CN109055343A - A kind of novel algin catenase AlyB10 and its application - Google Patents

A kind of novel algin catenase AlyB10 and its application Download PDF

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CN109055343A
CN109055343A CN201811117210.3A CN201811117210A CN109055343A CN 109055343 A CN109055343 A CN 109055343A CN 201811117210 A CN201811117210 A CN 201811117210A CN 109055343 A CN109055343 A CN 109055343A
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algin
algin catenase
alyb10
catenase
gly
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王存良
李尚勇
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    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • 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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    • Y02P20/59—Biological synthesis; Biological purification

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Abstract

The present invention relates to a kind of with the endo-type algin catenase of hot recovery characteristics and its application.The algin catenase is a kind of novel algin catenase AlyB10, and amino acid sequence is as shown in SEQ ID NO.1.Algin catenase provided by the invention is the novel algin catenase of engineer a kind of, and N-terminal contains one section of catalytic domain (Ser1‑Phe292), C-terminal contains one section of carbohydrate binding domain (Met293‑Gly398), the algin catenase sequence similarity of amino acid sequence and existing known function is only 76%.Algin catenase AlyB10 of the present invention has hot recovery characteristics, is boiling after ten minutes, is placed on 10 DEG C and is incubated for 30 minutes, can restore its 52.5% activity.The enzyme effect mode is inscribe, and degradation principal product is algin two, trisaccharide.Algin catenase property of the present invention is stablized, and yield is big, has certain industrial applications potential quality.

Description

A kind of novel algin catenase AlyB10 and its application
Technical field
The present invention relates to a kind of with the endo-type algin catenase AlyB10 of hot recovery characteristics and its application, belongs to life Object technical field.
Background technique
Algin is the important component of brown alga cell wall, by the α-L- mannuronic acid of epimer each other (Mannuronic acid, M) and β-D- guluronic acid (Guluronic acid, G) pass through made of Isosorbide-5-Nitrae glucosides key connection Linear polysaccharide.In general, algin is processed by kelps such as kelp, sargassum, bulk kelps.China is algae culturing big country, Algin yield accounts for 70% of Gross World Product or more.Algin is widely used in the industries such as chemical industry, medicine, food.Difference is poly- Right algin oligosaccharide has different biological activities.It recent studies have shown that, the poly- M sections of oligosaccharides medicines prepared using algin The aggregation and cytotoxicity of the inhibition beta-amyloyd cell of object GV-971 energy multiple target point, are completed the clinical trial of three phases；Poly- G oligosaccharides The inhibition more drug resistance pathogenic bacteria of various clinical can be used with Antibiotic combination.Compare traditional acid-base method degradation algin, enzyme process Algin of degrading has the advantages such as mild condition, easy to control, environmentally protective.Therefore, finding, there is the algin of special nature to split Enzyme is solved, and studies its degradation condition and final product is of great significance.
Algin catenase is catalyzed the fracture of algin intramolecular Isosorbide-5-Nitrae glycosidic bond by beta-elimination reaction, generates non-reduced Property end have conjugated double bond unsaturated oligosaccharides.Traditional algin catenase is from marine animal alimentary canal or marine bacteria Fermented liquid supernatant in be directly separated extraction, be limited to natural algin catenase low output, develop the algin of genetic engineering Lyases is imperative.
(http://www.cazy.org/) has the several hundred algins predicted in polysaccharide degrading enzyme database at present Lyase gene, part of gene are recombinantly expressed in Escherichia coli or saccharomycete, and have studied its zymologic property. But the algin catenase thermostabilization of most of reports and heat are restorative poor, are easy inactivation, are unable to reach industrial applications Requirement.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of algin catenase with hot recovery characteristics and its answer With.The amino acid similarity of the enzyme and existing algin catenase is only 76%.Its N-terminal is catalytic domain, and C-terminal contains one section of carbon water Compound binding domain, the region can significantly increase the characteristic that its heat is restored.The optimal reactive temperature of the enzyme is 50 DEG C；Enzyme solution boils After ten minutes, 10 DEG C are placed on to be incubated for 30 minutes, its 52.5% activity can be restored.The enzyme degrades mode as inscribe, main product of degrading Object is algin unsaturation disaccharides and trisaccharide.
