CN109055344A

CN109055344A - A kind of algin catenase and its application with hot recovery characteristics

Info

Publication number: CN109055344A
Application number: CN201811117211.8A
Authority: CN
Inventors: 王存良; 李尚勇
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-09-25
Filing date: 2018-09-25
Publication date: 2018-12-21

Abstract

The present invention relates to a kind of algin catenase with hot recovery characteristics and its applications.The algin catenase is a kind of algin catenase AlyL05, and amino acid sequence is as shown in SEQ ID NO.1.The algin catenase sequence similarity of algin catenase amino acid sequence provided by the invention and existing known function is only 49%.Algin catenase AlyL05 of the present invention has hot recovery characteristics, is boiling after five minutes, is put on ice for being incubated for 30 minutes, can restore its 58.3% activity.The enzyme degrades principal product as unsaturated 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 algin catenase and its application with hot recovery characteristics

Technical field

The present invention relates to a kind of algin catenase with hot recovery characteristics and its applications, belong to field of biotechnology.

Background technique

Algin is the cell wall constituent of a variety of seaweed, 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.The algin oligosaccharide of different polymerization degree has different Biological activity.It recent studies have shown that, utilize the inhibition β of poly- M sections of oligosaccharides drugs " GV-971 " the energy multiple target point of algin preparation The aggregation and cytotoxicity of starch like cell, are completed the clinical trial of three phases；Poly- G oligosaccharides can use inhibition with Antibiotic combination The more drug resistance pathogenic bacteria of various clinical.Compare traditional acid-base method degradation algin, enzymic degradation algin has mild condition, easily The advantages such as control, environmentally protective.Therefore, the algin catenase with special nature is found, and studies its degradation condition and most 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 conch digestive system, marine bacteria It is directly separated extraction, is limited to natural algin catenase low output, the algin catenase for developing genetic engineering is imperative. It has now been found that more or less a hundred alginate lyase gene, portion gene are recombinantly expressed in Escherichia coli, and has studied Its zymologic property.But most of algin catenase thermostabilizations and heat are restorative poor, are easy inactivation, are unable to reach industrialization The requirement of application.

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 enzyme and reported algin catenase amino acid similarity are only 49%, are the completely new algin of a structure and function Lyases.The optimal reactive temperature of the enzyme is 45 DEG C, and optimal reaction pH is 8.2；Enzyme solution boils after five minutes, is put on ice for 30 Minute, its 58.3% activity can be restored.The enzyme degrades principal product as algin unsaturation disaccharides and trisaccharide.

On the one hand, the present invention provides a kind of algin catenase AlyL05 with hot recovery characteristics, amino acid sequence As shown in SEQ ID NO. 1.

SEQ ID NO.1:

MITATKGLLRSVVFTVLALGSALVENHSLAETLATTHILPESHQALPQGELLGDFNSLDPSKSPSENFDLLSW TLSLPTDLNKDLKADIIYEKALSDSFALKPLFYTSTDGGMVFACPNAGAKTSKNTKYARTELREMLRRGNTRIKTKG ITENNWVLNSAHGSVKRKAGAVEGSLEATLAVNRVSTTGDEKKVGRVIIGQIHATDDEPIRLYYRKLPGNDNGSIYF AHEINGGDDVWVNMVGSRSHTLENPEEGIALNEKFSYKITVKNNILFVTLIREGKQNLTKSFDMSKSGYNKNNQYMY FKAGVYNQNNSGSDKDYVQATFYHLENQH。

On the other hand, the present invention also provides a kind of corresponding nucleic acid of algin catenase AlyL05 with hot recovery characteristics Sequence, as shown in SEQ ID NO.2.

