CN110004134A - A kind of algin catenase mutant and its application - Google Patents

Abstract

The present invention relates to a kind of algin catenase mutant and applications, belong to enzyme engineering and genetic engineering field, 226th E is sported K on the basis of sequence SEQ ID NO.3 by the mutant, through being overexpressed, mutant enzyme activity improves 1.11 times, and crude enzyme liquid relatively truncates enzyme AlgL-T157N by Rate activity after purification and improves 1.03 times；It 55 DEG C of mutant optimum temperature, is basically unchanged in 1 h enzyme activity of pH 6.0-8.0 inside holding, there is stronger pH stability；Catalytic efficiency of the mutant to sodium alginate, Poly M and Poly GK _cat /K _m 10.22,8.59,2.97 times are improved than AlgL-T157N respectively.The method that the present invention is combined using structure sequence analysis and software assisting sifting, determines mutational site, obtains the mutant of catalytic activity raising by PCR rite-directed mutagenesis, lays the foundation for its further industrial applications.

Description

A kind of algin catenase mutant and its application

Technical field

The present invention relates to a kind of algin catenase mutant and its applications, belong to enzyme engineering and genetic engineering field.

Background technique

Algin catenase is a kind of lyases of alginate in degradation brown alga, main to be disappeared by the β-of 4-O- glycosidic bond Oligosaccharide aldehydic acid with unsaturated double-bond is generated except the depolymerization of catalytic reaction alginates, and between non-reducing end C-4 and C-5 Structure.Poly M, poly G and poly MG Specific lytic enzymes can be divided into according to the substrate Preference of algin catenase；Root According to the difference of the mode of action, and restriction endonuclease and excision enzyme can be divided into, restriction endonuclease mainly cuts the glucosides inside alginic acid polymer Key generates unsaturated oligosaccharide, and excision enzyme is then that brown alga oligose is further degraded to monosaccharide.

Go deep into now with what is studied brown alga oligose, more and more bioactivity are found, such as antitumor, anti-mistake Quick, antibacterial, blood coagulation, degeneration-resistant and anti-oxidant etc., wide application value promotes researcher to brown alga oligose preparation method Further exploration.Traditional brown alga oligose edman degradation Edman mainly has: acidolysis, alkaline hydrolysis, oxidative degradation, these biodegrading process not only hold Environmental pollution is easily caused, and obtained oligosaccharides bioactivity is low and effect is poor.On the contrary, enzyme process prepares brown alga oligose not only item Part is mild, and yield is high, vulnerable to control, free from environmental pollution, also has preferable oligosaccharides bioactivity, therefore gradually replace traditional Learning edman degradation Edman becomes main oligosaccharides preparation method.However currently screen mostly to be that substrate specificity is poor, enzymatic activity is low brown It is fewer and fewer to be applied to industrialized algin catenase product for phycocolloid lyases, so obtaining has high activity and extensive bottom The algin catenase of object specificity is mentioned at current research emphasis especially by genetic engineering and protein engineering means The catalytic activity of high algin catenase even more receives the favor of most researchers.

The method that the present invention is combined using structure sequence analysis and Discovery Studio software assisting sifting, determines Mutational site, and the mutant that catalytic activity significantly improves is obtained by PCR rite-directed mutagenesis, it is its further industrial applications It lays the foundation.

Summary of the invention

The object of the present invention is to provide a kind of algin catenase mutant and its application, using structure sequence analysis and The method that Discovery Studio software assisting sifting combines determines mutational site, and is urged by PCR rite-directed mutagenesis Change the mutant that activity significantly improves, lays the foundation for its further industrial applications.

Following technical scheme is used to achieve the above object:

Alginate lyase gene in the present inventionAlgL(nucleotide sequence is as shown in SEQ ID NO.1) is derived from and is isolated from boat Mountain group island seawaterPseudoalteromonasSp. zb7-4,1203 bp of overall length, encode 400 amino acid, and 1-31 Amino acid is signal peptide, and 32-131 amino acids are carbohydrate binding domain (i.e. CBM structural domain), 197-385 ammonia Base acid is catalyst structure domain.It is truncated between 157-158 amino acids, amputates 1-157 amino acids, obtain algin Lyases truncates enzyme AlgL-T157N, and amino acid sequence is as shown in SEQ ID NO.3.

