.0 ~ 8.0) and NaOH-Gly buffer (pH 8.0 ~ 10.0) were used to prepare 10mmol/L oNPG as the substrate. The pure enzyme was incubated at 50°C for 1 hour and tested at different time points. Samples were taken to measure the residual enzyme activity, and the enzyme activity without incubation was taken as 100% to calculate the relative enzyme activity under different pH. The results showed that the enzyme could maintain more than 95% activity at pH 5.0-6.0, as shown in Figure 3.
实施例4：重组β-半乳糖苷酶的应用Example 4: Application of recombinant β-galactosidase
利用重组β-半乳糖苷酶制备低聚半乳糖，其反应过程如下：配制200～400g/L乳糖为底物，调节pH至5.0～7.0，加入500～1000U/g底物的纯酶，于50℃下48～72h(反应至溶液中残余乳糖不再继续降解)。取部分反应液沸水浴10min终止反应后，8000r/min离心5min，取上清液稀释适当倍数，过0.22μm水系滤膜待测。Recombinant β-galactosidase is used to prepare galactooligosaccharides. The reaction process is as follows: prepare 200-400g/L lactose as substrate, adjust the pH to 5.0-7.0, add 500-1000U/g of pure enzyme as substrate, and 48 to 72 hours at 50°C (react until the residual lactose in the solution no longer degrades). Take part of the reaction solution and put it in a boiling water bath for 10 minutes to terminate the reaction, centrifuge at 8000r/min for 5 minutes, take the supernatant and dilute it to an appropriate multiple, and pass it through a 0.22μm water filter membrane for testing.
利用HPAEC-PAD测定酶解液中各组分含量。采用三元梯度洗脱程序，洗脱液A为0.25M氢氧化钠，洗脱液B为1.0M醋酸钠，洗脱液C为超纯水。流速0.5mL/min，柱温35℃，进样量10μL，以糖四电位波形检测。HPAEC-PAD was used to determine the content of each component in the enzymatic hydrolyzate. A ternary gradient elution program was adopted, eluent A was 0.25M sodium hydroxide, eluent B was 1.0M 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.
色谱分析结果如图4所示，底物转化率约为58％～70％，低聚半乳糖含量约为46％～63％(％总糖)。特别地，在最佳条件下(pH 6.5，50℃，400g/L乳糖，加酶量1000U/g底物，反应60h)底物转化率约为70.9％，产物中低聚半乳糖含量约占总糖的63.1％。The chromatographic analysis results are shown in Figure 4. The substrate conversion rate is about 58% to 70%, and the galacto-oligosaccharide content is about 46% to 63% (% total sugar). In particular, under optimal conditions (pH 6.5, 50°C, 400g/L lactose, enzyme amount 1000U/g substrate, reaction 60h) the substrate conversion rate is approximately 70.9%, and the galactooligosaccharide content in the product accounts for approximately 63.1% of total sugars.
对比例Comparative ratio
具体实施方式同实施例1～2及实施例4，区别在于，将来源于Paenibacillus macquariensis的基因替换成现有报道的、其他来源的β-半乳糖苷酶基因，按照实施例1～2中的方法构建基因工程菌，并培养制备得到纯酶液，按照实施例4中的方法分析产物情况。对比结果如表1所示。由表中数据可知，SEQ ID NO.1所示基因编码的β-半乳糖苷酶在底物转化率和低聚半 乳糖产率方面均优于现有报道中大多数β-半乳糖苷酶，具有十分广阔的应用前景。The specific implementation is the same as in Examples 1 to 2 and 4. The difference is that the gene derived from Paenibacillus macquariensis is replaced with the β-galactosidase gene from other sources reported in the past. According to the steps in Examples 1 to 2 Methods Genetically engineered bacteria were constructed and cultured to prepare pure enzyme liquid. The product was analyzed according to the method in Example 4. The comparison results are shown in Table 1. It can be seen from the data in the table that the β-galactosidase encoded by the gene shown in SEQ ID NO.1 is superior to most β-galactosidase in existing reports in terms of substrate conversion rate and galactooligosaccharide yield. , has very broad application prospects.