SEQ ID NO.2:

ATGATCACGGCCACGAAAGGCCTGCTGCGCAGCGTCGTCTTTACGGTCCTGGCCCTGGGCAGCGCCCTGGTCG AAAACCATAGCCTGGCCGAAACGCTGGCCACGACGCATATCCTGCCGGAAAGCCATCAGGCCCTGCCGCAGGGCGAA CTGCTGGGCGACTTTAACAGCCTGGACCCGAGCAAAAGCCCGAGCGAAAACTTTGACCTGCTGAGCTGGACGCTGAG CCTGCCGACGGACCTGAACAAAGACCTGAAAGCCGACATCATCTATGAAAAAGCCCTGAGCGACAGCTTTGCCCTGA AACCGCTGTTTTATACGAGCACGGACGGCGGCATGGTCTTTGCCTGCCCGAACGCCGGCGCCAAAACGAGCAAAAAC ACGAAATATGCCCGCACGGAACTGCGCGAAATGCTGCGCCGCGGCAACACGCGCATCAAAACGAAAGGCATCACGGA AAACAACTGGGTCCTGAACAGCGCCCATGGCAGCGTCAAACGCAAAGCCGGCGCCGTCGAAGGCAGCCTGGAAGCCA CGCTGGCCGTCAACCGCGTCAGCACGACGGGCGACGAAAAAAAAGTCGGCCGCGTCATCATCGGCCAGATCCATGCC ACGGACGACGAACCGATCCGCCTGTATTATCGCAAACTGCCGGGCAACGACAACGGCAGCATCTATTTTGCCCATGA AATCAACGGCGGCGACGACGTCTGGGTCAACATGGTCGGCAGCCGCAGCCATACGCTGGAAAACCCGGAAGAAGGCA TCGCCCTGAACGAAAAATTTAGCTATAAAATCACGGTCAAAAACAACATCCTGTTTGTCACGCTGATCCGCGAAGGC AAACAGAACCTGACGAAAAGCTTTGACATGAGCAAAAGCGGCTATAACAAAAACAACCAGTATATGTATTTTAAAGC CGGCGTCTATAACCAGAACAACAGCGGCAGCGACAAAGACTATGTCCAGGCCACGTTTTATCATCTGGAAAACCAGC ATTAA

On the other hand, the present invention also provides the preparation methods of algin catenase AlyL05 with hot recovery characteristics a kind of.

On the other hand, the application the present invention also provides the algin catenase AlyL05 in cracking algin.

On the other hand, the present invention also provides the algin catenase AlyL05 to prepare the application in brown alga oligose.

On the other hand, a method of cracking algin, selected algin catenase are AlyL05.

It is preferred that: reaction temperature is 20 ~ 70 DEG C in the cracking condition.Optimal reactive temperature is 45 DEG C.

It is preferred that: it is 5 ~ 10 that pH is reacted in the cracking condition.Optimal reaction pH is 8.2.

It is preferred that: incubation temperature is 0 ~ 50 DEG C in the hot recovery characteristics.Most suitable incubation temperature is 0 DEG C.

It is preferred that: incubation time is 1 ~ 45 min in the hot recovery characteristics.Most suitable incubation time is 30 min.

The utility model has the advantages that

1. algin catenase AlyL05 amino acid sequence of the invention and the algin catenase sequence similarity reported before It is lower, only 49%, it is the novel algin catenase of sequence.

2. algin catenase AlyL05 of the invention is after 5 L fermentation, every milliliter of fermentation broth enzyme activity is up to 790 U/ mL.Also, the characteristic that there is the enzyme heat to restore can be incubated on ice after boiling conditions and restore its activity.It is this novel Property can promote its industrial applications.

3. algin catenase AlyL05 of the present invention derives from marine bacteriaVibrio alginolyticus MCCC 1D00120.The present invention also provides a kind of methods for preparing the novel algin catenase, that is, utilize genetic engineering Technical method by AlyL05 gene heterologous recombination to Escherichia coli, and isolates and purifies it using nickel column, has studied its enzyme Learn property, find its characteristic that there is heat to restore, enzyme solution boils after five minutes, is put on ice for 30 minutes, can restore its 58.3% Activity.Catabolite analysis is carried out using the recombinase, which degrades principal product as algin disaccharides and trisaccharide.The present invention The algin catenase AlyL05 has good industrial applications prospect.

