阅读说明：本技术 一种新型β-半乳糖苷酶及其在降解牛奶中乳糖的应用 (Novel beta-galactosidase and application thereof in degrading lactose in milk ) 是由 夏玉军 于 2020-08-12 设计创作，主要内容包括：本发明涉及一种新型β-半乳糖苷酶及其在降解牛奶中乳糖的应用。本发明所述β-半乳糖苷酶氨基酸序列如SEQ ID NO.1所示。本发明所述的β-半乳糖苷酶产量可达306.3U/mL,最适反应温度和pH分别为40℃和7.5。本发明所述的β-半乳糖苷酶在6.0-9.0的pH范围内显示出广泛的pH稳定性,适用于牛奶中乳糖的水解。而且,将β-半乳糖苷酶添加至含有酵素的奶制品中,可生产一种适合儿童饮用的功能性奶制品饮料。本发明所述的β-半乳糖苷酶产量大,是生产无乳糖乳制品的理想选择。(The invention relates to novel beta-galactosidase and application thereof in degrading lactose in milk. The amino acid sequence of the beta-galactosidase is shown as SEQ ID NO. 1. The yield of the beta-galactosidase can reach 306.3U/mL, and the optimal reaction temperature and the optimal pH are respectively 40 ℃ and 7.5. The beta-galactosidase of the invention shows wide pH stability in the pH range of 6.0-9.0, and is suitable for the hydrolysis of lactose in milk. Moreover, the beta-galactosidase is added to the dairy product containing the ferment, so that a functional dairy drink suitable for children can be produced. The beta-galactosidase of the invention has high yield and is an ideal choice for producing lactose-free dairy products.)

1. A novel beta-galactosidase has an amino acid sequence shown in SEQ ID NO. 1.

2. The nucleotide sequence corresponding to the beta-galactosidase according to claim 1, wherein the nucleotide sequence is represented by SEQ ID No. 2.

3. The method for preparing and purifying beta-galactosidase according to claim 1.

4. Use of a beta-galactosidase according to claim 1 for degrading lactose in milk.

5. Use of the beta-galactosidase according to claim 1 for the preparation of lactose-reduced milk and dairy products.

6. A method for degrading lactose, wherein the beta-galactosidase selected is the beta-galactosidase of claim 1.

7. The method according to claim 6, wherein the degradation condition is a reaction temperature of 0 to 70 ℃ and an optimum reaction temperature of 40 ℃.

8. The method according to claim 6, wherein the degradation condition is a reaction pH of 5.5 to 10.5, and the optimum reaction pH is 7.5.

Technical Field

The invention relates to a novel beta-galactosidase and application thereof in degrading lactose in milk, belonging to the technical field of biology.

Background

Beta-galactosidase (EC3.2.1.23, lactase) is an important medical enzyme that regulates the hydrolysis process of lactose by cleaving the terminal non-reducing beta-D-galactose component. Beta-galactosidase can be a potential treatment option for lactose intolerance. Lactose intolerance is currently defined as a clinical syndrome characterized by pain, abdominal distension, flatulence and diarrhea after intake of lactose. Lactose is a disaccharide that is common in human nutrition at present, both for breast-fed infants and adults, but its digestion requires a specific enzyme, lactase. In adulthood, genetic programming of lactase activity affects most adults worldwide, and lactose intolerance exists in about 2/3 people worldwide due to lactase deficiency. Treatment of lactose intolerance mainly involves reduction or elimination of the dietary lactose content, but this is difficult to achieve because lactose is present in dairy products and even commonly used as a food additive. In addition to dietary restriction of lactose-containing foods, lactase may be a food supplement. Therefore, the method of beta-galactosidase hydrolysis of lactose or whey will become the main method for obtaining lactose-free milk products.

The ferment is a product containing specific bioactive components and prepared by fermenting animals, plants, fungi and the like serving as raw materials through microorganisms. The ferment beverage has various physiological effects, such as regulating stomach and intestine, improving digestion and absorption; enhancing immunity and improving resistance; eliminating free radicals and delaying senility. The lactase is added into the milk product containing the ferment, so that the lactase has better synergistic effect of promoting in-vivo metabolism and decomposing toxic substances. The lactase has wide action conditions, good adaptability and good stability, and can effectively and rapidly improve the biochemical reaction rate. Therefore, by adding beta-galactosidase to the enzyme-containing dairy product, a functional dairy drink suitable for children can be produced.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a novel beta-galactosidase Gal42 and a preparation method thereof. The optimal reaction temperature of the beta-galactosidase Gal42 is 40 ℃, and the optimal reaction pH is 7.5; it shows a broad pH stability in the pH range of 6.0-9.0 and is suitable for the hydrolysis of lactose in milk. The enzyme has high yield, and is an ideal choice for producing lactose-free dairy products.

