CN106884025A - A kind of enzymatic hydrolysis beam system for algin oligosaccharide method - Google Patents

A kind of enzymatic hydrolysis beam system for algin oligosaccharide method Download PDF

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CN106884025A
CN106884025A CN201710298634.3A CN201710298634A CN106884025A CN 106884025 A CN106884025 A CN 106884025A CN 201710298634 A CN201710298634 A CN 201710298634A CN 106884025 A CN106884025 A CN 106884025A
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algin
ser
gly
ala
val
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CN106884025B (en
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牟海津
杨敏
李丽
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Ocean University of China
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    • C12N9/88—Lyases (4.)
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    • 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

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Abstract

The present invention provides a kind of method of enzymatic hydrolysis beam system for algin oligosaccharide, wherein the algin catenase for using, its amino acid sequence is SEQ ID NO:3.The present invention utilizes the restructuring algin catenase of genetic modification, beam system is for algin oligosaccharide, compared with the method that existing Acid hydrolysis prepare algin oligosaccharide, overcome the low shortcoming of destructive big, efficiency, compared with the method that existing enzymatic hydrolysis prepares algin oligosaccharide, with the restructuring algin catenase of transformation, overcome enzymolysis time long, high cost, be unable to shortcoming of the beam system for the algin oligosaccharide of specific aggregation degree.Process of the present invention is simple, low cost and suitable for industrialized production, can in a short time reduce brown alga adhesiveness, and the total yield of made oligosaccharides is more than 90%.The effects such as prepared algin oligosaccharide has antitumor, anti-inflammatory, reducing blood lipid and enhance immunity, can be used for food and field of health care products, be with a wide range of applications.

Description

A kind of enzymatic hydrolysis beam system for algin oligosaccharide method
Technical field
The invention belongs to algin oligosaccharide preparing technical field, and in particular to a kind of enzymatic hydrolysis beam system is few for algin The method of sugar.
Background technology
Algin oligosaccharide (alginate oligosaccharides) be algin through the degree of polymerization obtained from hydrolysis it is small and The low molecular weight fraction of good water solubility.Because algin oligosaccharide has reducing blood lipid, antiviral, antitumor and anti-oxidant etc. various Physiologically active, can be widely used in the fields such as food, medicine and cosmetics, with huge application and Development volue.It is newest to grind Study carefully and show：The poly- M sections of oligosaccharides medicine " 971 " prepared with algin can suppress aggregation and the cytotoxicity of beta-amyloyd cell, positive to use In the second stage of clinical research of anti-alzheimer's disease；Poly- G oligosaccharides can cooperate with the clinical many resistance pathogenic bacteria of suppression with antibiotic.Cause This, the composition specific algin oligosaccharide of the special, degree of polymerization has significant application value and economic worth, realizes the height of this kind of oligosaccharides Effect prepares significant.
The preparation method of algin oligosaccharide mainly has Acid hydrolysis and enzymatic hydrolysis.Acid hydrolysis can in various degree destroy brown The structure of phycocolloid oligosaccharides, and enzymatic hydrolysis has the advantages that reaction condition is gentle, therefore, enzymatic hydrolysis prepares algin oligosaccharide and receives To extensive concern.Marine microorganism is the important sources of algin catenase, and some algin catenases are in succession from vibrios, alternating Separated in monad, Pseudoalteromonas.But due to the particularity of marine environment, the brown alga for causing marine microorganism to produce Glue lyases limited activity, beam system is unable to for algin oligosaccharide.Therefore, the algin cracking that selectivity is strong, vigor is high is found Enzyme is still the emphasis of researcher's concern.
The content of the invention
It is an object of the invention to provide a kind of enzymatic hydrolysis beam system for algin oligosaccharide method, by improved enzyme Target algin oligosaccharide can efficiently, be directionally prepared, the efficiency that solution prior art is present is low, be unable to beam system for algin The shortcoming of oligosaccharides.