It submits to truncate and carries out homologous modeling on enzyme AlgL-T157N amino acid sequence to SWISS MODEL line server, Protein model structure evaluation is carried out using Verify-3D, ERRAT and PROCHECK program in Saves server, is used Model and mannuronic acid tetrose small molecule Tetra are carried out molecular docking by AutoDock Tools, obtain docking result (see figure 1).The method combined using structure sequence analysis and Discovery Studio software assisting sifting, determines mutational site, On the basis of AlgL-T157N amino acid sequence SEQ ID NO.3, it is lysine by the 226th glutamic acid rite-directed mutagenesis, is urged Change the algin catenase mutant E226K that activity significantly improves, amino acid sequence is as shown in SEQ ID NO.4, nucleotide Sequence is as shown in SEQ ID NO.5.

Mutant E226K enzyme activity improves 1.11 times compared with AlgL-T157N, improves 1.03 times than work；Optimum temperature is 55 DEG C, 5 DEG C are improved compared with AlgL-T157N, residual enzyme activity has up to 90% or more after 1 h of pH 6.0-8.0 range inside holding Stronger pH stability.Determination of Kinetic Parameters discovery, E226K is to sodium alginate, Poly M and Poly G this 3 kinds of substratesK _m Value has dropped 89.69%, 88.14%, 61.11% respectively, shows that substrate affinity is remarkably reinforced；K _cat Respectively AlgL-T157N's 118.02%, 114.62%, 151.37%,K _cat / K _m Value improves 10.22,8.59,2.97 times compared with AlgL-T157N respectively.

Mutant E226K enzymatic hydrolysis is respectively adopted and acidolysis prepares brown alga oligose, and ESI-MS is carried out to brown alga oligose product Mass spectral analysis, as a result, it has been found that, the brown alga oligose that enzymatic isolation method obtains is unsaturated oligosaccharides, is made of monosaccharide, disaccharides and trisaccharide；Acidolysis The brown alga oligose that method obtains is saturation oligosaccharides, is made of monosaccharide and disaccharides, based on monosaccharide.It is found by researches that unsaturated oligosaccharides There is higher bioactivity than saturation oligosaccharides, thus enzymatic isolation method prepare brown alga oligose can preferably retain oligosaccharides biology it is living Property.

The present invention has the advantages that

Mutant E226K enzyme activity of the present invention improves 1.11 times compared with AlgL-T157N, improves 1.03 times than work；Mutant E226K optimum temperature is 55 DEG C, improves 5 DEG C compared with AlgL-T157N, the remnant enzyme activity after 1 h of pH 6.0-8.0 range inside holding Power has stronger pH stability up to 90% or more；And mutant is to sodium alginate, Poly M and Poly G this 3 kinds of substrates Catalytic efficiencyK _cat / K _m 10.22,8.59,2.97 times are improved than AlgL-T157N respectively.It is few using enzyme solution preparation brown alga Sugar can preferably retain the bioactivity of oligosaccharides.

Detailed description of the invention

The docking structure figure of Fig. 1 truncation enzyme AlgL-T157N and four glycan molecule Tetra of mannuronic acid.Note: Glu226 is Rite-directed mutagenesis site.

The recombinant plasmid nucleic acid electrophoresis figure of Fig. 2 truncation enzyme AlgL-T157N and mutant E226K.M:1 Kb DNA Ladder；Swimming lane 1:AlgL-T157N recombinant plasmid；Swimming lane 2:E226K recombinant plasmid.