In one aspect, the invention provides a novel beta-galactosidase Gal42, the amino acid sequence of which is shown in SEQ ID No. 1.

SEQ ID NO.1：

MINEKLPKIWRFEDDGLKPDDEVWDEIRMFKLDGIDVDTLNVFSWDLNQPDEETYDFTWLDEQIDRLYENGIYTCLDTSTDDHPDWMDKKYPDVLRVDYQGRKRDFGGRHNSCPNSTRSYKYDERMDDRLGERYKDHPGVLIWHVSNEYGGYCYCDNCDDSFRKWLQQKYGTLQNVNKDWNTRFWGHTFYDWDEIVPPNVLSEEWEGDSTNFQGISLDYRIFQSDSLLECFKLERDDLKKHTPNLPETTNLMGTYKELDYFKWGKEMHVVSWDNYPDYDTPVSFTDMDHDLMTALKSGQPFMLMEQTPSQQNWQPYNSLKRPGVMRLWSYQDVDRGDETVMFFQLRRSVGDCEKYHGDVIEHVGHENTRVFRETDDLGKELGNLSDDLLDDRVQDKVDIVFDWENRWDTELSSGPSKDLDYVKEVHNYYDDLFDENIPVDMIGVDEDLSKYEIVIDPVLYMVKSGYDDRVKEFVQNGGTFVTTFFSGIVNEHDLVTLGGYPGELRDLLGIWVEEIDDLPPEVKNQIVITNDTGRLTGTYECRLLFDIIHSEGDDVLDEYGSAFYFGTPVITRHTYGKGKTYYVGTCPDQDFLTRFMKTVCAEKEIDPLLNVPKGVEVTERRKRGESYFFIMNHNDSTVELEIGEGTHLLTGKELTGDTSLEDYRGEGTA

In another aspect, the invention also provides a nucleic acid sequence corresponding to the novel beta-galactosidase Gal42, which is shown as SEQ ID No. 2.

SEQ ID NO.2：

ATGATCAACGAAAAACTGCCGAAAATCTGGCGTTTCGAAGACGACGGTCTGAAACCGGACGACGAAGTTTGGGACGAAATCCGTATGTTCAAACTGGACGGTATCGACGTTGACACCCTGAACGTTTTCTCTTGGGACCTGAACCAGCCGGACGAAGAAACCTACGACTTCACCTGGCTGGACGAACAGATCGACCGTCTGTACGAAAACGGTATCTACACCTGCCTGGACACCTCTACCGACGACCACCCGGACTGGATGGACAAAAAATACCCGGACGTTCTGCGTGTTGACTACCAGGGTCGTAAACGTGACTTCGGTGGTCGTCACAACTCTTGCCCGAACTCTACCCGTTCTTACAAATACGACGAACGTATGGACGACCGTCTGGGTGAACGTTACAAAGACCACCCGGGTGTTCTGATCTGGCACGTTTCTAACGAGTACGGTGGTTACTGCTACTGCGACAACTGCGACGACTCTTTCCGTAAATGGCTGCAGCAGAAATACGGTACCCTGCAGAACGTTAACAAAGACTGGAACACCCGTTTCTGGGGTCACACCTTCTACGACTGGGACGAAATCGTTCCGCCGAACGTTCTGTCTGAAGAATGGGAAGGTGACTCTACCAACTTCCAGGGTATCTCTCTGGACTACCGTATCTTCCAGTCTGACTCTCTGCTGGAATGCTTCAAACTGGAACGTGACGACCTGAAAAAACACACCCCGAACCTGCCGGAAACCACCAACCTGATGGGTACCTACAAAGAACTGGACTACTTCAAATGGGGTAAAGAAATGCACGTTGTTTCTTGGGACAACTACCCGGACTACGACACCCCGGTTTCTTTCACCGACATGGACCACGACCTGATGACCGCTCTGAAATCTGGTCAGCCGTTCATGCTGATGGAACAGACCCCGTCTCAGCAGAACTGGCAGCCGTACAACTCTCTGAAACGTCCGGGTGTTATGCGTCTGTGGTCTTACCAGGACGTTGACCGTGGTGACGAAACCGTTATGTTCTTCCAGCTGCGTCGTTCTGTTGGTGACTGCGAAAAATACCACGGTGACGTTATCGAACACGTTGGTCACGAAAACACCCGTGTTTTCCGTGAAACCGACGACCTGGGTAAAGAACTGGGTAACCTGTCTGACGACCTGCTGGACGACCGTGTTCAGGACAAAGTTGACATCGTTTTCGACTGGGAAAACCGTTGGGACACCGAACTGTCTTCTGGTCCGTCTAAAGACCTGGACTACGTTAAAGAAGTTCACAACTACTACGACGACCTGTTCGACGAAAACATCCCGGTTGACATGATCGGTGTTGACGAAGACCTGTCTAAATACGAAATCGTTATCGACCCGGTTCTGTACATGGTTAAATCTGGTTACGACGACCGTGTTAAAGAATTCGTTCAGAACGGTGGTACCTTCGTTACCACCTTCTTCTCTGGTATCGTTAACGAACACGACCTGGTTACCCTGGGTGGTTACCCGGGTGAACTGCGTGACCTGCTGGGTATCTGGGTTGAAGAAATCGACGACCTGCCGCCGGAAGTTAAAAACCAGATCGTTATCACCAACGACACCGGTCGTCTGACCGGTACCTACGAATGCCGTCTGCTGTTCGACATCATCCACTCTGAAGGTGACGACGTTCTGGACGAATACGGTTCTGCTTTCTACTTCGGTACCCCGGTTATCACCCGTCACACCTACGGTAAAGGTAAAACCTACTACGTTGGTACCTGCCCGGACCAGGACTTCCTGACCCGTTTCATGAAAACCGTTTGCGCTGAAAAAGAAATCGACCCGCTGCTGAACGTTCCGAAAGGTGTTGAAGTTACCGAACGTCGTAAACGTGGTGAATCTTACTTCTTCATCATGAACCACAACGACTCTACCGTTGAACTGGAAATCGGTGAAGGTACCCACCTGCTGACCGGTAAAGAACTGACCGGTGACACCTCTCTGGAAGACTACCGTGGTGAAGGTACCGCTTAA

