CN110438111A - A kind of algin catenase and its application - Google Patents

A kind of algin catenase and its application Download PDF

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Publication number
CN110438111A
CN110438111A CN201910671029.5A CN201910671029A CN110438111A CN 110438111 A CN110438111 A CN 110438111A CN 201910671029 A CN201910671029 A CN 201910671029A CN 110438111 A CN110438111 A CN 110438111A
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algin catenase
enzyme
nacl
gly
ala
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江波
刘晓勇
张涛
孟青
陈静静
王心怡
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Shandong Haizhibao Seafood Co ltd
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Shandong Haizhibao Seafood Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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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Abstract

The invention discloses a kind of algin catenase and its applications, belong to field of biotechnology.Algin catenase degrading activity provided by the invention is high, and enzyme activity reaches 65U/mg；Property is stablized, and enzyme activity still keeps 98% or more of initial enzyme activity after storing 18 months in 4 DEG C；It, can specificity production brown alga oligose trisaccharide with very high product specificities.Gained algin catenase of the invention has important industrial application value and scientific research value.

Description

A kind of algin catenase and its application
Technical field
The present invention relates to a kind of algin catenase and its applications, belong to field of biotechnology.
Background technique
Algin (Alginate) is by beta-D-mannuronic acid (β-D-mannuronate) and α-L- guluronic acid The linear polysaccharide that (α-L-guluronate) two kinds of sugar units are polymerized by Isosorbide-5-Nitrae glycosidic bond, China is maximum in the world Algin production country, output are more than the 70% of total output.Brown alga oligose (Alginate Oligosaccharides, AOS) be algin catabolite, containing 2-20 sugared unit, molecular weight is small, and algin macromolecular can be overcome to cannot pass through The limitation of the various biological barriers of body, application range is wider, can show the bioactivity more more brilliant than brown alga glue polysaccharide, Such as anticoagulation reduces the effects of blood pressure and blood lipoid, anti-inflammatory, anti-oxidant, antitumor, immunological regulation, moreover it is possible to Bifidobacterium etc. be promoted to have The growth of beneficial microorganism.
Enzyme digestion reaction satisfies the requirements many advantages such as easy to control, substrate specificity is strong, yield is high, energy conservation and environmental protection, it has also become The main path of brown alga oligose (AOS) is prepared at present.Preparation AOS is digested as raw material using algin mainly to split by algin It solves what the effect of enzyme was realized, i.e., brown alga glue polysaccharide glycosidic bond is broken by beta-elimination reaction, and generation is newly-generated non-reduced The product oligosaccharides of unsaturated double-bond structure are formed between the C4,5 of end.
Algin catenase belongs to polysaceharide lyase family (EC 4.2.2), is divided by it to the degradation mode difference of substrate Poly- β-D-1,4- mannuronic acid lyases (EC 4.2.2.3) and poly- α-L-1,4- guluronic acid lyases (EC 4.2.2.11).Algin catenase is from a wealth of sources, is mainly derived from seaweed plant, marine bacteria, fungi and mollusk Deng, however enzyme preparation type is few and expensive at present, limits the application development of the enzyme.
Summary of the invention
The present invention provides a kind of novel algin catenase Aly01, and amino acid sequence is as shown in SEQ ID NO:1；It compiles The nucleotide sequence of the gene of the code algin catenase can be as shown in SEQ ID NO:2, wherein 1~78bp encoded signal Peptide.
The present invention also provides a kind of method for producing novel algin catenase Aly01 using Vibrio natriegen SK42.001, The following steps are included:
(1) seed culture: seed culture medium: sodium alginate 5, (NH4)2SO45, NaCl 30, MgS04·7H2O 1, K2HPO42, FeSO4·7H2O 0.01；Vibrio natriegen SK42.001 is accessed into seed culture medium, is trained in 28 DEG C, 200rpm shaking table Support 12 h；
(2) fermented and cultured: fermentation medium: sodium alginate 8, NH4Cl 5, NaCl 30, MgS04·7H2O 1, K2HPO4 2, FeSO4·7H2O 0.01；Fermentation condition are as follows: inoculum concentration 5%, 28 DEG C of temperature, revolving speed 200rpm fermentation 36h are obtained containing brown The fermentation liquid of phycocolloid lyases；
(3) it purifies: thallus is removed into fermentation liquid centrifugation and obtains algin catenase crude enzyme liquid, through 20%~80% ammonium sulfate Precipitation and separation destination protein, buffer dialysis, 16/10 ion-exchange chromatography of DEAE-FF, 75 Gel filtration of Superdex Analysis, finally by the pure enzyme solution body of Aly01 after purification, freeze-drying obtains enzyme powder.