Fig. 3 truncates the SDS-PAGE analysis of the expression and purity of enzyme AlgL-T157N and mutant E226K.M: albumen Molecular weight standards (180 kDa albumen Marker)；Swimming lane 1: recombinant bacterium supernatant is not induced；After swimming lane 2:IPTG induction AlgL-T157N supernatant；E226K supernatant after swimming lane 3:IPTG induction；Swimming lane 4: the egg of AlgL-T157N mesh after purification It is white；Swimming lane 5: E226K destination protein after purification.

The optimal reaction pH of Fig. 4 truncation enzyme AlgL-T157N and mutant E226K.

The optimal reactive temperature of Fig. 5 truncation enzyme AlgL-T157N and mutant E226K.

The pH stability of Fig. 6 truncation enzyme AlgL-T157N and mutant E226K.

The temperature stability of Fig. 7 truncation enzyme AlgL-T157N and mutant E226K.

The Electrospray Ionization Mass Spectrometry figure of Fig. 8 enzymolysis product.

The Electrospray Ionization Mass Spectrometry figure of Fig. 9 acid hydrolysate.

Figure 10 sodium alginate, enzymatic hydrolysis brown alga oligose product, acidolysis brown alga oligose product compare Hydroxyl Radical Scavenging.

Figure 11 sodium alginate, enzymatic hydrolysis brown alga oligose product, acidolysis brown alga oligose product act on ratio to ABTS radicals scavenging Compared with.

Figure 12 sodium alginate, enzymatic hydrolysis brown alga oligose product, acidolysis brown alga oligose product reducing power compare.

Specific embodiment

The building of 1 algin catenase mutant E226K of embodiment

Alginate lyase gene in the present inventionAlgL(nucleotide sequence is as shown in SEQ ID NO.1) is derived from and is isolated from boat Mountain group island seawaterPseudoalteromonasSp. zb7-4,1203 bp of overall length, encode 400 amino acid, and 1-31 Amino acid is signal peptide, and 32-131 amino acids are carbohydrate binding domain (i.e. CBM structural domain), 197-385 ammonia Base acid is catalyst structure domain.It is truncated between 157-158 amino acids, amputates 1-157 amino acids, obtain algin cracking Enzyme truncates enzyme AlgL-T157N, and sequence is as shown in SEQ ID NO.3.

By sequence alignment, it is found that AlgL-T157N and 4Q8K has identical amino acid sequence at catalyst structure domain position, Therefore docking result (Fig. 1) is combined on 4Q8K protein structure and catalytic mechanism Research foundation, utilizes Discovery Studio 2016 softwares carry out virtual alanine scanning and saturation mutation to the amino acid near AlgL-T157N catalytic chamber 8, determine Best mutating acid finally screens to have obtained mutant E226K.

Primer containing mutation sites is designed, with recombinant plasmid pET-22b (+)-AlgL-T157NFor template, using PCR Technology amplification mutation recombinant plasmid pET-22b (+)-E226K, conversion is extremelyE. coliIn BL21 (DE3), mutant recombination is obtained Engineering bacteria.Specific step is as follows:

To contain recombinant plasmid pET-22b (+)-AlgL-T157NLaboratory save bacterial strainE.coliTop10 is starting strain, Recombinant plasmid is extracted using OMEGA company plasmid kit.Specific mutation primers are designed, by the 226th of algin catenase the Position glutamic acid mutation is lysine, and positive anti-primer is as follows:

E226K-F:5’-AGGTTAAAAAGAGTTTACGCGTTGCTATGA-3 ',

E226K-R:5’-CGCGTAAACTCTTTTTAACCTTATACTCATGAC-3’。

Scribing line letter represents the codon of mutating acid lysine.With the pET-22b (+)-of extractionAlgL-T157NMatter Grain is template, carries out rite-directed mutagenesis, PCR amplification system: 5 × TransStart to AlgL-T157N gene using round pcr FastPfu Fly Buffer 10 μ L, High Pure dNTPs (2.5 mmol/L) 4 μ L, PCR Stimulant (5 ×) 5 μ L, MgSO₄ 1 μ L, pET-22b (+)-E226K1 μ L,E226K- F (10 μm of ol/L) 2 μ L,E226K-R (10 µ Mol/L) 1 μ L, ddH of 2 μ L, TransStart FastPfu Fly DNA Polymerase₂O is mended to 50 μ L.

