CN106884025B - Method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis - Google Patents

Abstract

Translated fromChinese

本发明提供一种酶法水解定向制备褐藻胶寡糖的方法，其中使用的褐藻胶裂解酶，其氨基酸序列为SEQ ID NO:3。本发明利用基因改造的重组褐藻胶裂解酶，定向制备褐藻胶寡糖，与现有酸法水解制备褐藻胶寡糖的方法相比，克服了破坏性大、效率低的缺点，与现有的酶法水解制备褐藻胶寡糖的方法相比，运用改造的重组褐藻胶裂解酶，克服了酶解时间长、成本高、不能定向制备特定聚合度的褐藻胶寡糖的缺点。本发明过程简单、成本低且适用于工业化生产，能够在短时间内降低褐藻胶粘度，所制寡糖的总得率在90％以上。所制备的褐藻胶寡糖有抗肿瘤、抗炎、降血脂和提高免疫力等功效，可用于食品及保健品领域，具有广泛的应用前景。

The present invention provides a method for directional preparation of alginate oligosaccharide by enzymatic hydrolysis, wherein the alginate lyase used therein has an amino acid sequence of SEQ ID NO:3. The invention utilizes genetically modified recombinant alginate lyase to directionally prepare alginate oligosaccharide, and compared with the existing method for preparing alginate oligosaccharide by acid hydrolysis, it overcomes the shortcomings of large destructiveness and low efficiency, and is in contrast to the existing method. Compared with the method for preparing alginate oligosaccharide by enzymatic hydrolysis, using the modified recombinant alginate lyase overcomes the disadvantages of long enzymatic hydrolysis time, high cost, and inability to orientately prepare alginate oligosaccharide with a specific degree of polymerization. The invention has simple process, low cost and is suitable for industrial production, can reduce the viscosity of alginate in a short time, and the total yield of the prepared oligosaccharide is over 90%. The prepared alginate oligosaccharide has anti-tumor, anti-inflammatory, blood lipid-lowering and immunity-improving effects, can be used in the fields of food and health care products, and has wide application prospects.

Description

Method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis

Technical Field

The invention belongs to the technical field of alginate oligosaccharide preparation, and particularly relates to a method for directionally preparing alginate oligosaccharide by enzymatic hydrolysis.

Background

The algin oligosaccharide (alginate oligosaccharides) is a low molecular weight fragment with small polymerization degree and good water solubility obtained by hydrolyzing algin, and can be widely applied to the fields of food, medicine, cosmetics and the like due to the various physiological activities of blood fat reduction, virus resistance, tumor resistance, oxidation resistance and the like, and has huge application and development values.

The preparation method of the algin oligosaccharide mainly comprises acid hydrolysis and enzyme hydrolysis. Acid hydrolysis can destroy the structure of alginate oligosaccharides to different degrees, and enzymatic hydrolysis has the advantages of mild reaction conditions and the like, so that the preparation of alginate oligosaccharides by enzymatic hydrolysis is widely concerned. The marine microorganism is an important source of alginate lyase, and some of the alginate lyase are separated from vibrio, alteromonas and pseudoalteromonas in sequence. However, due to the particularity of marine environment, the activity of alginate lyase produced by marine microorganisms is limited, and the alginate oligosaccharides cannot be directionally prepared. Therefore, the search for alginate lyase with strong specificity and high activity is still the focus of attention of researchers.

Disclosure of Invention

The invention aims to provide a method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis, which can efficiently and directionally prepare target alginate oligosaccharides by modified enzyme and solve the defects of low efficiency and incapability of directionally preparing alginate oligosaccharides in the prior art.

The invention firstly provides a method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis, which comprises the following steps:

1) preparing a substrate: mixing the algin raw material with water to prepare an enzymolysis substrate solution with the concentration of 5-8% and the pH value of neutral;

2) step-by-step enzymolysis: adding alginate lyase, stirring and performing enzymolysis for 1-2h at 40-50 ℃, adding 0.3% -0.8% of alginate lyase again, stirring and performing enzymolysis for 2-4 h;

3) preparing oligosaccharide: and after the enzymolysis is finished, filtering or centrifuging to remove dregs, concentrating supernate, and freeze-drying to obtain the algal oligosaccharide.