Present invention firstly provides a kind of enzymatic hydrolysis beam system for algin oligosaccharide method, including the steps：
1) substrate is prepared：Algin raw material is mixed with water, it is neutral enzymolysis to be configured to the pH value that concentration is 5%-8% Substrate solution；
2) stepwise discretization：Algin catenase is added, in enzymolysis 1-2h is stirred at 40-50 DEG C, 0.3%- is added again 0.8% algin catenase, stirring enzymolysis, total enzymolysis time is 2-4h；
3) prepared by oligosaccharide：After enzymolysis terminates, bits are filtered or are centrifuged off, supernatant concentration, freeze-drying obtain sea Algae oligosaccharide.
In above-mentioned method, wherein the amino acid sequence of algin catenase is SEQ ID NO:1；
In order to obtain the greater concentration of algin oligosaccharide of the degree of polymerization, the present invention is optimized to algin catenase, excellent The amino acid sequence of the algin catenase after change is SEQ ID NO:3；
Optimization provided by the present invention, can beam system for algin oligosaccharide algin catenase, its amino acid sequence It is SEQ ID NO:3：
MKVSCAVVLSACIASANATDNNGDGKADSIKENDLNAGYADGTYFYTAADGGMVFRCPIDGYKTSTNTSYTRTELRE MLRRGDTSIATQGVNGNNWVFGSAPASAREAAGGVDGVLRATLAVNHVTTTGDSGQVGRVIVGQIHANNDEPLRLYY RKLPGHSKGSVYIAHEPNGGSDSWYDMIGSRSSSASDPSDGIALDEVWSYEVKVVGNTLTVTIFRAGKDDVVQVVDM GNSGYDVADQYQYFKAGVYNQNNTGNASDYVQVTFYALEQSHD；
The gene of above-mentioned algin catenase is encoded, one kind nucleotide sequence is SEQ ID NO:4:
ATGAAAGTAAGTTGCGCTGTCGTACTGTCTGCTTGTATTGCCAGTGCCAACGCAGACAACAATGGCGATGGCAAGGC CGACTCCATCAAGGAAAATGACCTGAATGCAGGCTATGCAGATGGCACCTACTTCTATACTGCTGCCGATGGCGGCA TGGTGTTCCGCTGCCCGATCGATGGCTATAAAACATCGACCAACACGTCCTATACCCGCACCGAGCTGCGCGAGATG CTACGTCGTGGCGACACCAGCATTGCCACCCAGGGGGTCAATGGAAACAACTGGGTATTCGGCTCCGCACCCGCTTC GGCACGTGAAGCAGCCGGCGGTGTCGACGGTGTTTTACGCGCAACCCTCGCGGTAAACCATGTCACCACTACCGGAG ATAGCGGCCAGGTTGGACGGGTGATTGTTGGACAGATTCACGCCAACAACGACGAACCGCTGCGTCTTTACTACCGC AAGTTACCGGGCCACAGCAAAGGTTCTGTGTATATCGCCCATGAGCCAAACGGCGGCAGCGACAGCTGGTACGACAT GATTGGCAGCCGTTCCAGCAGCGCCTCGGACCCGTCCGACGGTATCGCACTGGATGAAGTCTGGAGCTACGAGGTCA AGGTTGTCGGTAACACCCTCACCGTGACCATCTTCCGTGCTGGTAAAGACGATGTGGTACAGGTTGTGGATATGGGC AACAGCGGTTACGACGTCGCCGACCAGTACCAGTACTTCAAGGCCGGGGTGTACAACCAGAACAACACCGGCAATGC CAGTGACTATGTCCAGGTGACCTTCTACGCCCTGGAGCAGTCGCACGATTAA。
The present invention using genetic modification restructuring algin catenase, beam system for algin oligosaccharide, with existing acid system water The method that solution prepares algin oligosaccharide is compared, and overcomes the low shortcoming of destructive big, efficiency, is prepared with existing enzymatic hydrolysis brown The method of phycocolloid oligosaccharides is compared, and with the restructuring algin catenase of transformation, overcomes enzymolysis time long, high cost, Bu Nengding To the shortcoming of the algin oligosaccharide for preparing specific aggregation degree.Process of the present invention is simple, low cost and suitable for industrialized production, energy Enough to reduce brown alga adhesiveness in a short time, the total yield of made oligosaccharides is more than 90%.Prepared algin oligosaccharide has anti- The effects such as tumour, anti-inflammatory, reducing blood lipid and enhance immunity, can be used for food and field of health care products, be with a wide range of applications.