The present invention also provides using novel algin catenase Aly01 specificity production brown alga oligose trisaccharide method, with Sodium alginate is substrate, and using NaCl as enzyme stabilizers, sodium alginate synthesis brown alga of degrading in the buffer system of pH6.5-9 is few Sugared trisaccharide.The preferred 100mM or more of dosage of the NaCl stabilizer.The method preferably carries out under the conditions of 25~40 DEG C, especially It is 35 DEG C.
Beneficial effects of the present invention:
Gained algin catenase degrading activity of the invention is high, and enzyme activity reaches 65U/mg；Property is stablized, and stores 18 in 4 DEG C Enzyme activity still keeps 98% or more of initial enzyme activity after month；It, can specificity production brown alga oligose three with very high product specificities Sugar.The characteristic of stable, the high product specificities of gained algin catenase property of the invention is reporting algin catenase correlation Have no that there is important industrial application value and scientific research value in document.
Biomaterial preservation
Vibrio natriegen Vibrio natriegens SK42.001 is preserved in Chinese Typical Representative culture on January 5th, 2017 Collection CCTCC, preservation address are the Wuhan Wuhan University, China, and deposit number is CCTCC NO:M2017011.
Detailed description of the invention
The Aly01 enzyme SDS-PAGE analysis of Fig. 1 after purification
Influence of Fig. 2 temperature to Aly01
Influence of Fig. 3 pH to Aly01
Influence of Fig. 4 NaCl to Aly01
Fig. 5 Aly01 degradation sodium alginate product analysis；DP2: poly- mannobiose aldehydic acid；DP3: poly- Gu Luosan uronic acid；； 1:Aly01 enzyme 4h reaction solution；2: substrate sodium alginate soln.
Specific embodiment
The production method of 1 algin catenase Aly01 of embodiment
The screening technique of A Vibrio natriegen Vibrio natriegens
(1) ooze is sampled near laminaria culture factory, Rongcheng City, Shandong Province, takes 1g sample in 50mL sterile water It is uniformly dispersed.
(2) 1mL supernatant is taken to be inoculated in 50mL screening fluid nutrient medium, 28 DEG C, 200rpm culture 2 days, dilution 10-6 And be coated on screening flat board culture medium, 28 DEG C are cultivated 2 days, and picking different shape single colonie obtains pure through multiple plate streaking Culture.
(3) picking different shape single colonie, is inoculated in fluid nutrient medium, and 28 DEG C, 200rpm culture 2 days take supernatant Bacterial strain algin catenase enzyme activity is measured, the higher bacterial strain of enzyme activity is chosen, entrusts China typical culture collection center preservation, and right Morphological feature, physiological and biochemical property and the 16S rDNA sequence of the bacterial strain are analyzed.
The identification of B Vibrio natriegen Vibrio natriegens
(1) flat-plate bacterial colony form
The flat-plate bacterial colony form of Vibrio natriegen SK42.001: lining and mushroom out on plating medium, and 28 DEG C of cultures are for 24 hours After grow single colonie, the rounded protrusion of bacterium colony, milky, it is wet it is slightly glutinous, surface is smooth, flush edge, diameter about 0.6~ 0.8cm。
(2) the thallus feature under Electronic Speculum
Thallus feature of the Vibrio natriegen SK42.001 under Electronic Speculum: thallus is short and small, and both ends blunt circle bends to arcuation, size It is 1.2~1.4 μm of 0.6~0.8 μ m.
