Efficient and stable α -galactosidase, and coding gene and application thereof
阅读说明：本技术 一种高效、稳定的α-半乳糖苷酶及其编码基因和应用 (Efficient and stable α -galactosidase, and coding gene and application thereof ) 是由 袁红莉 张真明 刘亮 杨金水 于 2018-08-02 设计创作，主要内容包括：本发明公开了一种高效、稳定的α-半乳糖苷酶及其编码基因和应用。本发明提供的蛋白质,是：由序列1自N末端第20至437位氨基酸残基组成的蛋白质；由序列1所示的氨基酸序列组成的蛋白质；由序列表的序列3第90-513位氨基酸残基组成的蛋白质；由序列3所示的氨基酸序列组成的蛋白质。本发明还保护所述蛋白质的应用,为：作为α-半乳糖苷酶；降解含α-半乳糖苷键物质。本发明提供的蛋白可满足饲料、食品等工业中对α-半乳糖苷酶热稳定性、pH稳定性和蛋白酶抗性的要求,具有更广泛的工业应用,为进一步工业化高产工程菌株的构建提供了材料。本发明具有非常好的研究前景和商业应用价值。(The invention discloses a high-efficiency and stable α -galactosidase, a coding gene and application thereof.A protein provided by the invention is a protein consisting of 20 th to 437 th amino acid residues from the N terminal of a sequence 1, a protein consisting of an amino acid sequence shown in the sequence 1, a protein consisting of 90 th to 513 th amino acid residues in a sequence 3 of a sequence table, and a protein consisting of an amino acid sequence shown in the sequence 3.)
1. A protein which is (a) or (b) or (c) or (d) or (e) or (f) below:
(a) protein consisting of 20 th to 437 th amino acid residues from the N terminal of a sequence 1 in a sequence table;
(b) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;
(c) protein consisting of 90 th-513 th amino acid residues in a sequence 3 in a sequence table;
(d) a protein consisting of an amino acid sequence shown in a sequence 3 in a sequence table;
(e) the amino acid sequence of (a) or (b) or (c) or (d) is substituted and/or deleted by one or more amino acid residues and/or added with a protein which has α -galactosidase activity and is derived from the amino acid sequence;
(f) and (C) a fusion protein obtained by connecting a tag to the N-terminus or/and the C-terminus of (a), (b), (C), (d) or (e).
2. A nucleic acid molecule encoding the protein of claim 1.
3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a DNA molecule as described in any one of (1) to (6) below:
(1) the coding region is shown as the DNA molecule from the 58 th to 1311 st nucleotides at the 5' end of the sequence 2 in the sequence table;
(2) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;
(3) the coding region is DNA molecule shown as 268 th to 1539 th nucleotides from 5' end of sequence 4 in the sequence table;
(4) the coding region is a DNA molecule shown as a sequence 4 in the sequence table;
(5) a DNA molecule which hybridizes with the DNA sequence defined in (1) or (2) or (3) or (4) under stringent conditions and encodes the protein of claim 1;
(6) a DNA molecule derived from Rapex baileyi, having 90% or more homology with the DNA sequence defined in (1) or 2), and encoding the protein of claim 1.
4. An expression cassette, recombinant expression vector or recombinant bacterium comprising the nucleic acid molecule of claim 2 or 3.
5. A primer pair for amplifying the full length or any fragment of the nucleic acid molecule of claim 2 or 3.
6. A method of producing the protein of claim 1, comprising: fermenting the recombinant bacterium according to claim 4 to express a nucleic acid molecule encoding the protein according to claim 1, thereby obtaining the protein according to claim 1.
7. The use of the protein of claim 1 in the following (I), (II), (III) or (IV):
as α -galactosidase;
(II) preparing a product with α -galactosidase function;
(III) degrading substances containing α -galactose glycosidic bond;
(IV) preparing a product with the function of degrading the substance containing α -galactose glycosidic bond.
