阅读说明：本技术 一种新型β-半乳糖苷酶GalA及其应用 (Novel β -galactosidase GalA and application thereof ) 是由 陈豪 董娟 陈鹏 狄国虎 吴祥根 于 2020-04-22 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GalA及其应用。所述β-半乳糖苷酶GalA氨基酸序列如SEQ ID NO.1所示。本发明提供一种所述β-半乳糖苷酶GalA的重组表达及纯化制备方法,发酵液酶活力可达3795.6U/mL,一步纯化纯度>97％,回收率为79.5％。本发明所述β-半乳糖苷酶GalA具有良好的热稳定性和储存稳定性,最适反应温度为50℃,在50℃下持续孵育24h仍保留89.7％的酶活力,室温下(30℃)储存30天,仍然能保留81.5％的酶活力。本发明所述β-半乳糖苷酶GalA活力高、稳定性好,降解乳糖能力强,具有良好的应用潜质。(The invention relates to a novel β -galactosidase GalA and application thereof, wherein an amino acid sequence of the β -galactosidase GalA is shown as SEQ ID No. 1. the invention provides a recombinant expression and purification preparation method of the β -galactosidase GalA, wherein the enzyme activity of a fermentation broth can reach 3795.6U/m L, the one-step purification purity is more than 97%, and the recovery rate is 79.5%.)

1. A novel β -galactosidase GalA, the amino acid sequence of which is shown in SEQ ID NO. 1.

2. The nucleotide sequence corresponding to β -galactosidase GalA according to claim 1, which is represented by seq id No. 2.

3. The process for the preparation and purification of β -galactosidase GalA according to claim 1.

4. Use of the β -galactosidase GalA of claim 1 for degrading lactose.

5. A method for degrading lactose in milk, wherein the β -galactosidase used is the β -galactosidase GalA of claim 1.

6. The method according to claim 5, wherein the degradation condition is a reaction temperature of 0 to 70 ℃ and an optimum reaction temperature of 50 ℃.

Technical Field

The invention relates to a novel β -galactosidase GalA and application thereof, belonging to the technical field of biology.

Background

β -galactosidase (EC 3.2.1.23) is a glycoside hydrolase that catalyzes lactose hydrolysis and transgalactosylation As a commonly used medical enzyme, β -galactosidase has been widely used in the dairy industry to break down lactose into galactose and glucose in addition, β -galactosidase can also produce galactooligosaccharides by transglycosylation during the hydrolysis of lactose or other structurally related galactosides β -galactosidase can be widely used in nutrition and food processing.

The hydrolytic activity of β -galactosidase has been used in the food industry for decades to help absorb undigested lactose. worldwide, particularly in east asia, many children and adults are facing the problem of lactose dyspepsia and lactase deficiency-induced intolerance.

Although β -galactosidase has been purified and identified from various organisms, such as bacteria, fungi, yeast, plants, and mammals, β -galactosidase available for commercial use is still scarce due to problems of yield, stability, degradation efficiency, etc. moreover, β -galactosidase currently marketed is expensive, of a single variety, and cannot meet the application under specific conditions, and thus, there is an urgent need to develop new β -galactosidase.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a novel β -galactosidase GalA and a preparation method thereof, the invention provides a recombinant expression and purification preparation method of β -galactosidase GalA, the enzyme activity of fermentation broth can reach 3795.6U/m L, the one-step purification purity is more than 97%, and the recovery rate is 79.5%.