On the one hand, the present invention provides a kind of algin catenase AlyB10 with hot recovery characteristics, amino acid sequence As shown in SEQ ID NO. 1.
SEQ ID NO.1:
STVLALGSALVDNSSLAETLATTHILPESHQALPQGELLGDFNSLDEIEENTLIPKSHTSLAEGVLLWYQKKE YELPGEKALSDSFALKPLFYTSTDGGMVFACPNAGAKTSKNTKYARTELREMLRRGNTTIKTKGITENNWVLNSAHG SVKRKAGAVEGSLEATLAVNRVSTTGDEEKVGRVIIGQITATDEEPIRLYYRKLPGNDNGSIYFAHEINGGDDVWVK MVGKVTLSDNTLTKVGHGKRNKSYKITVNKNILFVTLYMEGKQNATKSFDMSKSGYNKNNQYMYFMETGYSIDGNGG AVQGQQLYLWTTKTTNVNQNWVQISHGSGYYSYKKEGTSLCWDGGTGGAKRQAVTLEVCDSSNYDQHWKKVKVTSGT EIYRFEKRNAPGFSIDG
On the other hand, the present invention also provides a kind of corresponding nucleic acid sequences of algin catenase AlyB10 with hot recovery characteristics Column, as shown in SEQ ID NO.2.
SEQ ID NO.2:
AGCACCGTGCTCGCCCTCGGCAGCGCCCTCGTGGACAACAGCAGCCTCGCCGAGACCCTCGCCACCACCCACA TACTCCCCGAGAGCCACCAGGCCCTCCCCCAGGGCGAGCTCCTCGGCGACTTCAACAGCCTCGACGAGATAGAGGAG AACACCCTCATACCCAAGAGCCACACCAGCCTCGCCGAGGGCGTGCTCCTCTGGTACCAGAAGAAGGAGTACGAGCT CCCCGGCGAGAAGGCCCTCAGCGACAGCTTCGCCCTCAAGCCCCTCTTCTACACCAGCACCGACGGCGGCATGGTGT TCGCCTGCCCCAACGCCGGCGCCAAGACCAGCAAGAACACCAAGTACGCCAGGACCGAGCTCAGGGAGATGCTCAGG AGGGGCAACACCACCATAAAGACCAAGGGCATAACCGAGAACAACTGGGTGCTCAACAGCGCCCACGGCAGCGTGAA GAGGAAGGCCGGCGCCGTGGAGGGCAGCCTCGAGGCCACCCTCGCCGTGAACAGGGTGAGCACCACCGGCGACGAGG AGAAGGTGGGCAGGGTGATAATAGGCCAGATAACCGCCACCGACGAGGAGCCCATAAGGCTCTACTACAGGAAGCTC CCCGGCAACGACAACGGCAGCATATACTTCGCCCACGAGATAAACGGCGGCGACGACGTGTGGGTGAAGATGGTGGG CAAGGTGACCCTCAGCGACAACACCCTCACCAAGGTGGGCCACGGCAAGAGGAACAAGAGCTACAAGATAACCGTGA ACAAGAACATACTCTTCGTGACCCTCTACATGGAGGGCAAGCAGAACGCCACCAAGAGCTTCGACATGAGCAAGAGC GGCTACAACAAGAACAACCAGTACATGTACTTCATGGAGACCGGCTACAGCATAGACGGCAACGGCGGCGCCGTGCA GGGCCAGCAGCTCTACCTCTGGACCACCAAGACCACCAACGTGAACCAGAACTGGGTGCAGATAAGCCACGGCAGCG GCTACTACAGCTACAAGAAGGAGGGCACCAGCCTCTGCTGGGACGGCGGCACCGGCGGCGCCAAGAGGCAGGCCGTG ACCCTCGAGGTGTGCGACAGCAGCAACTACGACCAGCACTGGAAGAAGGTGAAGGTGACCAGCGGCACCGAGATATA CAGGTTCGAGAAGAGGAACGCCCCCGGCTTCAGCATAGACGGC
On the other hand, the present invention also provides the preparation methods of algin catenase AlyB10 with hot recovery characteristics a kind of.