(3) physiological and biochemical property
The physiological and biochemical property of Vibrio natriegen SK42.001: Vibrio natriegen is Gram-negative, aerobic growth, indoles Reaction negative, hydrolyzable gelatin and weakly hydrolyse aesculin, cannot hydrolyze arginine, urea and beta galactose glycosides, using Portugal Grape sugar, sucrose, starch, arabinose, mannose, cannot using fructose, maltose, synanthrin, xylose, galactolipin, sorbose, Xylitol.Specifically, Vibrio natriegen provided by the invention can utilize starch, maltose with gelatin hydrolysate.
The 16S rDNA of Vibrio natriegen SK42.001 is compared with the data in ncbi database, the results showed that with need sodium Vibrios homology is high.
The preparation of C algin catenase
Inclined-plane, seed, fermentation third stage culture production are carried out to Vibrio natriegen (Vibrio natriegens) SK42.001, Culture medium each component is in terms of g/L:
A, inclined-plane culture: slant medium: sodium alginate 5, (NH4)2SO45, NaCl 30, MgS04·7H2O 1, K2HPO42, FeSO4·7H2O 0.01, agar 15-20, natural pH are prepared using deionized water, 121 DEG C of sterilizing 20min；Tiltedly Face condition of culture are as follows: 25~30 DEG C of cultivation temperature, incubation time 1~3 day；
B, seed culture: seed culture medium: sodium alginate 5, (NH4)2SO45, NaCl 30, MgS04·7H2O 1, K2HPO42, FeSO4·7H2O 0.01, natural pH, is prepared using deionized water, 121 DEG C of sterilizing 20min；Seed culture condition Are as follows: in 28 DEG C, 200rpm shaking table culture 12h；
C, fermented and cultured: fermentation medium: sodium alginate 8, NH4Cl 5, NaCl 30, MgS04·7H2O 1, K2HPO42, FeSO4·7H2O 0.01, natural pH, is prepared using deionized water, 121 DEG C of sterilizing 20min；Fermentation condition are as follows: inoculum concentration 5%, 28 DEG C of temperature, revolving speed 200rpm shaker fermentation 36h, obtain the fermentation liquid containing algin catenase.After measured, fermentation liquid Enzyme activity is 4.5U/mL.When surveying enzyme activity, using the PB buffer of 50mM, pH7.0 as buffer system, using sodium alginate as substrate, with NaCl is enzyme stabilizers, and 35 DEG C of reaction 30min take fermented supernatant fluid DNS method to detect enzyme activity.Enzyme activity definition: raw per minute Enzyme amount needed for producing 1 μm of ol reduced sugar.
Thallus is removed into fermentation liquid centrifugation and obtains algin catenase crude enzyme liquid, through 20%~80% ammonium sulfate precipitation and separation Destination protein, buffer dialysis, 16/10 ion-exchange chromatography of DEAE-FF, 75 gel permeation chromatography of Superdex finally will Pure enzyme solution body (Fig. 1) freeze-drying of Aly01 after purification obtains enzyme powder, and purification is 7.63-8.17 times, and final yield is 56.5-61.3%.
2 sequence alignment of embodiment
After carrying out amino acid sequencing to enzyme, design primer is from Vibrio natriegen (Vibrio natriegens) SK42.001 base Because amplifying the gene of coding algin catenase in group, nucleotide sequence is as shown in SEQ ID NO:2.DNA sequence dna BLAST As a result: algin catenase provided by the invention and the algin in the source Vibrio alginolyticus FDAARGOS crack Enzyme dna sequence homology is nearest, but similitude only has 85%, there is 231 base differences, 8 Gap.
Amino acid sequence BLAST result: a certain Vibrio in algin catenase provided by the invention and ncbi database The algin catenase amino acid sequence homology for belonging to source is nearest, and similitude 93% has 39 amino acid differences, 0 Gap。
3 zymologic property of embodiment
Enzyme activity definition: enzyme amount needed for 1 μm of ol reduced sugar of production per minute.
Enzyme activity assay method: 1mL enzyme reaction solution (the PB buffer of 50mM, pH7.0) includes: 5mg sodium alginate, 300mM NaCl, 0.84 μ g algin catenase (embodiment 1 purifies obtained enzyme), 35 DEG C of reaction 30min take fermented supernatant fluid DNS Method detects enzyme activity.