In the above method, the amino acid sequence of the alginate lyase is SEQ ID NO. 1;

in order to obtain the alginate oligosaccharide with more concentrated polymerization degree, the invention optimizes the alginate lyase, and the amino acid sequence of the optimized alginate lyase is SEQ ID NO. 3;

the optimized algin lyase capable of directionally preparing algin oligosaccharide has an amino acid sequence of SEQ ID NO. 3:

MKVSCAVVLSACIASANADNNGDGKADSIKENDLNAGYADGTYFYTAADGGMVFRCPIDGYKTSTNTSYTRTELREMLRRGDTSIATQGVNGNNWVFGSAPASAREAAGGVDGVLRATLAVNHVTTTGDSGQVGRVIVGQIHANNDEPLRLYYRKLPGHSKGSVYIAHEPNGGSDSWYDMIGSRSSSASDPSDGIALDEVWSYEVKVVGNTLTVTIFRAGKDDVVQVVDMGNSGYDVADQYQYFKAGVYNQNNTGNASDYVQVTFYALEQSHD；

one nucleotide sequence of the gene for coding the algin lyase is SEQ ID NO:4:

ATGAAAGTAAGTTGCGCTGTCGTACTGTCTGCTTGTATTGCCAGTGCCAACGCAGACAACAATGGCGATGGCAAGGCCGACTCCATCAAGGAAAATGACCTGAATGCAGGCTATGCAGATGGCACCTACTTCTATACTGCTGCCGATGGCGGCATGGTGTTCCGCTGCCCGATCGATGGCTATAAAACATCGACCAACACGTCCTATACCCGCACCGAGCTGCGCGAGATGCTACGTCGTGGCGACACCAGCATTGCCACCCAGGGGGTCAATGGAAACAACTGGGTATTCGGCTCCGCACCCGCTTCGGCACGTGAAGCAGCCGGCGGTGTCGACGGTGTTTTACGCGCAACCCTCGCGGTAAACCATGTCACCACTACCGGAGATAGCGGCCAGGTTGGACGGGTGATTGTTGGACAGATTCACGCCAACAACGACGAACCGCTGCGTCTTTACTACCGCAAGTTACCGGGCCACAGCAAAGGTTCTGTGTATATCGCCCATGAGCCAAACGGCGGCAGCGACAGCTGGTACGACATGATTGGCAGCCGTTCCAGCAGCGCCTCGGACCCGTCCGACGGTATCGCACTGGATGAAGTCTGGAGCTACGAGGTCAAGGTTGTCGGTAACACCCTCACCGTGACCATCTTCCGTGCTGGTAAAGACGATGTGGTACAGGTTGTGGATATGGGCAACAGCGGTTACGACGTCGCCGACCAGTACCAGTACTTCAAGGCCGGGGTGTACAACCAGAACAACACCGGCAATGCCAGTGACTATGTCCAGGTGACCTTCTACGCCCTGGAGCAGTCGCACGATTAA。

the invention utilizes the recombinant algin lyase of gene modification to prepare algin oligosaccharide directionally, overcomes the defects of large destructiveness and low efficiency compared with the prior method for preparing algin oligosaccharide by acid hydrolysis, and overcomes the defects of long enzymolysis time, high cost and incapability of directionally preparing algin oligosaccharide with specific polymerization degree by utilizing the modified recombinant algin lyase compared with the prior method for preparing algin oligosaccharide by enzymatic hydrolysis. The method has the advantages of simple process, low cost, suitability for industrial production, capability of reducing the viscosity of the algin in a short time, and over 90 percent of total yield of the prepared oligosaccharide. The prepared alginate oligosaccharide has the effects of resisting tumor, resisting inflammation, reducing blood fat, improving immunity and the like, can be used in the fields of food and health care products, and has wide application prospect.

Drawings

FIG. 1: ESI-MS (ESI-MS) spectrum of alginate lyase enzymolysis product before modification

FIG. 2: comparing the gene sequences before and after modification;

FIG. 3: ESI-MS atlas of modified algin lyase zymolysis product;

FIG. 4: modified algin lyase enzymolysis product¹A C-NMR spectrum;

FIG. 5: the viscosity of the alginate substrate with different concentrations changes along with the enzymolysis time (A: the viscosity of the alginate degraded by the alginate lyase before modification changes along with the enzymolysis time; B: the viscosity of the recombinant alginate lyase after modification degrades the alginate changes along with the enzymolysis time).