On the other hand, the application the present invention also provides the algin catenase AlyB10 in cracking algin.
On the other hand, the present invention also provides the algin catenase AlyB10 to prepare the application in brown alga oligose.
On the other hand, a method of cracking algin, selected algin catenase are AlyB10.
It is preferred that: reaction temperature is 10 ~ 70 DEG C in the cracking condition.Optimal reactive temperature is 50 DEG C.
It is preferred that: incubation temperature is 0 ~ 50 DEG C in the hot recovery characteristics.Most suitable incubation temperature is 10 DEG C.
It is preferred that: incubation time is 1 ~ 45 minute in the hot recovery characteristics.Most suitable incubation time is 30 minutes.
The utility model has the advantages that
1. algin catenase AlyB10 of the invention is the composition sequence of engineer, N-terminal is catalytic domain, and C-terminal is carbon water Compound binding structural domain, amino acid sequence and existing algin catenase sequence similarity are only 76%, are a sequence Novel novel algin catenase.
2. algin catenase AlyB10 of the invention is after 5 L fermentation, every milliliter of fermentation broth enzyme activity is up to 470 U. Also, the characteristic that there is the enzyme heat to restore, can be incubated at low temperature after boiling conditions, it is amount of activated can to restore its.It is this Novel property can promote its industrial applications.
3. algin catenase AlyB10 gene order of the present invention is engineer, fully synthetic sequence.The present invention A kind of method for preparing the novel algin catenase is additionally provided, that is, the technical method of genetic engineering is utilized, by AlyB10 base It is isolated and purified because heterologous recombination is to Escherichia coli, and using nickel column, has studied its zymologic property, it is found that it has heat The characteristic of recovery, enzyme solution boil after ten minutes, place 30 minutes at 10 DEG C, can restore its 52.5% activity.Utilize the recombination Enzyme has carried out catabolite analysis, which degrades principal product as algin disaccharides and trisaccharide.Algin cracking of the present invention Enzyme AlyB10 has good industrial applications prospect.
Detailed description of the invention
Fig. 1 is algin catenase AlyB10 protein separation figure (M, Protein standards of the present invention；1, purify institute Obtain algin catenase AlyB10)；
Fig. 2 is the optimal reactive temperature of algin catenase AlyB10 of the present invention；
Fig. 3 is heat restorative influence of the different incubation temperatures on algin catenase AlyB10；
Fig. 4 is heat restorative influence of the different incubation times on algin catenase AlyB10；
Fig. 5 is the degradation mode that viscosimetry detects algin catenase AlyB10 of the present invention；
Fig. 6 is enzymolysis product (M, 2 sugar of brown alga oligose that thin-layer chromatography (TLC) method detects algin catenase AlyB10 of the present invention DP2,3 sugar DP3 standard items；0, substrate before digesting；1, product after enzymatic hydrolysis).
Specific embodiment
The engineer of 1 algin catenase AlyB10 of embodiment and sequence analysis
Alginate lyase gene of the present inventionalyB10For engineer, fully synthetic sequence (Huada gene company synthesis), Include 1,194 base sequences, encode 398 amino acid sequences, N-terminal is the conserved catalytic functional domain of algin catenase (Ser1-Phe292), its C-terminal devise one section include 106 amino acid carbohydrate binding domain area (Met293-Gly398), which can form one section of stable tertiary structure, so that the thermal stability and heat to enzyme restore Property has an impact.Utilize the conservative knot in National Center for Biotechnology Information (NCBI) Structure domain analysis Conserved domain (CDD) and Multiple sequence alignments Basic Local Alignment Search Tool (Blast) it finds, which includes the 2nd superfamily (Alginate lyase 2 of an algin catenase Superfamily conserved region), C-terminal include a carbohydrate binding domain.Blast analysis is found, with AlyB10 Amino acid sequence similarity is highest for the algin catenase of the 7th family (PL-7) of polysaceharide lyase and the amino acid of AlyB10 Sequence similarity is only up to 76%.