(1) influence of the temperature to enzyme activity: 1mL enzyme reaction solution (the PB buffer of 50mM, pH7.0) includes: 5mg alginic acid Sodium, 300mM NaCl, 0.84 μ g algin catenase；By enzyme reaction solution be respectively placed in 4 DEG C, 20 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 30min is reacted in 50 DEG C, 60 DEG C and 70 DEG C of water-bath, measures algin catenase enzyme activity at each temperature.As shown in Fig. 2, most suitable 35 DEG C of reaction temperature.
(2) influence of the pH to enzyme activity: 1mL enzyme reaction solution includes: 5mg sodium alginate, 300mM NaCl, 0.84 μ g brown alga Glue lyases, is respectively adopted the buffer (50mM) of different pH value: Acetic acid-sodium acetate buffer (pH 3.5,4.0,4.5, 5.0), citric acid salt buffer (pH 5.0,5.5,6.0,6.5), phosphate buffer (pH 6.0,6.5,7.0,7.5, 8.0), Tris- hydrochloride buffer (pH 7.5,8.0,8.5) and glycine-NaOH buffer (pH 8.5,9.0,9.5,10, 10.5,11)；By enzyme reaction solution in 35 DEG C of reaction 30min, the enzyme activity under each pH is measured.As shown in figure 3, algin catenase is aobvious Wider pH adaptability is shown, 90% or more vigor is able to maintain within the scope of pH6.5-9.
(3) influence of the NaCl to enzyme activity: 1mL enzyme reaction solution (the PB buffer of 50mM, pH7.0) includes: 5mg alginic acid Sodium, 0.84 μ g algin catenase add final concentration of 0,50,80,100,200,250,300,400,500,1000mM respectively NaCl measure the enzyme activity under various concentration NaCl in 35 DEG C of reaction 30min.Algin catenase to NaCl have it is stronger according to Lai Xing, it is active (Fig. 4) that NaCl concentration is more than or equal to 100mM Shi Caiyou obvious degradation.
(4) product specificities:
1mL enzyme reaction system: NaCl 300mM, 0.84 μ g of algin catenase, 10mg sodium alginate, with 50mM, PH7.0PB buffer constant volume, 35 DEG C of reaction 12h.It extracts reaction solution and is detected using thin-layer chromatography (TLC), method particularly includes: The silica gel plate for deciding certain size, it is several with Pencil marks on line in bottom edge side one line parallel with bottom edge of pencil drawing A equidistant points；Disaccharides (DP2) (1mg/mL), trisaccharide (DP3) are marked into (1mg/mL) sample, reaction solution, sodium alginate substrate (10mg/ ML it) takes 1 μ L point in mark point respectively, silica gel plate is placed in ventilation and is dried completely, the chromatography containing solvent is then put into Start to chromatograph in cylinder, until liquid is run to silica gel plate top；Silica gel plate is dried up completely with hair dryer after chromatography, later It is put into 15s in developing solution, is baked to colour developing in 120 DEG C of baking ovens after drying.
Product analysis: as shown in figure 5, algin catenase enzyme provided by the invention is degraded when substrate sodium alginate, seaweed The degradation rate of sour sodium is 100%, and almost all is degraded to trisaccharide.The oligosaccharides of algin catenase degradation can generate unsaturated double Key, compared to the trisaccharide standard specimen of saturation, the size of one water of molecular weight differences.Substrate sodium alginate selective degradation can be produced There is not been reported for the algin catenase of specific brown alga oligose (such as trisaccharide).
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection of the invention without departing from the spirit and scope of the present invention Range should subject to the definition of the claims.
SEQUENCE LISTING
<110>Shandong Haizhibao Seafood Co., Ltd.