Detailed Description

The present invention will be described in detail with reference to examples.

Example 1: preparation of alginate oligosaccharide by using alginase lyase before modification

Dissolving algin in water with pH value of 7 adjusted by NaOH to prepare 200mL of alginate solution with concentration of 5%, adding 2mL of alginate lyase (amino acid sequence is shown as SEQ ID NO:1, nucleotide sequence is shown as SEQ ID NO: 2), performing enzymolysis for 2h at 45 ℃ with warm bath stirring, adding 2mL of alginate lyase, and continuing performing enzymolysis for 2h at 45 ℃ with warm bath stirring. Centrifuging the enzymolysis solution at 8000rpm for 10min, removing residue, and collecting supernatant as enzymolysis product. And carrying out 4 times of alcohol precipitation on the enzymolysis product to obtain a product, and carrying out rotary evaporation and freeze drying on the product to obtain an alginate oligosaccharide crude product, wherein the yield is 43.9%. The product is detected by mass spectrum, and the result shows that the product comprises the algin oligosaccharide with the polymerization degree of 3-10 (figure 1).

SEQ ID NO.1：

MKVSCAVVLSACIASANASILNPGFESSFDNWVDTDPSALSGVANSGSKSAKVSGSGGRVEQEVPVSSNTNYRLTAYVRGAGTVGAQVGGSTFDSSASHSDWQPVSVEFNSGSASSITIFGSYNGGEGRFDDFALESLGTGSSSSSSSSSSSSSSGGDSCTSGSSLTIIAATDDGTNDGNGPANVLDGSFAAQSRWSSQGIKWITLDLGVPQTVQAIDIAWYKGNQRASFFEVETSADNSNWTVVLSGGQSSGTTADFERYDLADTSARYVRVTGSGNTANNWNSILEMDVIGCTESGSGSSSGGSSSGSSSSSSSSGGSSSGGSGGSSSGGSLDPNLPPSSNFDLSAWYLSVPTDNNGDGKADSIKENDLNAGYADGTYFYTAADGGMVFRCPIDGYKTSTNTSYTRTELREMLRRGDTSIATQGVNGNNWVFGSAPASAREAAGGVDGVLRATLAVNHVTTTGDSGQVGRVIVGQIHANNDEPLRLYYRKLPGHSKGSVYIAHEPNGGSDSWYDMIGSRSSSASDPSDGIALDEVWSYEVKVVGNTLTVTIFRAGKDDVVQVVDMGNSGYDVADQYQYFKAGVYNQNNTGNASDYVQVTFYALEQSHD。

