CN110257410A - A kind of gene encoding algin catenase - Google Patents

A kind of gene encoding algin catenase Download PDF

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CN110257410A
CN110257410A CN201910671672.8A CN201910671672A CN110257410A CN 110257410 A CN110257410 A CN 110257410A CN 201910671672 A CN201910671672 A CN 201910671672A CN 110257410 A CN110257410 A CN 110257410A
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gly
ala
asp
algin catenase
thr
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CN110257410B (en
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江波
张涛
孟青
陈静静
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Jiangnan University
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    • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
    • C12Y402/02003—Poly(beta-D-mannuronate) lyase (4.2.2.3)
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    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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    • Y02E50/10—Biofuels, e.g. bio-diesel

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Abstract

The invention discloses a kind of genes for encoding algin catenase, 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 gene encoding algin catenase
Technical field
The present invention relates to a kind of genes for encoding algin catenase, 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) It is the catabolite of algin, containing 2-20 sugared unit, molecular weight is small, algin macromolecular can be overcome to cannot pass through body each The limitation of kind biological barrier, application range is wider, can show the bioactivity more more brilliant than brown alga glue polysaccharide, such as anticoagulant Blood reduces the effects of blood pressure and blood lipoid, anti-inflammatory, anti-oxidant, antitumor, immunological regulation, moreover it is possible to promote Bifidobacterium etc. beneficial to micro- life The growth of object.
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 crack by algin What the effect of enzyme was realized, i.e., brown alga glue polysaccharide glycosidic bond is broken by beta-elimination reaction, and generate in newly-generated non-reducing end The product oligosaccharides of unsaturated double-bond structure are formed between C4,5.
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 12h；
(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 of degrading in the buffer system of pH6.5-9 synthesizes brown alga oligose 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 10-6And It is coated on screening flat board culture medium, 28 DEG C are cultivated 2 days, and picking different shape single colonie obtains pure training through multiple plate streaking Support object.
(3) picking different shape single colonie, is inoculated in fluid nutrient medium, and 28 DEG C, 200rpm culture 2 days take supernatant Bacterial strain enzyme activity is measured, the higher bacterial strain of enzyme activity is chosen, entrusts China typical culture collection center preservation, and to the form of the bacterial strain Feature, physiological and biochemical property and 16S rDNA sequence 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 utilize fructose, maltose, synanthrin, xylose, galactolipin, sorbose, wood Sugar alcohol.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, the enzyme of fermentation liquid Living is 4.5U/mL.Fermented supernatant fluid detects enzyme activity with DNS method, using the PB buffer of 50mM, pH7.0 as buffer system, with sea Mosanom is substrate, using 300mM NaCl as stabilizer, 35 DEG C of reaction temperature, reaction time 30min.Enzyme activity definition: 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 knot Fruit: the algin catenase of algin catenase provided by the invention and the source Vibrio alginolyticus FDAARGOS DNA sequence dna 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, 35 DEG C of reaction 30min take supernatant DNS method to detect 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；Enzyme reaction solution is respectively placed in 4 DEG C, 20 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 50 DEG C, react 30min in the water-baths of 60 DEG C and 70 DEG C, measurement algin catenase enzyme activity at each temperature.As shown in Fig. 2, most suitable anti- Answer 35 DEG C of temperature.
(2) influence of the pH to enzyme activity: 1mL enzyme reaction solution includes: 5mg sodium alginate, 300mM NaCl, 0.84 μ g algin 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 show compared with Wide pH adaptability, is able to maintain 90% or more vigor 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: it cuts out The silica gel plate for determining certain size, it is several with Pencil marks on line in bottom edge side one line parallel with bottom edge of pencil drawing Equidistant points；By disaccharides (DP2) (1mg/mL), trisaccharide (DP3) standard specimen (1mg/mL), 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, is then put into the chromatography cylinder containing solvent Start to chromatograph, until liquid is run to silica gel plate top；After chromatography silica gel plate is dried up completely with hair dryer, is put into later aobvious 15s in color liquid 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 special Determining the algin catenase of brown alga oligose (such as trisaccharide), there is not been reported.
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 model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of gene for encoding algin catenase
<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
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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
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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
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Lys Thr Gly Thr Glu Asp Tyr Tyr Ser Leu Val Gly Asp Met Thr Gly
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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
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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. encoding the gene of algin catenase, which is characterized in that nucleotide sequence is as shown in SEQ ID NO:2.
2. being used to prepare the enzyme preparation of brown alga oligose, which is characterized in that the algin containing the coding of gene described in claim 1 is split Solve enzyme.
3. carrying the carrier or plasmid of gene described in claim 1.
4. carrying the cell of gene described in claim 1.
5. carrying the genetic engineering bacterium of gene described in claim 1.
6. application of the gene described in claim 1 in production brown alga oligose.
7. application of the carrier or plasmid described in claim 3 in production brown alga oligose.
8. application of the cell described in claim 4 in production brown alga oligose.
9. application of the genetic engineering bacterium described in claim 5 in production brown alga oligose.
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