PCR response parameter: 98 DEG C of 2 min of initial denaturation；98 DEG C of denaturation 15 s, 55 DEG C of annealing 15 s, 72 DEG C of 2 min of extension, 30 circulations；72 DEG C of 5 min of heat preservation；4 DEG C of preservations.10 uL pcr amplification products are taken to be detected with 1% agarose gel electrophoresis (see figure 2).The DMT enzyme of 1 μ L is added into remaining PCR product, is uniformly mixed, keeps the temperature 1 h under the conditions of 37 DEG C, digest original mould Plate.2 μ L digestion products are taken to be added to 100 μ L E.coli In Top10 competent cell, ice bath 30 after slight piping and druming mixes min；42 DEG C of heat shock 45-90 s, in placing 2 min on ice；800 μ L LB culture mediums, 37 DEG C, 200 r/min culture 45- is added 60 min；6000 × g is centrifuged 2 min, draws 80 μ L supernatants and thallus is resuspended, and is coated on LB(containing 100 μ g/mL Amp⁺) flat On plate, 37 DEG C of 12 h of culture.The sub- sequence verification of picked clones.

For mutant E226K nucleotide sequence as shown in SEQ ID NO.5, corresponding amino acid sequence is shown in SEQ ID NO.4 institute Show.

The inducing expression and purifying of the truncation of embodiment 2 enzyme AlgL-T157N and mutant E226K

It extracts recombinant plasmid pET-22b (+)-AlgL-T157NWith pET-22b (+)-E226K, it is transferred to respectivelyE.coli In BL21 (DE3) competent cell, picking positive recombinant is in 5 mL LB (Amp⁺) in fluid nutrient medium, 37 DEG C of 200 r/ Min cultivates 12 h, then is seeded to 25 mL LB (Amp with 1% inoculum concentration⁺) in culture medium, 37 DEG C of 200 r/min cultivates OD₆₀₀ To between 0.6-0.8,0.2 mmol/L of IPTG(final concentration is added) 24 h are induced in 20 DEG C of 200 r/min, measure algin Crack enzyme activity.The LB culture medium of IPTG to be not added after being equally inoculated with as blank control.Measure mutant E226K crude enzyme liquid enzyme Living is 7.14 ± 0.09 U/mL, compared with ± 0.11 U/mL of AlgL-T157N(3.38) improve 1.11 times.

Algin catenase vigor is measured using DNS method.0.1 mL enzyme solution is taken to be added to 0.9 mL, 0.3% sodium alginate In substrate, 15 min are kept the temperature at 50 DEG C, 1.5 mL DNS are added and terminate reaction, are boiled 5 min colour developing, are measured after cooling OD₅₄₀.Enzyme activity unit definition: under certain reaction condition, every min catalysis sodium alginate is generated needed for 1 μm of ol reduced sugar Enzyme amount is as an enzyme activity unit U.

Enzyme activity calculation formula are as follows:

In formula:

U: enzyme activity, unit U/mL

△ A: the OD after subtracting blank control₅₄₀

N: extension rate

K: slope of standard curve

V: enzyme solution volume, mL

T: reaction time, min

194.14: the relative molecular weight of glucuronic acid

1000: mass conversion multiple.

Embodiment 3 truncates isolating and purifying for enzyme AlgL-T157N and mutant E226K

(1) DEAE anion column purifies: AKTA purification system purifying protein is used, first with DEAE FF (Hi Trap^TM, 5mL) prepacked column purifies albumen, slow with the McIlvaine that 5 mL/min flow velocitys and concentration are 50 mmol/L, pH 7.0 Fliud flushing balances pillar, with 0.22 μm of membrane filtration crude enzyme liquid.Obtained filtrate is uploaded into anion column, then with 2 mL/min stream Speed balance pillar, after 4-5 column volume of ready to balance, with pre-prepared 1 mol/L NaCl solution (50 mmol/L, pH 7.0 McIlvaine buffer) gradient elution pillar, detects albumen by UV detector, collects different gradient elutions Under protein peak, carry out Enzyme activity assay, and carry out SDS-PAGE detection purity of protein.