SEQ ID NO.2：

ATGAAAGTAAGTTGCGCTGTCGTACTGTCTGCTTGTATTGCCAGTGCCAACGCATCCATTCTTAACCCTGGCTTTGAAAGCAGCTTTGACAACTGGGTCGACACCGATCCTTCTGCCCTTTCCGGCGTTGCTAACAGTGGCAGCAAGTCCGCAAAAGTTTCCGGTAGCGGTGGTCGCGTCGAACAGGAAGTTCCCGTCAGTTCAAACACCAATTATCGTTTGACCGCTTACGTGCGTGGGGCCGGCACCGTCGGTGCACAGGTGGGCGGATCTACGTTCGATAGCAGCGCAAGTCATTCCGACTGGCAACCGGTGTCAGTGGAGTTCAATTCCGGTAGTGCCAGCAGCATTACCATCTTCGGTAGTTATAACGGTGGCGAAGGTCGCTTCGATGATTTCGCCCTGGAGAGCCTCGGTACCGGGTCCAGCTCATCCAGCAGTTCCTCCAGCAGCTCCTCCAGTTCCGGCGGCGACAGCTGCACTTCAGGTAGCAGCCTTACCATTATTGCCGCAACGGATGATGGCACTAACGACGGTAACGGCCCGGCAAATGTACTCGACGGCAGCTTCGCGGCACAATCTCGCTGGTCCTCTCAGGGCATCAAATGGATCACGCTAGATCTCGGTGTCCCCCAAACCGTGCAGGCCATTGATATCGCATGGTACAAGGGCAACCAGCGAGCCAGCTTCTTTGAGGTCGAGACTTCGGCCGACAATAGCAACTGGACCGTGGTCCTATCTGGCGGGCAGTCGAGCGGTACCACAGCGGATTTTGAACGCTATGATCTCGCGGACACCAGCGCTCGCTATGTTCGCGTCACCGGCAGTGGCAACACCGCCAACAACTGGAACAGCATTCTGGAAATGGATGTAATCGGCTGCACGGAGAGCGGCAGCGGTTCCAGCTCTGGCGGATCCTCTTCCGGTTCCAGTAGTTCCAGCAGCAGTTCAGGTGGCAGCTCCAGCGGTGGCTCTGGCGGTTCCAGCTCGGGCGGAAGCCTCGATCCGAACCTGCCCCCGTCCAGCAACTTCGACCTGAGCGCCTGGTACCTGAGCGTGCCTACCGACAACAATGGCGATGGCAAGGCCGACTCCATCAAGGAAAATGACCTGAATGCAGGCTATGCAGATGGCACCTACTTCTATACTGCTGCCGATGGCGGCATGGTGTTCCGCTGCCCGATCGATGGCTATAAAACATCGACCAACACGTCCTATACCCGCACCGAGCTGCGCGAGATGCTACGTCGTGGCGACACCAGCATTGCCACCCAGGGGGTCAATGGAAACAACTGGGTATTCGGCTCCGCACCCGCTTCGGCACGTGAAGCAGCCGGCGGTGTCGACGGTGTTTTACGCGCAACCCTCGCGGTAAACCATGTCACCACTACCGGAGATAGCGGCCAGGTTGGACGGGTGATTGTTGGACAGATTCACGCCAACAACGACGAACCGCTGCGTCTTTACTACCGCAAGTTACCGGGCCACAGCAAAGGTTCTGTGTATATCGCCCATGAGCCAAACGGCGGCAGCGACAGCTGGTACGACATGATTGGCAGCCGTTCCAGCAGCGCCTCGGACCCGTCCGACGGTATCGCACTGGATGAAGTCTGGAGCTACGAGGTCAAGGTTGTCGGTAACACCCTCACCGTGACCATCTTCCGTGCTGGTAAAGACGATGTGGTACAGGTTGTGGATATGGGCAACAGCGGTTACGACGTCGCCGACCAGTACCAGTACTTCAAGGCCGGGGTGTACAACCAGAACAACACCGGCAATGCCAGTGACTATGTCCAGGTGACCTTCTACGCCCTGGAGCAGTCGCACGATTAA。

Example 2 modification of alginate lyase gene and construction of recombinant vector aiming at low efficiency, wide distribution range of the enzymatic products and incapability of directionally preparing target products of alginate lyase before modification, the applicant designs an upstream primer F1 and a downstream primer R1 according to the complete sequence of the alginate lyase gene on the basis of analyzing the alginate lyase, and obtains the complete gene sequence by PCR amplification. The PCR conditions were: pre-denaturation at 94 ℃ for 3min, followed by 30 cycles at 94 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 2min, and final extension at 72 ℃ for 5 min. The first 54 bases and the last 768 bases of the gene complete sequence are respectively amplified by primers F1, R2, F2 and R1, two sections of PCR products are cut by AvrII enzyme and are connected by T4 ligase to form a gene modified sequence. The gene modified sequence and the Escherichia coli expression vector pProEXHTa are cut by Ecor I and Hind III enzyme, connected by T4 ligase and heat-shocked into Escherichia coliBL 21. Screening transformants with ampicillin resistance for sequencing analysis, the result shows that the total length of the whole nucleotide sequence of the alginate lyase gene is 822bp, and the nucleotide sequence is shown as SEQ ID NO:4 is shown in the specification; the encoded 273 amino acids have the amino acid sequence shown as SEQ ID NO. 3. The results of gene sequence alignment before and after modification are shown in FIG. 2.

Example 3 production of recombinant alginate lyase Using recombinant strains of Escherichia coli

The recombinant Escherichia coli strain is picked up and inoculated into 5mL of liquid LB culture medium containing 100ug/mL ampicillin, and shaking cultured for 12-16h at 37 ℃ and 170 r/min. Inoculating into 50mL liquid LB medium containing 100ug/mL ampicillin at 2%, shaking at 37 deg.C and 170r/min to OD600 of 0.5-0.8, adding IPTG with final concentration of 0.9mM, and further culturing at 23 deg.C and 150r/min for 24 h. Centrifuging at 1000rpm for 10min to obtain extracellular enzyme in supernatant.