Brief description of the drawings
Fig. 1：Algin catenase enzymolysis product ESI-MS collection of illustrative plates before transformation
Fig. 2：Gene order comparison result figure before and after transformation；
Fig. 3：Algin catenase enzymolysis product ESI-MS collection of illustrative plates after transformation；
Fig. 4：Algin catenase enzymolysis product after transformation1C-NMR collection of illustrative plates；
Fig. 5：The algin substrate viscosity of various concentrations with enzymolysis time change (A：Algin catenase degraded before transformation Brown alga adhesiveness is changed over time；B：Recombinate algin catenase degraded brown alga adhesiveness after transformation to change over time).
Specific embodiment
The present invention is described in detail with reference to embodiment.
Embodiment 1：Algin oligosaccharide is prepared using the algin enzymatic lysis enzyme before transformation
Algin is dissolved in the water of the pH=7 adjusted by NaOH, the alginate solution that 200mL concentration is 5% is prepared, plus (amino acid sequence is such as to enter 2mL algin catenases：SEQ ID NO：1, nucleotides sequence is classified as SEQ ID NO：2), in 45 DEG C of temperature Bath stirring enzymolysis 2h, adds 2mL algin catenases, continues at 45 DEG C of warm bath stirring enzymolysis 2h.Enzymolysis liquid through 8000rpm from Heart 10min, removes bits, and supernatant is enzymolysis product.Enzymolysis product obtains product through 4 times of alcohol precipitations, the rotated evaporation of product, Algin oligosaccharide crude product is obtained after freeze-drying, yield is 43.9%.As a result product shows that product includes poly- through Mass Spectrometer Method Right is the algin oligosaccharide (Fig. 1) of 3-10.
SEQ ID NO.1：
MKVSCAVVLSACIASANASILNPGFESSFDNWVDTDPSALSGVANSGSKSAKVSGSGGRVEQEVPVSSNTNYRLTAY VRGAGTVGAQVGGSTFDSSASHSDWQPVSVEFNSGSASSITIFGSYNGGEGRFDDFALESLGTGSSSSSSSSSSSSS SGGDSCTSGSSLTIIAATDDGTNDGNGPANVLDGSFAAQSRWSSQGIKWITLDLGVPQTVQAIDIAWYKGNQRASFF EVETSADNSNWTVVLSGGQSSGTTADFERYDLADTSARYVRVTGSGNTANNWNSILEMDVIGCTESGSGSSSGGSSS GSSSSSSSSGGSSSGGSGGSSSGGSLDPNLPPSSNFDLSAWYLSVPTDNNGDGKADSIKENDLNAGYADGTYFYTAA DGGMVFRCPIDGYKTSTNTSYTRTELREMLRRGDTSIATQGVNGNNWVFGSAPASAREAAGGVDGVLRATLAVNHVT TTGDSGQVGRVIVGQIHANNDEPLRLYYRKLPGHSKGSVYIAHEPNGGSDSWYDMIGSRSSSASDPSDGIALDEVWS YEVKVVGNTLTVTIFRAGKDDVVQVVDMGNSGYDVADQYQYFKAGVYNQNNTGNASDYVQVTFYALEQSHD。
SEQ ID NO.2：
ATGAAAGTAAGTTGCGCTGTCGTACTGTCTGCTTGTATTGCCAGTGCCAACGCATCCATTCTTAACCCTGGCTTTGA AAGCAGCTTTGACAACTGGGTCGACACCGATCCTTCTGCCCTTTCCGGCGTTGCTAACAGTGGCAGCAAGTCCGCAA AAGTTTCCGGTAGCGGTGGTCGCGTCGAACAGGAAGTTCCCGTCAGTTCAAACACCAATTATCGTTTGACCGCTTAC GTGCGTGGGGCCGGCACCGTCGGTGCACAGGTGGGCGGATCTACGTTCGATAGCAGCGCAAGTCATTCCGACTGGCA ACCGGTGTCAGTGGAGTTCAATTCCGGTAGTGCCAGCAGCATTACCATCTTCGGTAGTTATAACGGTGGCGAAGGTC GCTTCGATGATTTCGCCCTGGAGAGCCTCGGTACCGGGTCCAGCTCATCCAGCAGTTCCTCCAGCAGCTCCTCCAGT