(2) ni-sepharose purification: nickel column (HisTrap will be uploaded to by the albumen of DEAE preliminary purification^TM, 5mL) carry out it is pure Change, use NTA 0(20 mmol/L phosphate buffer, 0.5 mol/L NaCl, 10% glycerol) as equilibration buffer, and with NTA 0 is that lysate prepares 500 mmol/L imidazoles, elutes nickel column using prepared imidazole gradient, collects washing for various concentration De- albumen, measures enzyme activity, and carries out SDS-PAGE detection purity of protein.

(3) desalination: imidazoles is carried out to albumen using desalting column (HiTrap Desalting, 5mL), takes 1 mL nickel column pure Enzyme solution after change uploads desalting column, and using the McIlvaine buffer of 50 mmol/L, pH 7.0 as equilibrium liquid, flow velocity is set as 2 ML/min collects protein peak, carries out Enzyme activity assay, and carry out SDS-PAGE electrophoresis, obtains single band albumen (see figure 3).It is pure Measuring after change and truncating the Rate activity of enzyme AlgL-T157N and mutant E226K is respectively 77.81 U/mL, 157.93 U/mL, warp 1.03 times are improved than work after mutation.

The zymologic property of the truncation of embodiment 4 enzyme AlgL-T157N and mutant E226K

Optimal reaction pH measurement: enzyme AlgL-T157N and mutant E226K will be truncated and be added separately to by different pH buffers (the McIlvaine buffer that pH is 4.0 ~ 7.0, the Na that pH is 7.0 ~ 8.0₂HPO₄-NaH₂PO₄Buffer, pH are 8.0 ~ 11.0 Gly-NaOH buffer) prepare concentration be 3% sodium alginate substrate in reacted, reaction temperature be 50 DEG C, buffer Concentration is 50 mmol/L.Enzyme activity is measured using DNS method, the highest enzyme activity of light absorption value is set as 100%, is calculated under different pH Enzyme activity, measure its optimal reaction pH.As a result as Fig. 4 shows that the optimal reaction pH of two kinds of enzymes is 7.0.

Optimal reactive temperature measurement: under the conditions of optimal reaction pH, measure respectively at different temperatures AlgL-T157N and The highest enzyme activity of measurement is set as 100% by the residual enzyme activity of E226K.As a result as Fig. 5 shows that AlgL-T157N's is most suitable Reaction temperature is 50 DEG C, and the optimal reactive temperature of E226K is 55 DEG C.

PH Stability Determination: AlgL-T157N and E226K are mixed from the buffer of above-mentioned different pH in certain proportion It closes, 1 h is kept the temperature at 37 DEG C, measure its enzyme activity under the conditions of optimal reactive temperature and optimal reaction pH, most by measurement High enzymatic activity is defined as 100%, calculates the residual enzyme activity under different pH.As a result as shown in fig. 6, after keeping the temperature 1 h at 37 DEG C, The residual enzyme activity of AlgL-T157N and E226K illustrates that enzyme has within the scope of the pH up to 90% or more within the scope of pH 6.0-8.0 There is stronger stability.

Temperature stability measurement: in optimal reactive temperature and under the conditions of optimal reaction pH, measure respectively AlgL-T157N and E226K keeps the temperature the residual enzyme activity of 30 min at different temperatures, respectively using the enzyme activity that is not handled by as control.As a result If Fig. 7 is shown, two kinds of enzymes are preferable with stability inferior at 40 DEG C, and 40 DEG C or more enzyme activity are begun to decline.