The enzyme activity is measured by a DNS method, and the result shows that the enzyme activity of the recombinant alginate lyase after gene modification is 29.7U/mL, which is 5.2 times of the enzyme activity before gene modification.

EXAMPLE 4 production of alginate oligosaccharides Using recombinant alginate lyase

Dissolving algin in water with pH value of 7 regulated by NaOH to prepare 200mL of alginate solution with concentration of 5%, adding 0.8mL of recombinant algin lyase, performing enzymolysis for 1.5h at 45 ℃ in a warm bath under stirring, adding 0.4mL of recombinant algin lyase, and continuing performing enzymolysis for 1h at 45 ℃ in a warm bath under stirring. Centrifuging the enzymolysis solution at 8000rpm for 10min, removing residue, and collecting supernatant as enzymolysis product. And carrying out 4 times of alcohol precipitation on the enzymolysis product to obtain a target product, and carrying out rotary evaporation and freeze drying on the target product to obtain an alginate oligosaccharide crude product, wherein the yield is 73.7%.

ESI-MS was used to determine the degree of polymerization of oligosaccharides, and an ESI-MS spectrum showed that the final products of enzymatic hydrolysis were mainly trisaccharides, tetrasaccharides and pentasaccharides (FIG. 3). Compared with the algin lyase before modification, the modified recombinase can effectively degrade algin to obtain a target product with the polymerization degree of 3-5. The nuclear magnetic analysis is used for the action mode of the alginate lyase,¹the C-NMR spectrum showed that alginase acts exclusively on the polymannuronic acid fragment (FIG. 4).

EXAMPLE 5 reduction of alginate viscosity with alginate lyase

Dissolving the algin in water with pH value of 7 to prepare an algin solution with the concentration of 5-8%, respectively adding algin lyase before and after modification, stirring and performing enzymolysis for 6h at 45 ℃ in a warm bath, and observing the viscosity change in different time periods. The results show that the modified alginate lyase can reduce the viscosity of the alginate to 1/10 within 10min, while the alginate lyase before modification can reduce the viscosity of the alginate to 1/10 in 1h (fig. 5).

Sequence listing

<110> China oceanic university

<120> method for directionally preparing algin oligosaccharide by enzymatic hydrolysis

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Asn Ala Ser Ile Leu Asn Pro Gly Phe Glu Ser Ser Phe Asp Asn Trp

20 25 30

Val Asp Thr Asp Pro Ser Ala Leu Ser Gly Val Ala Asn Ser Gly Ser

35 40 45

Lys Ser Ala Lys Val Ser Gly Ser Gly Gly Arg Val Glu Gln Glu Val

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Pro Val Ser Ser Asn Thr Asn Tyr Arg Leu Thr Ala Tyr Val Arg Gly