Detailed description of the invention

Fig. 1 is algin catenase AlyL05 protein separation figure (M, Protein standards of the present invention；1, purify institute Obtain algin catenase AlyL05)；

Fig. 2 is the optimal reactive temperature of algin catenase AlyL05 of the present invention；

Fig. 3 is the optimal reaction pH of algin catenase AlyL05 of the present invention；

Fig. 4 is heat restorative influence of the different incubation temperatures on algin catenase AlyL05；

Fig. 5 is heat restorative influence of the different incubation times on algin catenase AlyL05；

Fig. 6 is enzymolysis product (M, 2 sugar of brown alga oligose that thin-layer chromatography (TLC) method detects algin catenase AlyL05 of the present invention DP2,3 sugar DP3 standard items；0, substrate before digesting；1, product after enzymatic hydrolysis)；

(175.1 be unsaturated brown to the enzymolysis product that Fig. 7 is mass spectrum ESI-MS method detection algin catenase AlyL05 of the present invention Phycocolloid monosaccharide, 193.1 be saturation algin monosaccharide, and 351.1 be unsaturated algin disaccharides, and 527.2 be unsaturated algin three Sugar).

Specific embodiment

The sequence of 1 algin catenase AlyL05 of embodiment is analyzed

Algin catenase AlyL05 of the present invention from Vibrio alginolyticus (Vibrio alginolyticus), bacterial strain purchase It buys from Chinese Sea Microbiological Culture Collection administrative center (MCCC), strain number is MCCC 1D00120.It is surveyed by pyrophosphoric acid Bacterial strain genome-wide screening is sequenced in sequence technology, is completed by Beijing shellfish is auspicious with Kanggong department.Utilize polysaccharide hydrolase database Carbohydrate-Active enZYmes Database analyzes (CAZy, http://www.cazy.org/), obtains one The polysaceharide lyase Polysaccharide Lyase family 7(PL-7 of prediction) family algin catenase sequence, life It is entitledalyL05.SequencealyL05Include 1,002 base sequence, encodes 333 amino acid sequences；The sequence is utilized National Center for Biotechnology Information (NCBI, https:// Www.ncbi.nlm.nih.gov/ in) conserved structure domain analysis Conserved domain (CDD, https: // ) and Multiple sequence alignments Basic Local Alignment Search Tool www.ncbi.nlm.nih.gov/cdd (Blast, https: //blast.ncbi.nlm.nih.gov/) discovery,The sequence includes that algin catenase 2 surpasses The conserved region of family (2 superfamily of Alginate lyase).Blast analysis finds,alyL05Nucleotide sequence with All genes compare in ncbi database, and similarity is only up to 76%.Institute in the amino acid sequence and ncbi database of AlyL05 There is amino acid sequence to be compared, is the unknown prediction albumen of biological function with the higher albumen of its similarity (> 90%) (Hypothetical protein), with the highest known functional protein of AlyL05 sequence similarity be fromGilvimarinus polysaccharolyticusThe algin catenase of the 7th family (PL-7) of polysaceharide lyase of bacterial strain It (WP_084005511), is only 49% with the amino acid sequence similarity of AlyL05.Therefore,alyL05For a novel brown alga Glue lyase gene.

The gene cloning and recombinant expression of 2 algin catenase AlyL05 of embodiment

The marine bacteria strain that will be bought in embodiment 1Vibrio alginolyticus MCCC 1D00120 is dedicated in marine bacteria In culture medium (2216E culture medium), 20 DEG C 180 rpm shake culture of incubator 10-12 hours, until cell density OD₆₀₀=1.0； Using genome extraction kit (being purchased from Dalian treasured biotech firm), the full-length genome of the bacterial strain is extracted.With restriction enzymeNcoI andXhoI(is purchased from Dalian treasured biotech firm) it is restriction enzyme site and designs restriction enzyme site protection base, remove termination codon Sub- TAA, design recombination primer are following (underscore is restriction endonuclease sites, and italic is restriction enzyme enzyme protection base):

Forward primer: SEQ ID NO.3:PalyL5EF:

5’- CATG CCATGGGTATGATCACGGCCACGAAAGG -3’ (Nco I)

Reverse primer: SEQ ID NO.4:PalyL5ER:

5’- CCG CTCGAGATGCTGGTTTTCCAGATGAT -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 5mL fluid nutrient medium), 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-alyL05.

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-alyL05, be stored in -80 DEG C it is spare.

The zymotechnique and method for preparing purified of 3 algin catenase AlyL05 of embodiment

It will be constructed in embodiment 2 and be stored in -80 DEG C of e. coli bl21 (DE3)/pET22b-alyL05 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 72 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 45 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 790 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 30 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 76.7%, purity of protein reach 95% with On, as shown in Figure 1.