The gene cloning and recombinant expression of 2 algin catenase AlyB10 of embodiment
It will be fully synthetic in embodiment 1alyB10Gene order is with restriction enzymeNcoI andXhoI(is raw purchased from Dalian treasured Object company) it is restriction enzyme site and designs restriction enzyme site protection base, design recombination primer is following, and (underscore is restriction enzyme Site, italic are restriction enzyme enzyme protection base):
Forward primer: SEQ ID NO.3:PalyB10EF:
5’- CATGCCATGGGTAGCACCGTGCTCGCCCTCGGCA -3’ (Nco I)
Reverse primer: SEQ ID NO.4:PalyB10ER:
5’- CCGCTCGAGGCCGTCTATGCTGAAGCCGG -3’ (Xho I)
The alginate lyase gene, high-fidelity DNA polymerase used in PCR are expanded using above-mentioned recombination primer PCR Primerstar HS is purchased from Dalian treasured biotech firm.Specific PCR amplification condition are as follows: 94 DEG C initial denaturation 3 minutes；94 DEG C of denaturation 30 Second, 55 DEG C are annealed 30 seconds, and 72 DEG C extend 1 minute, and totally 30 recycle；72 DEG C extend 5 minutes；4 DEG C are stablized 15 minutes.
By PCR product restriction enzymeNcoI andXhoI carries out double digestion, is recycled by agarose gel electrophoresis PCR product after digestion.It is same to use by purchase from pET22b (+) Plasmid DNA (100 ng) of Invitrogen company of the U.S. Restriction enzymeNcoI andXhoI carries out double digestion, carries out agarose gel electrophoresis and recycles the product segment after digestion. The description of product behaviour that enzyme used in digestion and substrate reactions system (temperature, time, DNA dosage etc.) are provided referring to the precious biology in Dalian Make.
By the PCR product and pET-22b (+) plasmid vector by double digestion processing, referring to (the purchase of DNA ligase specification From Dalian treasured biotech firm) it is attached；Connection product is converted to e.colistraindh5α (purchased from Dalian treasured biotech firm), It is coated on Luria-Bertani (LB) culture medium solid plate containing 50 μ g/mL ampicillins, 37 DEG C of cultures 16 are small Shi Hou, picking monoclonal；Monoclonal is forwarded in the LB liquid medium containing 50 μ g/mL ampicillins (50 mL point Bottom centrifuge tube loads 5 mL fluid nutrient mediums), 180 rpm shake cultures are stayed overnight in 37 DEG C of shaking tables.Reagent is extracted using plasmid Box (being purchased from Dalian treasured biotech firm) extracts the plasmid in bacterium solution according to product description, passes throughNcoI andXhoI double digestion mirror Determine positive colony and carries out sequencing；Selection and the extracted plasmid of the consistent monoclonal of objective gene sequence, are named For pET22b-AlyB10.
Referring to product description, by recombinant plasmid transformed to e. coli bl21 (DE3) (being purchased from Dalian treasured biotech firm), Will recombination large intestine bacterial strain be named as BL21 (DE3)/pET22b-AlyB10, be stored in -80 DEG C it is spare.