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

SEQ ID NO.1：

MINEKLPKIWRFEDDGLKPDDEVWDEIRMFKLDGIDVDTLNVFSWDLNQPDEETYDFTWLDEQIDRLYENGIYTCLDTSTDDHPDWMDKKYPDVLRVDYQGRKRDFGGRHNSCPNSTRSYKYDERMDDRLGERYKDHPGVLIWHVSNMYGGYCYCDNCDDSFRKWLQQKYGTLQNVNKDWNTRFWGHTFYDWDEIVPPNVLSEEWEGDSTNFQGISLDYRIFQSDSLLECFKLERDDLKKHTPNLPETTNLMGTYKELDYFKWGKEMHVVSWDNYPDYDTPVSFTDMDHDLMTALKSGQPFMLMEQTPSQQNWQPYNSLKRPGVMRLWSYQDVDRGDETVMFFQLRRSVGDCEKYHGDVIEHVGHENTRVFRETDDLGKELGNLSDDLLDDRVQDKVDIVFDWENRWDTELSSGPSKDLDYVKEVHNYYDDLFDENIPVDMIGVDEDLSKYEIVIDPVLYMVKSGYDDRVKEFVQNGGTFVTTFFSGIVNEHDLVTLGGYPGELRDLLGIWVEEIDDLPPEVKNQIVITNDTGRLTGTYECRLLFDIIHSEGDDVLDEYGSAFYFGTPVITRHTYGKGKTYYVGTCPDQDFLTRFMKTVCAEKEIDPLLNVPKGVEVTERRKRGESYFFIMNHNDSTVELEIGEGTHLLTGKELTGDTSLEDYRGEGTA

In another aspect, the invention also provides a corresponding nucleic acid sequence of the β -galactosidase GalA, which is shown as SEQ ID No. 2.

SEQ ID NO.2：

ATGATCAACGAAAAACTGCCGAAAATCTGGCGTTTCGAAGACGACGGTCTGAAACCGGACGACGAAGTTTGGGACGAAATCCGTATGTTCAAACTGGACGGTATCGACGTTGACACCCTGAACGTTTTCTCTTGGGACCTGAACCAGCCGGACGAAGAAACCTACGACTTCACCTGGCTGGACGAACAGATCGACCGTCTGTACGAAAACGGTATCTACACCTGCCTGGACACCTCTACCGACGACCACCCGGACTGGATGGACAAAAAATACCCGGACGTTCTGCGTGTTGACTACCAGGGTCGTAAACGTGACTTCGGTGGTCGTCACAACTCTTGCCCGAACTCTACCCGTTCTTACAAATACGACGAACGTATGGACGACCGTCTGGGTGAACGTTACAAAGACCACCCGGGTGTTCTGATCTGGCACGTTTCTAACATGTACGGTGGTTACTGCTACTGCGACAACTGCGACGACTCTTTCCGTAAATGGCTGCAGCAGAAATACGGTACCCTGCAGAACGTTAACAAAGACTGGAACACCCGTTTCTGGGGTCACACCTTCTACGACTGGGACGAAATCGTTCCGCCGAACGTTCTGTCTGAAGAATGGGAAGGTGACTCTACCAACTTCCAGGGTATCTCTCTGGACTACCGTATCTTCCAGTCTGACTCTCTGCTGGAATGCTTCAAACTGGAACGTGACGACCTGAAAAAACACACCCCGAACCTGCCGGAAACCACCAACCTGATGGGTACCTACAAAGAACTGGACTACTTCAAATGGGGTAAAGAAATGCACGTTGTTTCTTGGGACAACTACCCGGACTACGACACCCCGGTTTCTTTCACCGACATGGACCACGACCTGATGACCGCTCTGAAATCTGGTCAGCCGTTCATGCTGATGGAACAGACCCCGTCTCAGCAGAACTGGCAGCCGTACAACTCTCTGAAACGTCCGGGTGTTATGCGTCTGTGGTCTTACCAGGACGTTGACCGTGGTGACGAAACCGTTATGTTCTTCCAGCTGCGTCGTTCTGTTGGTGACTGCGAAAAATACCACGGTGACGTTATCGAACACGTTGGTCACGAAAACACCCGTGTTTTCCGTGAAACCGACGACCTGGGTAAAGAACTGGGTAACCTGTCTGACGACCTGCTGGACGACCGTGTTCAGGACAAAGTTGACATCGTTTTCGACTGGGAAAACCGTTGGGACACCGAACTGTCTTCTGGTCCGTCTAAAGACCTGGACTACGTTAAAGAAGTTCACAACTACTACGACGACCTGTTCGACGAAAACATCCCGGTTGACATGATCGGTGTTGACGAAGACCTGTCTAAATACGAAATCGTTATCGACCCGGTTCTGTACATGGTTAAATCTGGTTACGACGACCGTGTTAAAGAATTCGTTCAGAACGGTGGTACCTTCGTTACCACCTTCTTCTCTGGTATCGTTAACGAACACGACCTGGTTACCCTGGGTGGTTACCCGGGTGAACTGCGTGACCTGCTGGGTATCTGGGTTGAAGAAATCGACGACCTGCCGCCGGAAGTTAAAAACCAGATCGTTATCACCAACGACACCGGTCGTCTGACCGGTACCTACGAATGCCGTCTGCTGTTCGACATCATCCACTCTGAAGGTGACGACGTTCTGGACGAATACGGTTCTGCTTTCTACTTCGGTACCCCGGTTATCACCCGTCACACCTACGGTAAAGGTAAAACCTACTACGTTGGTACCTGCCCGGACCAGGACTTCCTGACCCGTTTCATGAAAACCGTTTGCGCTGAAAAAGAAATCGACCCGCTGCTGAACGTTCCGAAAGGTGTTGAAGTTACCGAACGTCGTAAACGTGGTGAATCTTACTTCTTCATCATGAACCACAACGACTCTACCGTTGAACTGGAAATCGGTGAAGGTACCCACCTGCTGACCGGTAAAGAACTGACCGGTGACACCTCTCTGGAAGACTACCGTGGTGAAGGTACCGCT