65 70 75 80

Ala Gly Thr Val Gly Ala Gln Val Gly Gly Ser Thr Phe Asp Ser Ser

85 90 95

Ala Ser His Ser Asp Trp Gln Pro Val Ser Val Glu Phe Asn Ser Gly

100 105 110

Ser Ala Ser Ser Ile Thr Ile Phe Gly Ser Tyr Asn Gly Gly Glu Gly

115 120 125

Arg Phe Asp Asp Phe Ala Leu Glu Ser Leu Gly Thr Gly Ser Ser Ser

130 135 140

Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Gly Gly Asp Ser Cys

145 150 155 160

Thr Ser Gly Ser Ser Leu Thr Ile Ile Ala Ala Thr Asp Asp Gly Thr

165 170 175

Asn Asp Gly Asn Gly Pro Ala Asn Val Leu Asp Gly Ser Phe Ala Ala

180 185 190

Gln Ser Arg Trp Ser Ser Gln Gly Ile Lys Trp Ile Thr Leu Asp Leu

195 200 205

Gly Val Pro Gln Thr Val Gln Ala Ile Asp Ile Ala Trp Tyr Lys Gly

210 215 220

Asn Gln Arg Ala Ser Phe Phe Glu Val Glu Thr Ser Ala Asp Asn Ser

225 230 235 240

Asn Trp Thr Val Val Leu Ser Gly Gly Gln Ser Ser Gly Thr Thr Ala

245 250 255

Asp Phe Glu Arg Tyr Asp Leu Ala Asp Thr Ser Ala Arg Tyr Val Arg

260 265 270

Val Thr Gly Ser Gly Asn Thr Ala Asn Asn Trp Asn Ser Ile Leu Glu

275 280 285

Met Asp Val Ile Gly Cys Thr Glu Ser Gly Ser Gly Ser Ser Ser Gly

290 295 300

Gly Ser Ser Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Gly Gly Ser

305 310 315 320

Ser Ser Gly Gly Ser Gly Gly Ser Ser Ser Gly Gly Ser Leu Asp Pro

325 330 335

Asn Leu Pro Pro Ser Ser Asn Phe Asp Leu Ser Ala Trp Tyr Leu Ser

340 345 350

Val Pro Thr Asp Asn Asn Gly Asp Gly Lys Ala Asp Ser Ile Lys Glu

355 360 365

Asn Asp Leu Asn Ala Gly Tyr Ala Asp Gly Thr Tyr Phe Tyr Thr Ala

370 375 380

Ala Asp Gly Gly Met Val Phe Arg Cys Pro Ile Asp Gly Tyr Lys Thr

385 390 395 400

Ser Thr Asn Thr Ser Tyr Thr Arg Thr Glu Leu Arg Glu Met Leu Arg

405 410 415

Arg Gly Asp Thr Ser Ile Ala Thr Gln Gly Val Asn Gly Asn Asn Trp

420 425 430

Val Phe Gly Ser Ala Pro Ala Ser Ala Arg Glu Ala Ala Gly Gly Val

435 440 445

Asp Gly Val Leu Arg Ala Thr Leu Ala Val Asn His Val Thr Thr Thr

450 455 460

Gly Asp Ser Gly Gln Val Gly Arg Val Ile Val Gly Gln Ile His Ala

465 470 475 480

Asn Asn Asp Glu Pro Leu Arg Leu Tyr Tyr Arg Lys Leu Pro Gly His

485 490 495

Ser Lys Gly Ser Val Tyr Ile Ala His Glu Pro Asn Gly Gly Ser Asp

500 505 510

Ser Trp Tyr Asp Met Ile Gly Ser Arg Ser Ser Ser Ala Ser Asp Pro

515 520 525

Ser Asp Gly Ile Ala Leu Asp Glu Val Trp Ser Tyr Glu Val Lys Val

530 535 540

Val Gly Asn Thr Leu Thr Val Thr Ile Phe Arg Ala Gly Lys Asp Asp

545 550 555 560

Val Val Gln Val Val Asp Met Gly Asn Ser Gly Tyr Asp Val Ala Asp

565 570 575

Gln Tyr Gln Tyr Phe Lys Ala Gly Val Tyr Asn Gln Asn Asn Thr Gly

580 585 590

Asn Ala Ser Asp Tyr Val Gln Val Thr Phe Tyr Ala Leu Glu Gln Ser

595 600 605

His Asp

610

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atgaaagtaa gttgcgctgt cgtactgtct gcttgtattg ccagtgccaa cgcatccatt 60