<120>a kind of algin catenase and its application
<130> BAA180804A
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 521
<212> PRT
<213>vibrio marinopraesens SK42.001
<400> 1
Met Lys His Ile Phe Phe Lys Ser Leu Leu Ala Ser Ser Ile Leu Leu
1 5 10 15
Ala Val Gly Cys Asn Ser Thr Ala Thr Ala Lys Ala Asp Phe Pro Asn
20 25 30
Asn Gln Glu Thr Gly Val Asp Ile Leu Thr Pro Val Ala Ile Thr Ala
35 40 45
Ser Ser His Asp Gly Asn Val Pro Glu Asn Leu Leu Asp Gln Asp Ile
50 55 60
Met Thr Arg Trp Ala Ala Asn Gly Asp Gly Glu Trp Ala Met Leu Asp
65 70 75 80
Tyr Gly Ser Val Tyr Gly Phe Asp Ala Ile Gln Ala Ser Phe Ser Lys
85 90 95
Gly Asn Glu Arg Val Thr Ser Phe Asp Val Gln Phe Ser Thr Asp Gly
100 105 110
Glu Asn Trp Val Thr Val Ile Glu Gly Ala Gln Ser Ser Gly Arg Ala
115 120 125
Leu Gly Leu Glu Arg Phe Gln Phe Glu Pro Ala Val Lys Ala Arg Tyr
130 135 140
Val Arg Tyr Val Gly His Gly Asn Thr Lys Asn Gln Trp Asn Ala Val
145 150 155 160
Thr Glu Met Ala Ala Val Asn Cys Gly Ile Asn Ala Cys Pro Ala Ser
165 170 175
His Val Ile Thr Asp Asp Val Val Lys Ala Glu Ala Thr Met Ile Ala
180 185 190
Ala Met Lys Ala Lys Glu Lys Ala Gln Lys Glu Leu Leu Lys Asn Asn
195 200 205
Arg Lys Gly Asp Phe Gly Glu Pro Ile Val Arg Pro Cys Gly Thr Thr
210 215 220
Val Thr Cys Asp Leu Thr Lys Ala Met Pro Ser Pro Thr Leu Pro Ala
225 230 235 240
Val Pro Leu Ala Lys Asn Ala Pro Gly Gln Asn Phe Asp Leu Thr Arg
245 250 255
Trp Lys Leu Thr Thr Pro Phe Asp His Asp Lys Asp Gly Arg Ala Asp
260 265 270
Asp Ile Asp Glu Trp Asp Met Ala Asn Gly Phe Gln His Pro Asp Ile
275 280 285
Phe Tyr Thr Ala Asp Asp Gly Gly Met Val Phe Lys Ser Tyr Val Lys
290 295 300
Gly Ala Arg Thr Ser Lys Asn Thr Lys Tyr Ala Arg Thr Glu Leu Arg
305 310 315 320
Thr Met Leu Arg Ala Gly Glu Lys Ser His Ser Thr Lys Gly Val Asn
325 330 335
Pro Asn Asn Trp Val Phe Ser Ser Ala Pro Val Glu Asp Gln Lys Ala
340 345 350
Ala Gly Gly Val Asp Gly Thr Leu Glu Ala Thr Leu Lys Ile Asp His
355 360 365
Ala Thr Thr Thr Gly Gln Ser His Glu Val Gly Arg Phe Ile Ile Gly
370 375 380
Gln Ile His Asp Lys Asp Asp Glu Pro Ile Arg Leu Tyr Tyr Arg Lys
385 390 395 400
Leu Pro Asp Gln Pro Thr Gly Thr Val Tyr Phe Ala His Glu Lys Thr
405 410 415
Lys Thr Gly Thr Glu Asp Tyr Tyr Ser Leu Val Gly Asp Met Thr Gly
420 425 430
Glu Ile Gly Asn Asp Gly Ile Ala Leu Gly Glu Lys Phe Ser Tyr Ile
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Ile Asp Val Lys Gly Asn Thr Met Thr Val Thr Val Lys Arg Asp Gly
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Lys Asp Asp Val Val Gln Val Val Asp Met Ser Asp Ser Gly Tyr Asp
465 470 475 480
Glu Gly Gly Arg Tyr Met Tyr Phe Lys Ala Gly Val Tyr Asn Gln Asn
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Asp Gln Ser Phe Gly Lys Tyr Gln Gly
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<210> 2
<211> 1566
<212> DNA
<213>vibrio marinopraesens SK42.001
<400> 2