The catalytic kinetic parameter of 5 algin catenase of embodiment measures

The specific method is as follows for catalytic kinetic parameter measurement:

Different final concentrations (0.3,0.45,0.6,1,2,3 mg/mL) are prepared respectively with the McIlvaine buffer of 50 mmol/L Sodium alginate, algin (Poly M) and guluronic acid sodium (Poly G) substrate.Measure AlgL-T157N With enzyme activity of E226K under the conditions of 45 DEG C.Using Lineweaver-Burk method, using reaction rate inverse as ordinate, substrate Inverse concentration is abscissa mapping, is calculatedK _m 、V _max 、K _cat AndK _cat /K _m 。

Catalytic kinetic parameter measurement result is shown in Table 1, and compared with truncating enzyme AlgL-T157N, mutant E226K is to brown alga Sour sodium, Poly M and Poly G this 3 kinds of substratesK _m Value has dropped 89.69%, 88.14%, 61.11% respectively, illustrates that mutation causes The substrate affinity of enzyme significantly improves.Pass through catalytic constantK _catIt was found that catalytic constant is respectively AlgL-T157N after mutation 118.02%, 114.62%, 151.37%, 3 kinds of substratesK _cat / K _m Value has been respectively increased 10.22,8.59,2.97 times.

The catalytic kinetic parameter of the truncation of table 1 enzyme AlgL-T157N and mutant E226K

The preparation and constituent analysis of 6 brown alga oligose of embodiment

It is prepared by the enzymatic hydrolysis of brown alga oligose.By sodium alginate with the McIlvaine buffer of 50 mmol/L, pH7.0 at dense The reaction substrate that degree is 1.2%.After 40 DEG C of 5 min of preheating, the enzyme solution that a certain amount of (0.5 U/mL) is added is reacted, enzymatic hydrolysis 24 H takes out 0.9 mL and digests substrate, and 0.1 mL enzyme solution is added and measures OD under the conditions of 40 DEG C₂₃₅, it is basically unchanged to light absorption value, substrate Enzymatic hydrolysis completely, 5 min of boiling water bath terminate reaction.Removing protein is extracted repeatedly with Sevage method, and centrifugation obtains supernatant and rotated Concentration precipitates oligosaccharides, and sample drying is spare, phenolsulfuric acid standard measure with dehydrated alcohol.

It is prepared by the acidolysis of oligosaccharides.The sodium alginate substrate that compound concentration is 1.5%, with hydrochloric acid tune pH to 4.0, in 120 DEG C of items 4 h of acidolysis under part, is cooled to room temperature after acidolysis, then adjusts pH to 7.0, and centrifugation removal precipitating obtains supernatant concentrated by rotary evaporation, Oligosaccharides is precipitated with dehydrated alcohol, and sample drying is spare, using phenolsulfuric acid standard measure.

ESI-MS analysis.Enzymolysis product and acid hydrolysate are analyzed by mass spectrometry respectively.Mass Spectrometry Conditions are ionization mode: ESI-, high-resolution full scan mode: m/z 100-800.Result is digested as shown in figure 8, ESI-MS detects 3 kinds of degree of polymerization oligosaccharides Presence, respectively monosaccharide 175.0327 (M-1Na+), disaccharides 351.0565 (M+H+-2Na+), 373.0385 (M-1Na+), three 527.0886 (M+2H+-3Na+) of sugar, 549.0706 (M+H+-2Na+), 571.0525 (M-1Na+), for unsaturated oligosaccharides.Acidolysis As a result as shown in figure 9, detecting the oligosaccharides of 2 kinds of degree of polymerization, respectively monosaccharide 193.0348 (M-1Na+), 369.0678 (M of disaccharides + H+-2Na+), 391.0500 (M-1Na+), for be saturated oligosaccharides.

7 brown alga oligose bioactivity of embodiment compares

By the brown alga oligose product that sodium alginate, the obtained brown alga oligose product of enzymatic hydrolysis, acidolysis obtain carry out inoxidizability (hydroxyl from By base scavenging effect, ABTS radicals scavenging effect, reducing power) compare, as a result (Figure 10,11,12) show that is digested is brown Algae oligosaccharides inoxidizability is better than acid hydrolysate and unhydrolysed sodium alginate polysaccharide.As it can be seen that enzymatic isolation method prepares brown alga oligose energy Preferably retain the bioactivity of oligosaccharides, this may be to have higher bioactivity due to digesting obtained unsaturated oligosaccharides.