TCCGGCGGCGACAGCTGCACTTCAGGTAGCAGCCTTACCATTATTGCCGCAACGGATGATGGCACTAACGACGGTAA CGGCCCGGCAAATGTACTCGACGGCAGCTTCGCGGCACAATCTCGCTGGTCCTCTCAGGGCATCAAATGGATCACGC TAGATCTCGGTGTCCCCCAAACCGTGCAGGCCATTGATATCGCATGGTACAAGGGCAACCAGCGAGCCAGCTTCTTT GAGGTCGAGACTTCGGCCGACAATAGCAACTGGACCGTGGTCCTATCTGGCGGGCAGTCGAGCGGTACCACAGCGGA TTTTGAACGCTATGATCTCGCGGACACCAGCGCTCGCTATGTTCGCGTCACCGGCAGTGGCAACACCGCCAACAACT GGAACAGCATTCTGGAAATGGATGTAATCGGCTGCACGGAGAGCGGCAGCGGTTCCAGCTCTGGCGGATCCTCTTCC GGTTCCAGTAGTTCCAGCAGCAGTTCAGGTGGCAGCTCCAGCGGTGGCTCTGGCGGTTCCAGCTCGGGCGGAAGCCT CGATCCGAACCTGCCCCCGTCCAGCAACTTCGACCTGAGCGCCTGGTACCTGAGCGTGCCTACCGACAACAATGGCG ATGGCAAGGCCGACTCCATCAAGGAAAATGACCTGAATGCAGGCTATGCAGATGGCACCTACTTCTATACTGCTGCC GATGGCGGCATGGTGTTCCGCTGCCCGATCGATGGCTATAAAACATCGACCAACACGTCCTATACCCGCACCGAGCT GCGCGAGATGCTACGTCGTGGCGACACCAGCATTGCCACCCAGGGGGTCAATGGAAACAACTGGGTATTCGGCTCCG CACCCGCTTCGGCACGTGAAGCAGCCGGCGGTGTCGACGGTGTTTTACGCGCAACCCTCGCGGTAAACCATGTCACC ACTACCGGAGATAGCGGCCAGGTTGGACGGGTGATTGTTGGACAGATTCACGCCAACAACGACGAACCGCTGCGTCT TTACTACCGCAAGTTACCGGGCCACAGCAAAGGTTCTGTGTATATCGCCCATGAGCCAAACGGCGGCAGCGACAGCT GGTACGACATGATTGGCAGCCGTTCCAGCAGCGCCTCGGACCCGTCCGACGGTATCGCACTGGATGAAGTCTGGAGC TACGAGGTCAAGGTTGTCGGTAACACCCTCACCGTGACCATCTTCCGTGCTGGTAAAGACGATGTGGTACAGGTTGT GGATATGGGCAACAGCGGTTACGACGTCGCCGACCAGTACCAGTACTTCAAGGCCGGGGTGTACAACCAGAACAACA CCGGCAATGCCAGTGACTATGTCCAGGTGACCTTCTACGCCCTGGAGCAGTCGCACGATTAA。
The transformation of the alginate lyase gene of embodiment 2 and the structure of recombinant vector
For algin catenase enzymolysis efficiency is low, enzymolysis product has a very wide distribution, be unable to beam system for target before transformation Product, applicant is analyzed on the basis of algin catenase enzyme again, and upstream is designed according to alginate lyase gene complete sequence Primers F 1 and anti-sense primer R1, are expanded with PCR and obtain gene complete sequence.PCR conditions are：94 DEG C of predegeneration 3min, then with 94 DEG C 30s, 55 DEG C of 30s, 72 DEG C of 2min carry out 30 circulations, finally extend 5min at 72 DEG C.Distinguished with primers F 1, R2 and F2, R1 Preceding 54 bases and rear 768 bases of amplification gene complete sequence, two sections of PCR primers digestions of Avr II are connected with T4 ligases Constitutivegene transforms sequence.Genetic modification sequence and coli expression carrier pProEXHTa Ecor I and the digestions of Hind III, Connected with T4 ligases, heat shock is transferred in e. coli bl21.Transformant of the screening with amicillin resistance is sequenced Analysis, as a result shows the full nucleotide sequence total length 822bp of alginate lyase gene, nucleotide sequence such as SEQ ID NO：4 It is shown；274 amino acid of coding, amino acid sequence such as SEQ ID NO:Shown in 3.Gene order comparison result before and after transformation is such as Fig. 2.