On the other hand, the invention also provides a preparation and purification method of the β -galactosidase GalA.

In another aspect, the invention also provides the application of the β -galactosidase GalA in degrading lactose.

On the other hand, the β -galactosidase used in the method for degrading lactose in milk is GalA.

Preferably: the reaction temperature in the degradation condition is 0-70 ℃. The optimum reaction temperature is 50 ℃.

Has the advantages that:

1. the β -galactosidase GalA is novel β -galactosidase, the highest amino acid sequence similarity with the β -galactosidase is only 88.87% in a database of Genbank, and the β -galactosidase is β -galactosidase with novel sequence and structure.

2. The invention provides a method for preparing β -galactosidase, namely, a genetic engineering technical method is utilized, a gene sequence of β -galactosidase GalA is heterologously recombined and expressed to escherichia coli, after fermentation, the enzyme activity of fermentation broth reaches 3795.6U/m L.

3. The β -galactosidase GalA disclosed by the invention has good thermal stability and storage stability, high enzyme activity can be still remained after continuous incubation for 24 hours at 50 ℃, the β -galactosidase GalA is suitable for large-scale continuous degradation, more than 80% of enzyme activity can be still remained after storage for 30 days at normal temperature, the storage is convenient, and the application potential is good.

In conclusion, the β -galactosidase GalA has novel sequence, high yield, simple preparation, stable property and good industrial application potential.

Drawings

FIG. 1 is a diagram showing the separation and purification of the protein of β -galactosidase GalA of the present invention (M, protein standard; 1, β -galactosidase GalA obtained by purification);

FIG. 2 is a graph showing the thermostability of β -galactosidase GalA incubated at 50 ℃ in accordance with the present invention;

FIG. 3 is a graph showing the storage stability of β -galactosidase GalA of the present invention at room temperature of 30 ℃;

FIG. 4 is a diagram of thin layer chromatography (T L C) for detecting the degradation products of β -galactosidase GalA in milk (1, glucose standard; 2, galactose standard; 3, lactose substrate; 4, polyoligosaccharide standard; 5, lactose degradation 10min, 6, lactose degradation 20min, 7, lactose degradation 40min, 8, lactose degradation 60 min).

Detailed Description