The zymotechnique and method for preparing purified of 3 algin catenase AlyB10 of embodiment
It will be constructed in embodiment 2 and be stored in -80 DEG C of e. coli bl21 (DE3)/pET22b-AlyB10 in LB solid plate Upper scribing line, after 37 DEG C are cultivated 16 hours, picking monoclonal；Monoclonal is forwarded to the LB liquid containing 50 μ g/mL ampicillins In body culture medium (500 mL conical flasks load 50 mL fluid nutrient mediums), cultivate in the shaking table of 37 DEG C of 180 rpm to OD600= 0.6.5 L fermentors load Terrific Broth (TB) culture medium of 60% (3 L), and sterilization treatment in advance；In fermentor 50 μ g/mL ampicillins are added, bacterium solution cultured in conical flask is seeded in 5 L fermentors according to 2% inoculum concentration. Adjusting initial ventilatory capacity is 50 L/h, and initial speed is 350 rpm, and at 37 DEG C, dissolved oxygen is controlled in 15-40% for temperature control；Work as bacterium Body grows into OD600When=5.0, the inducer isopropylthio-β-D- thiogalactoside (IPTG) of final concentration of 0.1 mM is added, 25 DEG C of 60 h of induction.Algin catenase activity determination method are as follows: 900 μ l, 0.3% algin substrate is added in 100 μ l enzyme solutions (20 mM phosphate buffers, pH=7.6), react 10 min at 50 DEG C, with spectrophotometric determination A235 numerical value.Enzyme activity Power is defined as 1 ml enzyme solution A235 numerical value is caused to increase by 0.1 being an enzyme activity unit.Every milliliter of fermentation broth enzyme activity is up to 470 U。
After fermentation stops, 12000 rpm are centrifuged 10 min, abandon thallus, collect supernatant；3 L fermented supernatant fluids point 10 batches Secondary (300 mL every time) is splined on 5 mL nickel ion affinity chromatograph columns, is eluted using 50 mM imidazoles, removes foreign protein, recycles The imidazoles of 150 mM elutes, and collects elution fraction.Obtained component after the imidazoles of 150 mM is eluted, dialysis removal imidazoles, packing Be stored in -20 DEG C it is spare.By nickel ion bio-affinity purifying, protein recovery reaches 54.4%, purity of protein reach 95% with On, as shown in Figure 1.
The optimal reactive temperature of 4 algin catenase AlyB10 of embodiment measures
900 μ l, 0.3% algin substrate (20 mM phosphoric acid are added in purifying gained 100 μ l of algin catenase in embodiment 3 Salt buffer, pH=7.6), 10 min are reacted under different temperatures (10-70 DEG C), with spectrophotometric determination A235 numerical value, with Highest enzyme activity is 100%, calculates the enzyme activity of AlyB10 under different temperatures.As shown in Fig. 2, algin catenase The optimal reactive temperature of AlyB10 is 50 DEG C.
The 5 restorative measurement of algin catenase AlyB10 heat of embodiment
The purifying gained pure enzyme of algin catenase in embodiment 3 is boiled into 10 min in 100 DEG C of boiling water, then takes out and puts immediately It sets and is incubated for 30 min at 0,10,20,30,40,50 DEG C, detect its remaining enzyme activity, not boil the vigor of enzyme solution for 100%； Gained enzyme activity is directly detected after boiling as negative control；Detect the influence restorative to its heat of different incubation temperatures；Such as Shown in Fig. 3, after algin catenase AlyB10 is boiled 10 min, be placed on 10 DEG C and be incubated for 30 minutes, can restore its 52.5% Activity.Pure enzyme boils 10 min in 100 DEG C of boiling water, be placed on immediately after taking-up 30 DEG C of incubation different times (0,1,5,10, 20,30 min), detect the influence restorative to enzyme of different incubation times.As shown in figure 4,10 DEG C of incubation different times, the heat of enzyme Restorative to gradually increase, in 30 min, heat is restorative reaches peak, then extends the restorative increase trend of incubation time heat not Obviously.
The viscosimetry measurement of 6 algin catenase AlyB10 of embodiment degradation mode
The determination of degradation mode is carried out using Ubbelohde viscometer, the 0.3% algin bottom 9.9 ml is added in 0.1 ml enzyme solution (30 U) Object, 50 DEG C of reaction different times (1,5,10,30,60 min) boil 10 min and terminate reaction.At room temperature, crow is utilized Family name's viscosimeter calculates the product viscosity of different enzymolysis times.Meanwhile the suction of the product of different enzymolysis times is measured using A235 method Light varience measures corresponding enzyme activity.By measurement result (Fig. 5) it is found that enzyme digestion reaction initial stage (1-5 minutes), product are viscous Degree sharply declines, and the unsaturated double-bond that the enzymolysis product of A235 method detection at this time is formed does not sharply increase, and illustrates AlyB10 Mode of action be inscribe.