8. Use of the nucleic acid molecule of claim 2 or 3, the expression cassette of claim 4, the recombinant expression vector or the recombinant bacterium as (i), (ii) or (iii) below:
preparing the protein of claim 1;
(ii) preparing a product having α -galactosidase function;
(iii) preparing a product having a function of degrading a material containing α -galactosyl bonds.
9. The use according to claim 7 or 8, wherein the substance containing α -galactoside bonds is an oligosaccharide containing α -galactoside bonds.
10. The use of claim 9, wherein: the oligosaccharide is melibiose, raffinose or stachyose.
Technical Field
The invention belongs to the technical field of biology, and relates to high-efficiency and stable α -galactosidase, and a coding gene and application thereof.
Background
α -galactosidase (α -galactosidase, EC 3.2.1.22), also known as melibiase, specifically hydrolyzes α -galactoside linkages of the non-reducing end of oligosaccharides or polysaccharides, and is therefore used in a large number of applications in the food, sugar, paper, feed and medical fields.
In the field of food and feed processing, α -galactosidase can be used as a food and feed additive, hydrolyzes α -galacto-oligosaccharide in anti-nutritional factors such as raffinose, stachyose, melibiose and the like in bean products and bean pulp, thereby relieving phenomena such as diarrhea, abdominal distension, stomachache and the like caused by the anti-nutritional factors, promoting the bean products to be better absorbed and utilized by human bodies, improving the food intake of monogastric animals, and further improving the production performance of the animals.
In the sugar industry, α -galactosidase can hydrolyze raffinose in molasses, reduce molasses viscosity, promote sucrose crystal precipitation, and improve raw material utilization rate and sucrose quality, and the reduction of viscosity can also reduce cost caused by reheating in the sugar production process, save energy, and greatly improve equipment processing capacity and turnover rate.
In the paper industry, α -galactosidase is used to hydrolyze galactomannan from pulp and remove α -galactose from the backbone, thus, the bleaching of softwood pulp is enhanced by the combined treatment of the pulp with enzyme preparations such as α -galactosidase, mannanase and xylanase.
In the medical field, α -galactosidase can be used for treating Fabry disease (Fabry disease) and blood group conversion.A deletion of α -galactosidase from lysosomes in humans causes accumulation of glycophospholipids and ultimately affects the function of pericardium, kidney and central nervous system and gastrointestinal tract, and exogenous intake of α -galactosidase can effectively remove α -galactose residues at the ends of the glycophospholipids, thereby alleviating Fabry disease.A most outer end of a sugar chain on the surface of a B-type blood erythrocyte has a α -1, 3 glycosidic bond-linked galactose residue compared with O-type blood, and the conversion between B-type blood and O-type blood can be achieved by removing the α -galactose residue with α -galactosidase.
α -galactosidase is widely present in animals, plants and microorganisms, particularly, the highest yield in microorganisms, α -galactosidase of plant origin is low and enzyme activity is unstable, α -galactosidase of animal origin is mostly from pancreas, the yield is limited by its origin, and microorganisms used as vectors for producing α -galactosidase have the advantages of high yield, stable quality, strong production controllability and suitability for industrialization, which are considered as the main approaches for the modern development of α -galactosidase preparations.
The optimal action condition, pH and temperature stability of α -galactosidase determine the application range and commercial value of the galactosidase in practical production, the common α -galactosidase has poor thermal stability and pH stability, which limits the application of the galactosidase in food and feed processing, while the α -galactosidase with thermal stability and pH stability is not researched much, the α -galactosidase reported at present can show higher activity on a model substrate p-nitrophenyl- α -D-galactopyranoside (pNPG), but has not high hydrolysis activity on natural substrates such as stachyose, raffinose and melibiose, and the like.
Disclosure of Invention
The invention aims to provide high-efficiency and stable α -galactosidase, and a coding gene and application thereof.