The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

SEQUENCE LISTING

<110>University of Fuzhou

<120>a kind of algin catenase mutant and its application

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Tyr Val Ser Asp Thr Asp Glu Ser Gly Phe Asn Asp Ser Val Ala Asn

115 120 125

Asn Gly Ile Phe Asp Val Tyr Val Arg Leu Arg Asn Thr Ser Gly Asn

130 135 140

Glu Glu Lys Phe Ala Leu Gly Thr Met Thr Ser Gly Glu Thr Phe Asn

145 150 155 160

Leu Arg Val Val Asn Asn Tyr Gly Asp Val Glu Val Thr Ala Phe Gly

165 170 175

Asn Ser Phe Gly Ile Pro Val Glu Asp Asp Ser Gln Ser Tyr Phe Lys

180 185 190

Phe Gly Asn Tyr Leu Gln Ser Gln Asp Pro Tyr Thr Leu Asp Lys Cys

195 200 205

Gly Glu Ala Gly Asn Ser Asn Ser Phe Lys Asn Cys Phe Glu Asp Leu

210 215 220

Gly Ile Thr Glu Ser Lys Val Thr Met Thr Asn Val Ser Tyr Thr Arg

225 230 235 240

Glu Thr Asn

<210> 5

<211> 732

<212> DNA

<213>artificial sequence

<400> 5

agtaatgatg gtggctcaga tggcggcagc gataactcaa atggttcaac aattcctagc 60

agcataacca gtggtagcat ttttgattta gaaggtgata acccaaatcc tctcgttgac 120

gatagcacct tagtgtttgt gccgttagag gcacaacata ttacgcctaa tggtaatggc 180

tggcgtcatg agtataaggt taaaaagagt ttacgcgttg ctatgactca aacctatgaa 240

gtgttcgaag ctacggtaaa agttgagatg tctgatggcg gaaaaacaat tatatcgcag 300

caccatgcta gcgataccgg cactatatct aaagtgtatg tgtcggatac tgatgaatcg 360

ggctttaatg atagcgtagc gaacaacgga atttttgatg tgtacgtacg tttacgtaat 420

accagcggta atgaagaaaa atttgctttg ggtacaatga ccagcggtga gacatttaac 480

ttgcgggtag ttaataacta cggcgatgta gaggttacgg cattcggtaa ctcgttcggt 540

ataccagtag aggatgattc gcagtcatac tttaagtttg gtaactacct gcaatcgcaa 600

gacccataca cattagataa atgtggtgag gccggaaact ctaactcgtt taaaaactgt 660

tttgaggatt taggcattac agagtcaaaa gtgacgatga ccaatgtgag ttatacgcgt 720

gaaactaatt aa 732

<210> 6

<211> 30

<212> DNA

<213>artificial sequence

<400> 6

aggttaaaaa gagtttacgc gttgctatga 30

<210> 7

<211> 33

<212> DNA

<213>artificial sequence

<400> 7

cgcgtaaact ctttttaacc ttatactcat gac 33

Claims (3)

1. a kind of algin catenase mutant, it is characterised in that: the mutant is to truncate enzyme AlgL- in algin catenase It is lysine, amino acid sequence by the 226th glutamic acid rite-directed mutagenesis on the basis of T157N amino acid sequence SEQ ID NO.3 As shown in SEQ ID NO.4, nucleotide sequence is as shown in SEQ ID NO.5.

2. a kind of algin catenase mutant according to claim 1, it is characterised in that: the algin catenase is cut Short enzyme AlgL-T157N is in alginate lyase geneAlgLOn the basis of the amino acid sequence SEQ ID NO.2 of coding,? It is truncated between 157-158 amino acids, amputation 1-157 amino acids obtain.

3. a kind of algin catenase mutant as described in claim 1 is preparing the application in brown alga oligose.