7 algin catenase AlyB10 enzymolysis product thin layer chromatography analysis of embodiment
The purifying gained pure enzyme of algin catenase AlyB10 (10-20 U) in embodiment 3 is incubated at 30 DEG C with 0.1% algin 6 h are educated, are then detected at High Performance Thin Layer Chromatography plate (HPTLC).Specifically: TLC is activating 2 h's in 100 DEG C of baking ovens in advance HPTLC chromatoplate is cut into the sample of the suitable size of 7 cm wide, will be incubated for front and back sample point sample at the origin, has been placed in solvent 20 min in the exhibition cylinder of (n-butanol: formic acid: water=4:5:1) dry up chromatoplate, immerse 2s in color developing agent (aniline diphenylamines), take It dries up out, high-temperature baking, until sample occurs.As shown in fig. 6, with standard items migration rate it was found that, algin catenase It is algin disaccharides (DP2) and trisaccharide (DP3) that AlyB10, which digests principal product,.
Sequence table
<110>Wang Cunliang
<120>a kind of novel algin catenase AlyB10 and its application
<141> 2018-09-25
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 398
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Ser Thr Val Leu Ala Leu Gly Ser Ala Leu Val Asp Asn Ser Ser Leu
1 5 10 15
Ala Glu Thr Leu Ala Thr Thr His Ile Leu Pro Glu Ser His Gln Ala
20 25 30
Leu Pro Gln Gly Glu Leu Leu Gly Asp Phe Asn Ser Leu Asp Glu Ile
35 40 45
Glu Glu Asn Thr Leu Ile Pro Lys Ser His Thr Ser Leu Ala Glu Gly
50 55 60
Val Leu Leu Trp Tyr Gln Lys Lys Glu Tyr Glu Leu Pro Gly Glu Lys
65 70 75 80
Ala Leu Ser Asp Ser Phe Ala Leu Lys Pro Leu Phe Tyr Thr Ser Thr
85 90 95
Asp Gly Gly Met Val Phe Ala Cys Pro Asn Ala Gly Ala Lys Thr Ser
100 105 110
Lys Asn Thr Lys Tyr Ala Arg Thr Glu Leu Arg Glu Met Leu Arg Arg
115 120 125
Gly Asn Thr Thr Ile Lys Thr Lys Gly Ile Thr Glu Asn Asn Trp Val
130 135 140
Leu Asn Ser Ala His Gly Ser Val Lys Arg Lys Ala Gly Ala Val Glu
145 150 155 160
Gly Ser Leu Glu Ala Thr Leu Ala Val Asn Arg Val Ser Thr Thr Gly
165 170 175
Asp Glu Glu Lys Val Gly Arg Val Ile Ile Gly Gln Ile Thr Ala Thr
180 185 190
Asp Glu Glu Pro Ile Arg Leu Tyr Tyr Arg Lys Leu Pro Gly Asn Asp
195 200 205
Asn Gly Ser Ile Tyr Phe Ala His Glu Ile Asn Gly Gly Asp Asp Val
210 215 220
Trp Val Lys Met Val Gly Lys Val Thr Leu Ser Asp Asn Thr Leu Thr
225 230 235 240
Lys Val Gly His Gly Lys Arg Asn Lys Ser Tyr Lys Ile Thr Val Asn
245 250 255
Lys Asn Ile Leu Phe Val Thr Leu Tyr Met Glu Gly Lys Gln Asn Ala
260 265 270
Thr Lys Ser Phe Asp Met Ser Lys Ser Gly Tyr Asn Lys Asn Asn Gln
275 280 285
Tyr Met Tyr Phe Met Glu Thr Gly Tyr Ser Ile Asp Gly Asn Gly Gly
290 295 300
Ala Val Gln Gly Gln Gln Leu Tyr Leu Trp Thr Thr Lys Thr Thr Asn
305 310 315 320
Val Asn Gln Asn Trp Val Gln Ile Ser His Gly Ser Gly Tyr Tyr Ser
325 330 335
Tyr Lys Lys Glu Gly Thr Ser Leu Cys Trp Asp Gly Gly Thr Gly Gly
340 345 350
Ala Lys Arg Gln Ala Val Thr Leu Glu Val Cys Asp Ser Ser Asn Tyr
355 360 365
Asp Gln His Trp Lys Lys Val Lys Val Thr Ser Gly Thr Glu Ile Tyr
370 375 380
Arg Phe Glu Lys Arg Asn Ala Pro Gly Phe Ser Ile Asp Gly
385 390 395
<210> 2
<211> 1194
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
agcaccgtgc tcgccctcgg cagcgccctc gtggacaaca gcagcctcgc cgagaccctc 60
gccaccaccc acatactccc cgagagccac caggccctcc cccagggcga gctcctcggc 120