cttaaccctg gctttgaaag cagctttgac aactgggtcg acaccgatcc ttctgccctt 120

tccggcgttg ctaacagtgg cagcaagtcc gcaaaagttt ccggtagcgg tggtcgcgtc 180

gaacaggaag ttcccgtcag ttcaaacacc aattatcgtt tgaccgctta cgtgcgtggg 240

gccggcaccg tcggtgcaca ggtgggcgga tctacgttcg atagcagcgc aagtcattcc 300

gactggcaac cggtgtcagt ggagttcaat tccggtagtg ccagcagcat taccatcttc 360

ggtagttata acggtggcga aggtcgcttc gatgatttcg ccctggagag cctcggtacc 420

gggtccagct catccagcag ttcctccagc agctcctcca gttccggcgg cgacagctgc 480

acttcaggta gcagccttac cattattgcc gcaacggatg atggcactaa cgacggtaac 540

ggcccggcaa atgtactcga cggcagcttc gcggcacaat ctcgctggtc ctctcagggc 600

atcaaatgga tcacgctaga tctcggtgtc ccccaaaccg tgcaggccat tgatatcgca 660

tggtacaagg gcaaccagcg agccagcttc tttgaggtcg agacttcggc cgacaatagc 720

aactggaccg tggtcctatc tggcgggcag tcgagcggta ccacagcgga ttttgaacgc 780

tatgatctcg cggacaccag cgctcgctat gttcgcgtca ccggcagtgg caacaccgcc 840

aacaactgga acagcattct ggaaatggat gtaatcggct gcacggagag cggcagcggt 900

tccagctctg gcggatcctc ttccggttcc agtagttcca gcagcagttc aggtggcagc 960

tccagcggtg gctctggcgg ttccagctcg ggcggaagcc tcgatccgaa cctgcccccg 1020

tccagcaact tcgacctgag cgcctggtac ctgagcgtgc ctaccgacaa caatggcgat 1080

ggcaaggccg actccatcaa ggaaaatgac ctgaatgcag gctatgcaga tggcacctac 1140

ttctatactg ctgccgatgg cggcatggtg ttccgctgcc cgatcgatgg ctataaaaca 1200

tcgaccaaca cgtcctatac ccgcaccgag ctgcgcgaga tgctacgtcg tggcgacacc 1260

agcattgcca cccagggggt caatggaaac aactgggtat tcggctccgc acccgcttcg 1320

gcacgtgaag cagccggcgg tgtcgacggtgttttacgcg caaccctcgc ggtaaaccat 1380

gtcaccacta ccggagatag cggccaggtt ggacgggtga ttgttggaca gattcacgcc 1440

aacaacgacg aaccgctgcg tctttactac cgcaagttac cgggccacag caaaggttct 1500

gtgtatatcg cccatgagcc aaacggcggc agcgacagct ggtacgacat gattggcagc 1560

cgttccagca gcgcctcgga cccgtccgac ggtatcgcac tggatgaagt ctggagctac 1620

gaggtcaagg ttgtcggtaa caccctcacc gtgaccatct tccgtgctgg taaagacgat 1680

gtggtacagg ttgtggatat gggcaacagc ggttacgacg tcgccgacca gtaccagtac 1740

ttcaaggccg gggtgtacaa ccagaacaac accggcaatg ccagtgacta tgtccaggtg 1800

accttctacg ccctggagca gtcgcacgat taa 1833

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Met Lys Val Ser Cys Ala Val Val Leu Ser Ala Cys Ile Ala Ser Ala

1 5 10 15

Asn Ala Asp Asn Asn Gly Asp Gly Lys Ala Asp Ser Ile Lys Glu Asn

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Asp Leu Asn Ala Gly Tyr Ala Asp Gly Thr Tyr Phe Tyr Thr Ala Ala

35 40 45

Asp Gly Gly Met Val Phe Arg Cys Pro Ile Asp Gly Tyr Lys Thr Ser

50 55 60

Thr Asn Thr Ser Tyr Thr Arg Thr Glu Leu Arg Glu Met Leu Arg Arg

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Gly Asp Thr Ser Ile Ala Thr Gln Gly Val Asn Gly Asn Asn Trp Val

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Phe Gly Ser Ala Pro Ala Ser Ala Arg Glu Ala Ala Gly Gly Val Asp

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Gly Val Leu Arg Ala Thr Leu Ala Val Asn His Val Thr Thr Thr Gly

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Asp Ser Gly Gln Val Gly Arg Val Ile Val Gly Gln Ile His Ala Asn

130 135 140

Asn Asp Glu Pro Leu Arg Leu Tyr Tyr Arg Lys Leu Pro Gly His Ser

145 150 155 160

Lys Gly Ser Val Tyr Ile Ala His Glu Pro Asn Gly Gly Ser Asp Ser

165 170 175

Trp Tyr Asp Met Ile Gly Ser Arg Ser Ser Ser Ala Ser Asp Pro Ser

180 185 190

Asp Gly Ile Ala Leu Asp Glu Val Trp Ser Tyr Glu Val Lys Val Val

195 200 205

Gly Asn Thr Leu Thr Val Thr Ile Phe Arg Ala Gly Lys Asp Asp Val

210 215 220

Val Gln Val Val Asp Met Gly Asn Ser Gly Tyr Asp Val Ala Asp Gln

225 230 235 240

Tyr Gln Tyr Phe Lys Ala Gly Val Tyr Asn Gln Asn Asn Thr Gly Asn

245 250 255

Ala Ser Asp Tyr Val Gln Val Thr Phe Tyr Ala Leu Glu Gln Ser His

260 265 270

Asp

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atgaaagtaa gttgcgctgt cgtactgtct gcttgtattg ccagtgccaa cgcagacaac 60