The optimal reactive temperature of 4 algin catenase AlyL05 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=7.3), 10 min are reacted at 20-70 DEG C of different temperatures, with spectrophotometric determination A235 numerical value, with most High enzymatic activity is 100%, calculates the enzyme activity of AlyL05 under different temperatures.As shown in Fig. 2, algin catenase AlyL05 Optimal reactive temperature be 45 DEG C.

The optimal reaction pH of 5 algin catenase AlyL05 of embodiment is measured

900 μ l, 0.3% algin substrate (20 mM are added in the pure 100 μ l of enzyme of purifying gained algin catenase in embodiment 3 PB buffer, pH=7.3), 10 min are reacted in 4 kinds of different buffers, with spectrophotometric determination A235 numerical value, with highest Enzyme activity is 100%, calculates the enzyme activity of AlyL05 under condition of different pH.The 4 kinds of buffers used are respectively 50 mM phosphorus Phthalate buffer, glycine-NaOH buffer, Na₂HPO₄Citrate buffer solution, Tris-HCl buffer.As shown in figure 3, brown The optimal reaction pH of phycocolloid lyases AlyL05 is 8.2.

The 5 restorative measurement of algin catenase AlyL05 heat of embodiment

The purifying gained pure enzyme of algin catenase in embodiment 3 is boiled into 5 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. 4, after algin catenase AlyL05 is boiled 5 min, it is placed on (0 DEG C) of ice water compound and is incubated for 30 minutes, it can be extensive Its multiple 58.3% activity, enzyme boil 5 min in 100 DEG C of boiling water, and it is different to be placed in ice water (0 DEG C) incubation after taking-up immediately Time (0,1,5,15,30,45 min) detects the influence restorative to enzyme of different incubation times.As shown in figure 5, being incubated in ice water Different time is educated, the heat of enzyme is restorative to be gradually increased, and heat is restorative in 30 min reaches peak, then extends incubation time heat Restorative increase trend is unobvious.

6 algin catenase AlyL05 enzymolysis product thin layer chromatography analysis of embodiment

The purifying gained pure enzyme of algin catenase AlyL05 (10-20 U) in embodiment 3 is incubated at 45 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 AlyL05, which digests principal product,.

The analysis of 7 algin catenase AlyL05 enzymolysis product mass spectrum (ESI-MS) of embodiment

It will be incubated for gained sample in embodiment 6 and carry out desalting processing, and mixed in equal volume with acetonitrile, ESI- anion one is carried out Grade Mass Spectrometer Method, determines the molecular weight of enzymolysis product.Ion mode first mass spectrometric is the result shows that (Fig. 7), the pure enzyme of purifying gained Oligosaccharides principal product after AlyL05 degradation algin is unsaturated disaccharides and trisaccharide.

Sequence table

<110>Wang Cunliang

<120>a kind of algin catenase and its application with hot recovery characteristics

<141> 2018-09-25

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 333

<212> PRT

<213>Vibrio alginolyticus (Vibrio alginolyticus)