gacttcaaca gcctcgacga gatagaggag aacaccctca tacccaagag ccacaccagc 180
ctcgccgagg gcgtgctcct ctggtaccag aagaaggagt acgagctccc cggcgagaag 240
gccctcagcg acagcttcgc cctcaagccc ctcttctaca ccagcaccga cggcggcatg 300
gtgttcgcct gccccaacgc cggcgccaag accagcaaga acaccaagta cgccaggacc 360
gagctcaggg agatgctcag gaggggcaac accaccataa agaccaaggg cataaccgag 420
aacaactggg tgctcaacag cgcccacggc agcgtgaaga ggaaggccgg cgccgtggag 480
ggcagcctcg aggccaccct cgccgtgaac agggtgagca ccaccggcga cgaggagaag 540
gtgggcaggg tgataatagg ccagataacc gccaccgacg aggagcccat aaggctctac 600
tacaggaagc tccccggcaa cgacaacggc agcatatact tcgcccacga gataaacggc 660
ggcgacgacg tgtgggtgaa gatggtgggc aaggtgaccc tcagcgacaa caccctcacc 720
aaggtgggcc acggcaagag gaacaagagc tacaagataa ccgtgaacaa gaacatactc 780
ttcgtgaccc tctacatgga gggcaagcag aacgccacca agagcttcga catgagcaag 840
agcggctaca acaagaacaa ccagtacatg tacttcatgg agaccggcta cagcatagac 900
ggcaacggcg gcgccgtgca gggccagcag ctctacctct ggaccaccaa gaccaccaac 960
gtgaaccaga actgggtgca gataagccac ggcagcggct actacagcta caagaaggag 1020
ggcaccagcc tctgctggga cggcggcacc ggcggcgcca agaggcaggc cgtgaccctc 1080
gaggtgtgcg acagcagcaa ctacgaccag cactggaaga aggtgaaggt gaccagcggc 1140
accgagatat acaggttcga gaagaggaac gcccccggct tcagcataga cggc 1194
<210> 3
<211> 34
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
catgccatgg gtagcaccgt gctcgccctc ggca 34
<210> 4
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ccgctcgagg ccgtctatgc tgaagccgg 29

Claims (8)

1. a kind of algin catenase with hot recovery characteristics, amino acid sequence is as shown in SEQ ID NO.1.
2. nucleotide sequence corresponding to algin catenase as described in claim 1, the nucleotide sequence such as SEQ ID Shown in NO.2.
3. application of the algin catenase as described in claim 1 in cracking macromolecular algin.
4. algin catenase as described in claim 1 is preparing the application in brown alga oligose.
5. a kind of method for cracking algin, characterized in that selected algin catenase has to be described in claim 1 The algin catenase of hot recovery characteristics.
6. method as claimed in claim 5, characterized in that reaction temperature is 10 ~ 70 DEG C in cracking condition, optimal reactive temperature It is 50 DEG C.
7. method as claimed in claim 5, characterized in that incubation temperature is 0 ~ 50 DEG C in the hot recovery characteristics, most suitable to incubate Educating temperature is 10 DEG C.
8. method as claimed in claim 5, characterized in that incubation time is 1 ~ 45 minute in the hot recovery characteristics, most suitable Incubation time is 30 minutes.
CN201811117210.3A 2018-09-25 2018-09-25 A kind of novel algin catenase AlyB10 and its application Withdrawn CN109055343A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112980822A (en) * 2021-03-02 2021-06-18 中国科学院青岛生物能源与过程研究所 High-catalytic-activity alginate lyase mutant and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112980822A (en) * 2021-03-02 2021-06-18 中国科学院青岛生物能源与过程研究所 High-catalytic-activity alginate lyase mutant and application thereof
CN112980822B (en) * 2021-03-02 2022-06-07 中国科学院青岛生物能源与过程研究所 High-catalytic-activity alginate lyase mutant and application thereof

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