CN106884025B - Method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis - Google Patents
Method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02011—Poly(alpha-L-guluronate) lyase (4.2.2.11), i.e. alginase II
Abstract
The invention provides a method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis, wherein the amino acid sequence of the used alginate lyase is SEQ ID NO. 3. 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.
Description
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 coli BL 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
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>610
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Met Lys Val Ser Cys Ala Val Val Leu Ser Ala Cys Ile Ala Ser Ala
1 5 10 15
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
50 55 60
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
<210>2
<211>1833
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
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
<210>3
<211>273
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
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
20 25 30
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
65 70 75 80
Gly Asp Thr Ser Ile Ala Thr Gln Gly Val Asn Gly Asn Asn Trp Val
85 90 95
Phe Gly Ser Ala Pro Ala Ser Ala Arg Glu Ala Ala Gly Gly Val Asp
100 105 110
Gly Val Leu Arg Ala Thr Leu Ala Val Asn His Val Thr Thr Thr Gly
115 120 125
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
<210>4
<211>822
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
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 tcgcacgatt aa 822
Claims (5)
1. A method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis is characterized by comprising 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: after enzymolysis, filtering or centrifuging to remove dregs, concentrating supernate, and freeze-drying to obtain algal oligosaccharide;
the amino acid sequence of the alginate lyase is SEQ ID NO. 3.
2. The method of claim 1, wherein the alginate lyase has the nucleotide sequence of SEQ ID NO. 4.
3. The algin lyase is characterized in that the amino acid sequence of the algin lyase is SEQ ID NO. 3.
4. A coding gene, which encodes the alginate lyase of claim 3.
5. Use of the alginate lyase of claim 3 in the preparation of alginate oligosaccharides.
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| CN109136303A (en) * | 2018-08-31 | 2019-01-04 | 中国海洋大学 | A kind of preparation method of the seaweed diet fiber rich in algin oligosaccharide |
| CN109182413A (en) * | 2018-08-31 | 2019-01-11 | 中国海洋大学 | A kind of method of the small molecule potassium alginate of beam system for high guluronic acid content |
| CN109221811A (en) * | 2018-09-28 | 2019-01-18 | 福州大学 | A kind of preparation method of feeding additive aquatic animal brown alga oligose |
| CN110257452A (en) * | 2019-01-30 | 2019-09-20 | 南京工业大学 | A method of the separating-purifying brown alga oligose monomer from enzymolysis liquid |
| CN110423787B (en) * | 2019-08-09 | 2022-03-04 | 中国海洋大学 | Preparation method of uniform brown algae trisaccharide |
| CN111197065A (en) * | 2020-02-24 | 2020-05-26 | 江南大学 | Method for producing algin hydrolysate |
| CN112715964B (en) * | 2020-12-16 | 2022-04-19 | 中国科学院南海海洋研究所 | Spirulina oligosaccharide and application thereof in preparation of preparation for regulating intestinal health function |
| CN114287521B (en) * | 2021-12-27 | 2023-06-20 | 中国海洋大学 | Compound enzymolysis preparation method of laminarin for replacing fish meal for cottonseed protein compound feed and application thereof |
| CN114507656B (en) * | 2022-03-02 | 2023-07-07 | 中国海洋大学 | Method for preparing fucoidan rich in guluronic acid |
| CN115948373B (en) * | 2022-11-11 | 2023-07-25 | 深圳润康生态环境股份有限公司 | Algin lyase mutant Pl7AaM and application thereof |
| CN117230051B (en) * | 2023-11-16 | 2024-01-30 | 深圳润康生态环境股份有限公司 | Algin lyase mutant Pl7MaM and preparation method and application thereof |
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