Embodiment 3 is using E. coli recombinant stain production restructuring algin catenase
Picking E. coli recombinant stain, is seeded in LB liquid mediums of the 5mL containing 100ug/mL ampicillins, 37 DEG C 170r/min shaken cultivations 12-16h.Liquid LBs of the 50mL containing 100ug/mL ampicillins is inoculated in by 2% inoculum concentration to train Support in base, 37 DEG C of 170r/min shaken cultivations to OD600 are 0.5-0.8, add the IPTG, 23 DEG C of 150r/ of final concentration of 0.9mM Min continues to cultivate to 24h.1000rpm is centrifuged 10min, obtains supernatant ectoenzyme.
Enzyme activity is surveyed using DNS methods, is as a result shown, be 29.7U/ through the restructuring algin catenase enzyme activity after genetic modification ML, is 5.2 times of enzyme activity before genetic modification.
Embodiment 4 produces algin oligosaccharide with restructuring algin catenase
Algin is dissolved in the water of the pH=7 adjusted by NaOH, the alginate solution that 200mL concentration is 5% is prepared, plus Enter 0.8mL restructuring algin catenases, in 45 DEG C of warm bath stirring enzymolysis 1.5h, add 0.4mL restructuring algin catenases, after Continue in 45 DEG C of warm bath stirring enzymolysis 1h.Enzymolysis liquid is centrifuged 10min through 8000rpm, removes bits, and supernatant is enzymolysis product.Enzyme Solution product obtains target product through 4 times of alcohol precipitations, and algin oligosaccharide crude product is obtained after the rotated evaporation of target product, freeze-drying, Yield is 73.7%.
The oligosaccharide degree of polymerization is determined with ESI-MS, ESI-MS collection of illustrative plates shows, enzymolysis end-product is mainly trisaccharide, tetrose and five Sugared (Fig. 3).Compared with the algin catenase before transformation, improved recombinase can effectively degrade algin, be polymerized Spend the target product for 3-5.With the nmr analysis algin catenase mode of action,1C-NMR collection of illustrative plates shows the single-minded work of algin enzyme For polymannuronate fragment (Fig. 4).
The algin catenase reduction brown alga adhesiveness of embodiment 5
Algin is dissolved in the water of pH=7, the alginate solution that concentration is 5%-8% is configured to, transformation is separately added into Preceding and improved algin catenase, in 45 DEG C of warm bath stirring enzymolysis 6h, viscosity B coefficent is observed in different time sections.Result shows Show, in 10min, brown alga adhesiveness can be reduced to original 1/10 by improved algin catenase, and before transforming Algin catenase needs the 1h 1/10 original (Fig. 5) could will be reduced to by brown alga adhesiveness.