atgaagcata ttttcttcaa aagcttgtta gcttcttcaa tcctattggc tgttggttgt 60
aacagcactg caactgcgaa ggctgatttc ccaaacaatc aagaaaccgg cgttgacatt 120
ctaactcctg ttgcaatcac ggcgagtagc catgatggta atgtgcctga gaacttactt 180
gaccaagata ttatgactcg ctgggcagcg aacggtgacg gtgagtgggc aatgttggat 240
tacggctcag tttatgggtt cgatgcaatc caagcgtcgt ttagtaaagg taatgaacgt 300
gtcacgtcat ttgatgttca gttcagcaca gatggtgaaa actgggtaac ggttattgaa 360
ggtgcacaaa gctctggtcg tgctcttggt ctggaacgct tccagttcga gcctgcggta 420
aaagctcgtt atgtacgtta cgttggccac ggcaatacca aaaaccaatg gaacgctgtt 480
actgaaatgg ccgcggttaa ctgtggaatc aatgcgtgcc cggcaagcca tgtcattacc 540
gatgatgttg ttaaagctga agcgactatg attgctgcaa tgaaggctaa ggaaaaagcg 600
caaaaggaac tccttaaaaa taatcgcaaa ggtgatttcg gagaaccaat cgtccgtcct 660
tgcgggacga cagtgacgtg tgacctaact aaagcaatgc catccccaac gctaccggct 720
gttccactag ctaagaatgc accaggccaa aactttgacc tgacgcgctg gaaactgaca 780
acgcctttcg atcacgacaa agacggccgc gctgatgata ttgatgagtg ggatatggca 840
aacggcttcc agcacccaga tatcttctac acagctgatg atggcggcat ggttttcaag 900
agctatgtaa aaggtgcacg tacctctaaa aatactaagt acgcacgtac agagttgcgc 960
actatgctgc gtgcgggtga gaagtctcac agtacaaaag gtgtaaatcc aaataactgg 1020
gtattcagct cagcgccggt agaagatcag aaagcagcgg gtggggtaga tggcacgctt 1080
gaggcaactc tgaagattga ccatgcaacc acaacgggtc agtcacacga agttggccgt 1140
ttcattatcg gtcagattca tgacaaagat gatgagccaa ttcgccttta ctaccgtaag 1200
ctaccagacc agccaacagg tacggtttac ttcgctcacg aaaaaaccaa aacaggtact 1260
gaagattact acagcctggt tggtgatatg actggtgaaa tcggtaacga tggtatcgcg 1320
ctaggtgaaa aattcagcta catcattgat gtaaaaggca acacgatgac agttacggta 1380
aaacgtgacg gtaaagatga tgttgtacaa gtcgtagata tgagtgacag tggttatgat 1440
gagggtggcc gatacatgta cttcaaggcc ggtgtttata accagaatat gtacggcaat 1500
ccagatgatt acgctcaagc aactttctac aagctagatc aatcttttgg taagtaccaa 1560
ggctag 1566

Claims (9)

1. a kind of algin catenase, which is characterized in that its amino acid sequence is as shown in SEQ ID NO:1；Alternatively, in SEQ ID One or several amino acid are replaced, missed or added on the basis of sequence shown in NO:1.
2. encoding the gene of algin catenase described in claim 1, which is characterized in that nucleotide sequence such as SEQ ID NO: Shown in 2.
3. a kind of method for preparing algin catenase described in claim 1, which is characterized in that utilize Vibrio natriegen SK42.001 produces algin catenase, the Vibrio natriegen Vibrio natriegens SK42.001, January 15 in 2017 It is preserved in China typical culture collection center CCTCC day, preservation address is the Wuhan Wuhan University, China, and deposit number is CCTCC NO:M2017011.
4. according to the method described in claim 3, fermenting it is characterized in that, Vibrio natriegen SK42.001 seed culture fluid is accessed Culture medium separates and collects algin catenase after fermented culture from fermented supernatant fluid.