The protein provided by the invention is obtained from Rapexlateus leucadendrus (Irpexlateus) and is named as ILgalA protein, and is (a), (b), (c), (d), (e) or (f) as follows:
(a) protein consisting of 20 th to 437 th amino acid residues from the N terminal of a
(b) a protein consisting of an amino acid sequence shown in a
(c) protein consisting of 90 th-513 th amino acid residues in a
(d) a protein consisting of an amino acid sequence shown in a
(e) the amino acid sequence of (a) or (b) or (c) or (d) is substituted and/or deleted by one or more amino acid residues and/or added with a protein which has α -galactosidase activity and is derived from the amino acid sequence;
(f) and (C) a fusion protein obtained by connecting a tag to the N-terminus or/and the C-terminus of (a), (b), (C), (d) or (e).
The labels may be as shown in table 1.
TABLE 1
Label (R)
Residue of
Sequence of
Poly-Arg
5-6 (usually 5)
RRRRR
Poly-His
2-10 (generally 6)
FLAG
8
DYKDDDDK
Strep-
8
WSHPQFEK
c-
10
EQKLISEEDL
The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and performing biological expression. The coding gene of the protein can be obtained by deleting one or more codons of amino acid residues in a DNA sequence shown in a
Nucleic acid molecules encoding such proteins are also within the scope of the invention.
The nucleic acid molecule is a DNA molecule as described in any one of (1) to (6) below:
(1) the coding region is shown as the DNA molecule from the 58 th to 1311 st nucleotides at the 5' end of the
(2) the coding region is a DNA molecule shown as a
(3) the coding region is DNA molecule shown as 268 th to 1539 th nucleotides from 5' end of
(4) the coding region is a DNA molecule shown as a
(5) a DNA molecule which hybridizes with the DNA sequence defined in (1) or (2) or (3) or (4) under stringent conditions and encodes the protein;
(6) a DNA molecule which is derived from the rakanka albicans, has more than 90% homology with the DNA sequence defined in (1) or 2) and encodes the protein.
The stringent conditions can be hybridization and washing with 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution at 65 ℃ in DNA or RNA hybridization experiments.
The DNA molecule may be produced by natural variation or artificial mutation.
The expression cassette, the recombinant expression vector or the recombinant bacterium containing the nucleic acid molecule all belong to the protection scope of the invention.
The recombinant expression vector may be a plasmid, cosmid, phage, or viral vector.
The recombinant expression vector can be specifically recombinant plasmid pPICZ α A-ILgalA, namely recombinant plasmid obtained by inserting DNA molecules shown by 250 th to 1542 th nucleotides from 5' tail end of
The recombinant bacterium can be specifically a linearized recombinant plasmid pPICZ α A-ILgalA introduced into Pichia pastoris x-33 to obtain recombinant yeast x-33-pPICZ α A-ILgalA, and the linearized recombinant plasmid pPICZ α A-ILgalA can be specifically a linearized plasmid obtained by digesting the recombinant plasmid pPICZ α A-ILgalA with restriction enzyme SacI.
Primer pairs for amplifying the full length or any fragment of the nucleic acid molecule are within the scope of the invention.
The invention also provides a method for preparing the protein, which comprises the following steps: and fermenting the recombinant bacteria to express the nucleic acid molecule for encoding the protein to obtain the protein.
The method specifically comprises the following steps: culturing the recombinant bacteria, performing methanol induction in the culture process, and then centrifuging to collect supernatant.
The method specifically comprises the following steps: culturing the recombinant bacteria to OD by adopting BMMY culture medium600nmAfter that, the mixture was cultured at 30 ℃ with shaking at 250rpm (methanol was added to the system every 24 hours so that the concentration in the system was 1%), and then centrifuged for 10min to collect the supernatant.