<400> 1

Met Ile Thr Ala Thr Lys Gly Leu Leu Arg Ser Val Val Phe Thr Val

1 5 10 15

Leu Ala Leu Gly Ser Ala Leu Val Glu Asn His Ser Leu Ala Glu Thr

20 25 30

Leu Ala Thr Thr His Ile Leu Pro Glu Ser His Gln Ala Leu Pro Gln

35 40 45

Gly Glu Leu Leu Gly Asp Phe Asn Ser Leu Asp Pro Ser Lys Ser Pro

50 55 60

Ser Glu Asn Phe Asp Leu Leu Ser Trp Thr Leu Ser Leu Pro Thr Asp

65 70 75 80

Leu Asn Lys Asp Leu Lys Ala Asp Ile Ile Tyr Glu Lys Ala Leu Ser

85 90 95

Asp Ser Phe Ala Leu Lys Pro Leu Phe Tyr Thr Ser Thr Asp Gly Gly

100 105 110

Met Val Phe Ala Cys Pro Asn Ala Gly Ala Lys Thr Ser Lys Asn Thr

115 120 125

Lys Tyr Ala Arg Thr Glu Leu Arg Glu Met Leu Arg Arg Gly Asn Thr

130 135 140

Arg Ile Lys Thr Lys Gly Ile Thr Glu Asn Asn Trp Val Leu Asn Ser

145 150 155 160

Ala His Gly Ser Val Lys Arg Lys Ala Gly Ala Val Glu Gly Ser Leu

165 170 175

Glu Ala Thr Leu Ala Val Asn Arg Val Ser Thr Thr Gly Asp Glu Lys

180 185 190

Lys Val Gly Arg Val Ile Ile Gly Gln Ile His Ala Thr Asp Asp Glu

195 200 205

Pro Ile Arg Leu Tyr Tyr Arg Lys Leu Pro Gly Asn Asp Asn Gly Ser

210 215 220

Ile Tyr Phe Ala His Glu Ile Asn Gly Gly Asp Asp Val Trp Val Asn

225 230 235 240

Met Val Gly Ser Arg Ser His Thr Leu Glu Asn Pro Glu Glu Gly Ile

245 250 255

Ala Leu Asn Glu Lys Phe Ser Tyr Lys Ile Thr Val Lys Asn Asn Ile

260 265 270

Leu Phe Val Thr Leu Ile Arg Glu Gly Lys Gln Asn Leu Thr Lys Ser

275 280 285

Phe Asp Met Ser Lys Ser Gly Tyr Asn Lys Asn Asn Gln Tyr Met Tyr

290 295 300

Phe Lys Ala Gly Val Tyr Asn Gln Asn Asn Ser Gly Ser Asp Lys Asp

305 310 315 320

Tyr Val Gln Ala Thr Phe Tyr His Leu Glu Asn Gln His

325 330

<210> 2

<211> 1002

<212> DNA

<213>Vibrio alginolyticus (Vibrio alginolyticus)

<400> 2

atgatcacgg ccacgaaagg cctgctgcgc agcgtcgtct ttacggtcct ggccctgggc 60

agcgccctgg tcgaaaacca tagcctggcc gaaacgctgg ccacgacgca tatcctgccg 120

gaaagccatc aggccctgcc gcagggcgaa ctgctgggcg actttaacag cctggacccg 180

agcaaaagcc cgagcgaaaa ctttgacctg ctgagctgga cgctgagcct gccgacggac 240

ctgaacaaag acctgaaagc cgacatcatc tatgaaaaag ccctgagcga cagctttgcc 300

ctgaaaccgc tgttttatac gagcacggac ggcggcatgg tctttgcctg cccgaacgcc 360

ggcgccaaaa cgagcaaaaa cacgaaatat gcccgcacgg aactgcgcga aatgctgcgc 420

cgcggcaaca cgcgcatcaa aacgaaaggc atcacggaaa acaactgggt cctgaacagc 480

gcccatggca gcgtcaaacg caaagccggc gccgtcgaag gcagcctgga agccacgctg 540

gccgtcaacc gcgtcagcac gacgggcgac gaaaaaaaag tcggccgcgt catcatcggc 600

cagatccatg ccacggacga cgaaccgatc cgcctgtatt atcgcaaact gccgggcaac 660

gacaacggca gcatctattt tgcccatgaa atcaacggcg gcgacgacgt ctgggtcaac 720

atggtcggca gccgcagcca tacgctggaa aacccggaag aaggcatcgc cctgaacgaa 780

aaatttagct ataaaatcac ggtcaaaaac aacatcctgt ttgtcacgct gatccgcgaa 840

ggcaaacaga acctgacgaa aagctttgac atgagcaaaa gcggctataa caaaaacaac 900

cagtatatgt attttaaagc cggcgtctat aaccagaaca acagcggcag cgacaaagac 960

tatgtccagg ccacgtttta tcatctggaa aaccagcatt aa 1002

<210> 3

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

catgccatgg gtatgatcac ggccacgaaa gg 32

<210> 4

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

ccgctcgaga tgctggtttt ccagatgat 29

Claims

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 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 20 ~ 70 DEG C in cracking condition, optimal reactive temperature It is 45 DEG C.

7. method as claimed in claim 5, characterized in that reacting pH in cracking condition is 5 ~ 10, and optimal reaction pH is 8.2.

8. 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 0 DEG C.

9. method as claimed in claim 5, characterized in that incubation time is 1 ~ 45 min in the hot recovery characteristics, most suitable Incubation time is 30 min.