aatggcgatg gcaaggccga ctccatcaag gaaaatgacc tgaatgcagg ctatgcagat 120

ggcacctact tctatactgc tgccgatggc ggcatggtgt tccgctgccc gatcgatggc 180

tataaaacat cgaccaacac gtcctatacc cgcaccgagc tgcgcgagat gctacgtcgt 240

ggcgacacca gcattgccac ccagggggtc aatggaaaca actgggtatt cggctccgca 300

cccgcttcgg cacgtgaagc agccggcggt gtcgacggtg ttttacgcgc aaccctcgcg 360

gtaaaccatg tcaccactac cggagatagc ggccaggttg gacgggtgat tgttggacag 420

attcacgcca acaacgacga accgctgcgt ctttactacc gcaagttacc gggccacagc 480

aaaggttctg tgtatatcgc ccatgagcca aacggcggca gcgacagctg gtacgacatg 540

attggcagcc gttccagcag cgcctcggac ccgtccgacg gtatcgcact ggatgaagtc 600

tggagctacg aggtcaaggt tgtcggtaac accctcaccg tgaccatctt ccgtgctggt 660

aaagacgatg tggtacaggt tgtggatatg ggcaacagcg gttacgacgt cgccgaccag 720

taccagtact tcaaggccgg ggtgtacaac cagaacaaca ccggcaatgc cagtgactat 780

gtccaggtga ccttctacgc cctggagcag tcgcacgattaa 822

Claims (5)

Translated fromChinese

1.一种酶法水解定向制备褐藻胶寡糖的方法，其特征在于，所述的方法包括如下的步骤：1. a method for directional preparation of alginate oligosaccharide by enzymatic hydrolysis, is characterized in that, described method comprises the steps:1)底物配制：将褐藻胶原料与水混合，配制成浓度为5％-8％的pH值为中性的酶解底物溶液；1) Substrate preparation: mix the alginate raw material with water, and prepare a neutral enzymatic hydrolysis substrate solution with a concentration of 5%-8%;2)分步酶解：加入褐藻胶裂解酶，于40-50℃下搅拌酶解1-2h，再次加入0.3％-0.8％的褐藻胶裂解酶，搅拌酶解，总酶解时间为2-4h；2) Step-by-step enzymatic hydrolysis: add alginate lyase, stir for 1-2h at 40-50°C, add 0.3%-0.8% algin lyase again, stir for enzymolysis, the total enzymolysis time is 2- 4h;3)低聚糖制备：酶解结束后，过滤或离心除去渣子，上清液浓缩、冷冻干燥得到海藻低聚糖；3) Preparation of oligosaccharides: after enzymatic hydrolysis, filter or centrifuge to remove slag, and the supernatant is concentrated and freeze-dried to obtain algal oligosaccharides;所述的褐藻胶裂解酶的氨基酸序列为SEQ ID NO:3。The amino acid sequence of the alginate lyase is SEQ ID NO:3.2.如权利要求1所述的方法，其特征在于，所述的褐藻胶裂解酶，其编码基因的核苷酸序列为SEQ ID NO:4。2 . The method of claim 1 , wherein the alginate lyase has a nucleotide sequence encoding the gene of SEQ ID NO: 4. 3 .3.一种褐藻胶裂解酶，其特征在于，所述的褐藻胶裂解酶的氨基酸序列为SEQ ID NO:3。3. An algin lyase, wherein the amino acid sequence of the algin lyase is SEQ ID NO: 3.4.一种编码基因，其特征在于，所述的基因编码权利要求3所述的褐藻胶裂解酶。4. An encoding gene, characterized in that the gene encodes the alginate lyase of claim 3.5.权利要求3所述的褐藻胶裂解酶在制备褐藻胶寡糖中的应用。5. The application of the alginate lyase of claim 3 in the preparation of alginate oligosaccharides.

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