5. according to the method described in claim 4, it is characterized in that, the formula of the fermented and cultured are as follows: sodium alginate 8g/L, NH4Cl 5g/L, NaCl 30g/L, MgS04·7H2O 1g/L, K2HPO42g/L, FeSO4·7H2O 0.01g/L。
6. method according to claim 4 or 5, which comprises the following steps:
(1) seed culture
Seed culture medium: sodium alginate 5, (NH4)2SO45, NaCl 30, MgS04·7H2O 1, K2HPO42, FeSO4·7H2O 0.01；Vibrio natriegen SK42.001 is accessed into seed culture medium, in 28 DEG C, 200rpm shaking table culture 12h；
(2) fermented and cultured
Fermentation medium: sodium alginate 8, NH4Cl 5, NaCl 30, MgS04·7H2O 1, K2HPO42, FeSO4·7H2O 0.01；Fermentation condition are as follows: inoculum concentration 5%, 28 DEG C of temperature, revolving speed 200rpm fermentation 36h obtain the hair containing algin catenase Zymotic fluid；
(3) it purifies: thallus is removed into fermentation liquid centrifugation and obtains algin catenase crude enzyme liquid, is separated through 20%~80% ammonium sulfate Destination protein, buffer dialysis, ion-exchange chromatography, gel permeation chromatography are precipitated, enzyme solution after purification is obtained, it will enzyme after purification Liquid, freeze-drying obtain enzyme powder.
7. a kind of method using the production brown alga oligose trisaccharide of algin catenase described in claim 1, which is characterized in that with sea Mosanom is substrate.
8. the method according to the description of claim 7 is characterized in that using NaCl as enzyme stabilizers, in the buffer system of pH6.5-9 In, 25~40 DEG C of temperature condition decline solution sodium alginates generate brown alga oligose trisaccharide.
9. method according to claim 7 or 8, which is characterized in that the dosage of the NaCl stabilizer is 100mM or more.
CN201910671029.5A 2019-07-24 2019-07-24 A kind of algin catenase and its application Withdrawn CN110438111A (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110331137A (en) * 2019-06-03 2019-10-15 中国海洋大学 A kind of algin catenase and preparation method thereof
CN111100825A (en) * 2020-02-27 2020-05-05 中国科学院烟台海岸带研究所 Bacillus and application thereof in industry
CN111197065A (en) * 2020-02-24 2020-05-26 江南大学 Method for producing algin hydrolysate
WO2021012959A1 (en) * 2019-07-24 2021-01-28 江南大学 Alginate lyase and application thereof
CN113481187A (en) * 2021-05-18 2021-10-08 江南大学 Alginate lyase mutant and application thereof
CN114457062A (en) * 2022-03-02 2022-05-10 山东海之宝海洋科技有限公司 Alginate lyase for preparing alginate oligosaccharide and application thereof
CN116555094A (en) * 2023-04-20 2023-08-08 山东省农业科学院 Polysaccharide degrading bacteria of vibrio alginolyticus and culture method and application thereof

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110331137A (en) * 2019-06-03 2019-10-15 中国海洋大学 A kind of algin catenase and preparation method thereof
WO2021012959A1 (en) * 2019-07-24 2021-01-28 江南大学 Alginate lyase and application thereof
US11993795B2 (en) 2019-07-24 2024-05-28 Jiangnan University Alginate lyase and application thereof
CN111197065A (en) * 2020-02-24 2020-05-26 江南大学 Method for producing algin hydrolysate
CN111100825A (en) * 2020-02-27 2020-05-05 中国科学院烟台海岸带研究所 Bacillus and application thereof in industry
CN111100825B (en) * 2020-02-27 2021-05-11 中国科学院烟台海岸带研究所 Bacillus and application thereof in industry
CN113481187A (en) * 2021-05-18 2021-10-08 江南大学 Alginate lyase mutant and application thereof
CN113481187B (en) * 2021-05-18 2023-07-25 江南大学 Algin lyase mutant and application thereof
CN114457062A (en) * 2022-03-02 2022-05-10 山东海之宝海洋科技有限公司 Alginate lyase for preparing alginate oligosaccharide and application thereof
CN114457062B (en) * 2022-03-02 2024-03-26 山东海之宝海洋科技有限公司 Algin lyase for preparing alginate oligosaccharides and application thereof
CN116555094A (en) * 2023-04-20 2023-08-08 山东省农业科学院 Polysaccharide degrading bacteria of vibrio alginolyticus and culture method and application thereof
CN116555094B (en) * 2023-04-20 2023-12-15 山东省农业科学院 Polysaccharide degrading bacteria of vibrio alginolyticus and culture method and application thereof

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Application publication date: 20191112