表1不同来源的β-半乳糖苷酶的产物情况Table 1 Products of β-galactosidase from different sources
Figure PCTCN2022140522-appb-000001
Figure PCTCN2022140522-appb-000001
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed above in terms of preferred embodiments, they are not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications 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 (10)

  1. 一种制备低聚半乳糖的方法，其特征在于，所述方法为以乳糖为底物，利用氨基酸序列如SEQ ID NO.2所示的β-半乳糖苷酶催化底物生成低聚半乳糖，编码所述β-半乳糖苷酶的核苷酸序列如SEQ ID NO.1所示。A method for preparing galactooligosaccharide, characterized in that the method uses lactose as a substrate, and utilizes a β-galactosidase catalytic substrate with an amino acid sequence as shown in SEQ ID NO.2 to generate galactooligosaccharide , the nucleotide sequence encoding the β-galactosidase is shown in SEQ ID NO.1.
  2. 根据权利要求1所述的方法，其特征在于，按照不少于500U/g底物的量添加所述β-半乳糖苷酶。The method according to claim 1, characterized in that the β-galactosidase is added in an amount of not less than 500 U/g substrate.
  3. 根据权利要求1或2所述的方法，其特征在于，所述底物为乳糖，乳糖浓度为200～400g/L。The method according to claim 1 or 2, characterized in that the substrate is lactose, and the lactose concentration is 200-400g/L.
  4. 根据权利要求3所述的方法，其特征在于，在45～55℃、pH 5.0～7.0下反应至乳糖不再降解。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. 根据权利要求4所述的方法，其特征在于，将SEQ ID NO.1所示的核苷酸连接至表达载体上，转入大肠杆菌中获得重组大肠杆菌。The method according to claim 4, characterized in that the nucleotide shown in SEQ ID NO. 1 is connected to an expression vector and transferred into E. coli to obtain recombinant E. coli.
  6. 根据权利要求5所述的方法，其特征在于，将重组大肠杆菌在TB培养基中，于25～37℃发酵24～72h得到培养液，将培养液经离心后取上清即为β-半乳糖苷酶粗酶液。The method according to claim 5, characterized in that the recombinant Escherichia coli is fermented in TB culture medium at 25-37°C for 24-72 hours to obtain a culture liquid, and the culture liquid is centrifuged and the supernatant is taken to obtain β-half Lactosidase crude enzyme solution.
  7. 核苷酸序列如SEQ ID NO.1所示的β-半乳糖苷酶基因、含有核苷酸序列如SEQ ID NO.1所示的β-半乳糖苷酶基因的重组质粒或重组菌、含有所述重组菌的微生物制剂在制备β-半乳糖苷酶中的应用。The β-galactosidase gene with the nucleotide sequence shown in SEQ ID NO.1, the recombinant plasmid or recombinant bacterium containing the β-galactosidase gene with the nucleotide sequence shown in SEQ ID NO.1, Application of the microbial preparation of the recombinant bacteria in the preparation of β-galactosidase.
  8. 根据权利要求7所述的应用，其特征在于，将所述重组质粒转入宿主细胞中得到重组菌，将重组菌在TB培养基中发酵产酶。The application according to claim 7, characterized in that the recombinant plasmid is transferred into a host cell to obtain a recombinant bacterium, and the recombinant bacterium is fermented in a TB medium to produce enzymes.
  9. 含有氨基酸序列如SEQ ID NO.2所示的β-半乳糖苷酶的酶制剂在制备低聚半乳糖中的应用。Application of an enzyme preparation containing β-galactosidase with an amino acid sequence as shown in SEQ ID NO. 2 in the preparation of galactooligosaccharides.
  10. 根据权利要求9所述的应用，其特征在于，所述酶制剂是将表达氨基酸序列如SEQ ID NO.2所示的β-半乳糖苷酶的重组菌在TB培养基中发酵得到的粗酶液分离纯化后制备得到酶制剂。The application according to claim 9, characterized in that the enzyme preparation is a crude enzyme obtained by fermenting a recombinant bacterium expressing β-galactosidase with an amino acid sequence as shown in SEQ ID NO. 2 in a TB culture medium The enzyme preparation was prepared after liquid separation and purification.
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