The method specifically comprises the following steps: culturing the recombinant bacteria to OD by adopting BMMY culture medium600nmAfter that, the mixture was cultured at 30 ℃ for 144 hours with shaking at 250rpm (methanol was added to the system every 24 hours so that the concentration in the system was 1%), and then centrifuged at 12000rpm for 10 minutes, and the supernatant was collected.
The method further comprises the steps of: and (4) dialyzing the supernatant to remove salt, and then performing Ni column affinity chromatography purification.
The invention also protects the application of the protein, which is (I), (II), (III) or (IV) as follows:
as α -galactosidase;
(II) preparing a product with α -galactosidase function;
(III) degrading substances containing α -galactose glycosidic bond;
(IV) preparing a product with the function of degrading the substance containing α -galactose glycosidic bond.
The invention also protects the application of the gene, the expression cassette, the recombinant expression vector or the recombinant bacterium, which is (i), (ii) or (iii) as follows:
preparing said protein;
(ii) preparing a product having α -galactosidase function;
(iii) preparing a product having a function of degrading a material containing α -galactosyl bonds.
The substance containing α -galactose glycosidic bond is oligosaccharide containing α -galactose glycosidic bond.
The oligosaccharide is melibiose, raffinose or stachyose.
The protein provided by the invention has the specific activity of α -galactosidase of 1012U/mg, the reaction temperature can be 22-90 ℃, preferably 60-70 ℃, and most preferably 70 ℃, the protein has excellent temperature stability, the enzyme activity can still be kept above 90% when the protein is kept at 50 ℃ or 60 ℃ for 10h, the reaction pH can be 3.2-7.5, preferably 4.0-5.5, and most preferably 4.8, the protein has excellent pH stability, the enzyme activity can be kept above 60% when the protein is kept at pH 3-11 for 2h, the enzyme activity can be kept above 80% when the protein is kept at pH 7-11, the activities of melibiose, raffinose and stachyose can respectively reach 644, 755U/mg, wherein the activities of the protein in water are respectively highest in the currently reported α -galactosidase, the highest in the activity of 1.0mg/mL protease, trypsin and stachyose can respectively keep above 90% when the protein is incubated with 1.0mg/mL of pepsin, trypsin and stachyose, the activity of the protein in the currently reported α -galactosidase can also be kept above 90% when the protein is incubated with 1.7-7K, and stachyose, the enzyme can be above 30.7-3-7.
The α -galactosidase prepared by the invention has better pH stability, thermal stability and protease resistance, the protein provided by the invention can meet the requirements of α -galactosidase on thermal stability, pH stability and protease resistance in industries such as feed, food and the like, has wider industrial application, and provides a material for further constructing industrial high-yield engineering strains.
Drawings
FIG. 1 is an electrophoretogram in example 2.
FIG. 2 shows the results of the optimum pH measurement in example 4.
FIG. 3 shows the results of pH stability measurement in example 4.
FIG. 4 shows the results of the optimum reaction temperature in example 4.
FIG. 5 shows the results of temperature stability in example 4.
FIG. 6 shows the results of protease resistance in example 4.
FIG. 7 shows the results of example 5.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Irpexliteus (Irpexliteus): CGMCC No. 5.809, pPICZ α A vector, Invitrogen, product No. 69909 Pichia pastoris (Pichia pastoris) X-33, Pichia pastoris X-33 for short, Invitrogen, product No. C18000.
Melibiose: SIGMA company, product code: 63630. raffinose: SIGMA, product coding: r0250. stachyose: TCI company, product code: s0397.
BMGY medium: peptone 2g/100ml, yeast extract 1g/100ml, YNB 1.34g/100ml,
BMMY medium: peptone 2g/100ml, yeast extract 1g/100ml, 1.34g/100ml YNB, 1% (volume ratio) methanol, 4X 10-5g/100ml Biotin, the balance being phosphate buffer (pH6.0, 0.1 mol/L); sterilizing at 121 deg.C for 20 min.
pNPG, all known as 4-nitrophenyl- α -D-galactopyranoside.