On the other hand, the invention also provides a preparation and purification method of the beta-galactosidase Gal 42.

On the other hand, the invention also provides the application of the beta-galactosidase Gal42 in degrading lactose in milk.

On the other hand, the beta-galactosidase selected by the method for degrading lactose in milk is Gal 42.

Preferably: the reaction temperature in the degradation condition is 0-70 ℃. The optimum reaction temperature is 40 ℃.

Preferably: the reaction pH value in the degradation condition is 5.5-10.5. The optimum reaction pH was 7.5.

Has the advantages that:

1. the beta-galactosidase Gal42 of the present invention is a novel beta-galactosidase cloned from marine bacteria and has an amino acid sequence with only 89.02% similarity (Identity) to the similar sequence in Genbank.

2. The invention provides a method for preparing beta-galactosidase Gal42, namely, a genetic engineering technical method is utilized, a gene sequence of Gal42 is expressed to escherichia coli in a heterogenous recombination mode, after fermentation, the enzyme activity of a fermentation solution is as high as 306.3U/mL, most of produced proteins exist in a soluble form, and the beta-galactosidase Gal42 has potential of industrial production. The enzyme purification method is simple, and one-step affinity purification is carried out on the enzyme by utilizing a nickel column.

3. The beta-galactosidase Gal42 of the invention has excellent physicochemical properties, the optimal reaction temperature and pH are respectively 40 ℃ and 7.5, the pH value of the beta-galactosidase Gal42 shows wide pH stability in the pH range of 6.0-9.0, and the beta-galactosidase Gal42 is suitable for the hydrolysis of lactose in milk. The beta-galactosidase of the invention has high yield, is an ideal choice for producing lactose-free dairy products, and has good industrial application prospect.

Drawings

FIG. 1 shows the separation and purification scheme of the beta-galactosidase Gal42 protein of the present invention (M, protein standard; 1, purified beta-galactosidase Gal 42);

FIG. 2 is a temperature and pH adaptation analysis of β -galactosidase Gal42 of the present invention (optimum reaction temperature for A, β -galactosidase Gal 42; temperature stability for B, β -galactosidase Gal 42; optimum reaction pH for C, β -galactosidase Gal 42; pH stability for D, β -galactosidase Gal 42);

FIG. 3 shows the detection of the enzymatic hydrolysate of beta-galactosidase Gal42 in lactose and milk by Thin Layer Chromatography (TLC) (A, TLC analysis of Gal42 for lactose hydrolysis; B, TLC analysis of Gal42 for milk hydrolysis; M1, galacto-oligosaccharide standard; M2, galactose standard; 0-8 for degrading lactose or milk by the enzyme for 0min, 1min, 10min, 30min, 60min, 360min, 480 min).

Detailed Description