SEQUENCE LISTING
<110>Chinese Marine University
<120>A kind of enzymatic hydrolysis beam system for algin oligosaccharide method
<130>
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Ser Thr Asn Thr Ser Tyr Thr Arg Thr Glu Leu Arg Glu Met Leu Arg
65 70 75 80
Arg Gly Asp Thr Ser Ile Ala Thr Gln Gly Val Asn Gly Asn Asn Trp
85 90 95
Val Phe Gly Ser Ala Pro Ala Ser Ala Arg Glu Ala Ala Gly Gly Val
100 105 110
Asp Gly Val Leu Arg Ala Thr Leu Ala Val Asn His Val Thr Thr Thr
115 120 125
Gly Asp Ser Gly Gln Val Gly Arg Val Ile Val Gly Gln Ile His Ala
130 135 140
Asn Asn Asp Glu Pro Leu Arg Leu Tyr Tyr Arg Lys Leu Pro Gly His
145 150 155 160
Ser Lys Gly Ser Val Tyr Ile Ala His Glu Pro Asn Gly Gly Ser Asp
165 170 175
Ser Trp Tyr Asp Met Ile Gly Ser Arg Ser Ser Ser Ala Ser Asp Pro
180 185 190
Ser Asp Gly Ile Ala Leu Asp Glu Val Trp Ser Tyr Glu Val Lys Val
195 200 205
Val Gly Asn Thr Leu Thr Val Thr Ile Phe Arg Ala Gly Lys Asp Asp
210 215 220
Val Val Gln Val Val Asp Met Gly Asn Ser Gly Tyr Asp Val Ala Asp
225 230 235 240
Gln Tyr Gln Tyr Phe Lys Ala Gly Val Tyr Asn Gln Asn Asn Thr Gly
245 250 255
Asn Ala Ser Asp Tyr Val Gln Val Thr Phe Tyr Ala Leu Glu Gln Ser
260 265 270
His Asp
<210> 4
<211> 822
<212> DNA
<213> 4
<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 (10)

1. a kind of enzymatic hydrolysis beam system for algin oligosaccharide method, it is characterised in that described method include following step Suddenly：
1) substrate is prepared：Algin raw material is mixed with water, it is neutral enzymolysis substrate to be configured to the pH value that concentration is 5%-8% Solution；
2) stepwise discretization：Algin catenase is added, in enzymolysis 1-2h is stirred at 40-50 DEG C, adds 0.3%-0.8%'s again Algin catenase, stirring enzymolysis, total enzymolysis time is 2-4h；
3) prepared by oligosaccharide：After enzymolysis terminates, bits are filtered or are centrifuged off, supernatant concentration, that freeze-drying obtains marine alga is low Glycan.
2. the method for claim 1, it is characterised in that the amino acid sequence of described algin catenase is SEQ ID NO:1。
3. the method for claim 1, it is characterised in that described algin catenase, the nucleotides of its encoding gene Sequence is SEQ ID NO:2.
4. the method for claim 1, it is characterised in that the amino acid sequence of described algin catenase is SEQ ID NO:3。
5. the method for claim 1, it is characterised in that described algin catenase, the nucleotides of its encoding gene Sequence is SEQ ID NO:4.
6. the method for claim 1, it is characterised in that the amino acid sequence of described algin catenase is SEQ ID NO:1。
7. the method for claim 1, it is characterised in that described step 1) enzymolysis substrate solution in algin it is dense It is 5%-8% to spend.
8. a kind of algin catenase, it is characterised in that the amino acid sequence of described algin catenase is SEQ ID NO: 3。
9. a kind of encoding gene, it is characterised in that the algin catenase described in described gene code claim 8.
10. application of the algin catenase described in claim 8 in algin oligosaccharide is prepared.
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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
CN110423787A (en) * 2019-08-09 2019-11-08 中国海洋大学 A kind of preparation method of homogeneity brown alga trisaccharide
CN111197065A (en) * 2020-02-24 2020-05-26 江南大学 Method for producing algin hydrolysate
CN112715964A (en) * 2020-12-16 2021-04-30 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
CN114287521A (en) * 2021-12-27 2022-04-08 中国海洋大学 Compound enzymolysis preparation method of laminarin compounded with cottonseed protein and used for feeding to replace fish meal and application of laminarin
CN114507656A (en) * 2022-03-02 2022-05-17 中国海洋大学 Method for preparing brown algae tetrasaccharide rich in guluronic acid
CN115786318A (en) * 2022-12-20 2023-03-14 中国海洋大学 Alginate lyase truncation Algt1 and application thereof
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CN117230051A (en) * 2023-11-16 2023-12-15 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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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
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
CN110423787A (en) * 2019-08-09 2019-11-08 中国海洋大学 A kind of preparation method of homogeneity brown alga trisaccharide
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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
CN112715964A (en) * 2020-12-16 2021-04-30 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
CN114287521A (en) * 2021-12-27 2022-04-08 中国海洋大学 Compound enzymolysis preparation method of laminarin compounded with cottonseed protein and used for feeding to replace fish meal and application of laminarin
CN114507656A (en) * 2022-03-02 2022-05-17 中国海洋大学 Method for preparing brown algae tetrasaccharide rich in guluronic acid
CN115948373A (en) * 2022-11-11 2023-04-11 深圳润康生态环境股份有限公司 Alginate lyase mutant Pl7AaM and application thereof
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