CN107460179A - A kind of polysaccharide degrading enzyme and its encoding gene and application - Google Patents
A kind of polysaccharide degrading enzyme and its encoding gene and application Download PDFInfo
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Abstract
A kind of polysaccharide degrading enzyme and its encoding gene and application and a whole set of amalgamation and expression, purify the method obtained, the entitled Chond EI of polysaccharide degrading enzyme, encoding gene obtains Edwardsiella tarda (E.tarda) environment separation bacterial strain, it is the protein with the amino acid residue sequence of sequence 2 in sequence table, the amino acid residue sequence of sequence 2 is either passed through to the substitution of one or several amino acid residues, missing or addition have the active protein as derived from sequence 2 identical with the amino acid residue sequence of sequence 2, the polysaccharide degrading enzyme enzyme Chond EI prepared by the tarda clone of ocean separation and expression and purification, with Product Activity height, stability is good, it is with short production cycle, the characteristics of cost is low, it is easier to realize industrialized production.
Description
Technical field
The present invention relates to a kind of polysaccharide degrading enzyme and its encoding gene and application.Belong to biotechnology genetic engineering field.
Background technology
Each tissue generally existing glycosaminoglycan (glycosaminoglycan, GAG) and proteoglycans in animal body
(proteoglycan) polysaccharide component such as, they are in the environment relative stability and the normal physiological function of cell for maintaining cell
Play an important role.Glycosaminoglycan is also known as mucopolysaccharide, mainly including hyaluronic acid (hyaluronic acid, HA), heparin
(heparin), chondroitin sulfate (chondroitin sulfate, CS) etc. is a kind of by hexosamine and uronic acid repetition disaccharides
The long-chain polymer of unit composition.Hyaluronic acid connects what is formed with D- glucuronic acids by N-acetyl-glucosamine with β -1,3- glycosidic bonds
Disaccharide unit is formed, be distributed in connective tissue, the vitreum of eyeball, cornea, cytoplasm, joint fluid, malignant tumor tissue and
In the cell membrane of some bacteriums；Chondroitin sulfate is connected by D- glucuronic acids and N- acetylgalactosamines sulfuric ester with β -1,3- glycosidic bonds
The disaccharide unit for connecing composition is formed, because the position difference of sulfuric ester is divided into chondroitin -4- sulfuric acid (chondroitin sulfate A (CSA)) and cartilage
Element -6- sulfuric acid (chondroitin sulfate C), it is the main component of cartilage, also contains in connective tissue, tendon, skin, heart valve, saliva
Have.The labyrinth of glycosaminoglycan determines the diversity of its function, the growth of human body and animal, tissue repair, cell recognition,
Immunological regulation, matter transportation, cell ageing, the treatment of cancer and diagnosis etc. all have close relation with it.
Protein and glycosaminoglycan form proteoglycans by covalent key connection.Proteoglycans is primarily present in cartilage, tendon
Deng in connective tissue, form cytoplasm, have it is stable, support, protection cell, holding water salt balance be with critical function.Carefully
The proteoglycans of cellular surface also participates in cell adherence, migration, propagation and differentiation function.
Polysaccharide degrading enzyme is cracked into oligosaccharides using one or more of polysaccharide as substrate specificity, has quite varied answer
Use prospect：
1) can be as a kind of toolenzyme for the structure and function for studying protein in body glycan.Polysaccharide degrading enzyme is to acting on bottom
Thing has selectivity and diversity, using this characteristic, can both detect corresponding product, again can to baroque polysaccharide into
Point carry out forming 26S Proteasome Structure and Function research, for example, combine various separation analysis means identification polysaccharide chains combined with target protein it is specific
Functional areas.
2) polysaccharide component that can produce low molecule amount is used for functional product and auxiliary pharmaceutical adjuvant.Polysaccharide in functional product and
There is extensive use in medicine, but due to the inhomogeneity of polysaccharide structures, make the same product of separate sources and different batches
Very big difference in activity be present.And there are some researches show the biological function of glycosaminoglycan is by having special knot in polysaccharide chain
The functional areas of different structure in same polysaccharide chain be present come what is realized in the functional areas of structure and the special interaction of specific protein,
Different functions can be exercised from different protein-interactings, therefore enzymatic isolation method preparation structure can be utilized homogeneous or relatively equal
One specific function area oligosaccharides, it is widely used in food, beverage, invigorant, high-grade health products, the cosmetics of super quality as additive
And medical supplies.For example, low-molecular weight chondroitin sulfate can be widely used in food, beverage, invigorant, height as additive
In shelves health products and the cosmetics of super quality, the chondroitin sulfate that low molecule amount is prepared using enzymatic isolation method is also to study both at home and abroad at present
One of focus.
3) polysaccharide degrading enzyme is a kind of novel medicinal enzyme.Main constituents of the glycosaminoglycan as extracellular matrix, protection
Body cell serves the effect of protective barrier from damage.But in disease treatment, fine and close extracellular matrix inhibits medicine
The permeability of thing (especially to some macromolecular drugs, such as insulin and antibody protein medicaments) in the tissue and effectively expansion
Dissipate, so as to reduce the onset time of medicine and curative effect.Clinically hyaluronidase can be used as auxiliary agent to promote other medicines
Absorption and diffusion；For liquid subcutaneous injection；The degraded that hyaluronic acid in plastic operation accident is filled for hyaluronic acid disappears
Remove；For treating the refractory skin related to glycosaminoglycan metabolic disorder accumulation, as diabetic keratopathy scleredema, chorionitis,
Keloid etc.；Chondroitinase (CSase) can be clinically used for chondroitin sulfate proteoglycan (CSPGs) of degrading, and CSPGs is maincenter
The important component of glial scar after nervous system (CNS) damage, can suppress neural axon regeneration.Using polysaccharase remove GAG or
Person disturbs its synthesis to make axon regeneration, promotes the regeneration of the neuraxis, neural regeneration after spinal cord injury and function reparation
(Nicole J et al.Chondroitinase ABC improves basic and skilled locomotion in
spinal cord injured cats.Experimental Neurology,2008,209(2):483-496)。
In summary, glycosaminoglycan digestive enzyme is not only the essential instrument of glycosaminoglycan structures functional study, and
Prepared in glycosaminoglycan activated oligosaccharide and have important application value in disease treatment.Abroad, grinding for chondroitinase
Study carefully that comparison is early, CSase ABC, CSase AC of commercialization etc. are more by Proteus vulgaris and Flavobacterium
Two kinds of bacterial strain productions of heparinum, its is expensive and produces by patent protection.Production strain growth is slow, CSase AC
The problems such as expression is low and isolates and purifies process complexity, this directly increases the cost of production enzyme, it is difficult to realizes industrialization
Using so the high efficiency recombinant expressed technology of exploitation newly is significant.
Innovation and creation content
To solve existing polysaccharide degrading enzyme production technology deficiency, the present invention provides a kind of polysaccharide degrading enzyme and its encoding gene
With application, and the method that a whole set of amalgamation and expression, purifying obtain, the solvable restructuring zymoprotein of a large amount of high-purities is obtained.
The entitled Chond EI of polysaccharide degrading enzyme provided by the invention, encoding gene obtain Edwardsiella tarda
(E.tarda) environment separation bacterial strain, it is the protein with the amino acid residue sequence of sequence 2 in sequence table, or by sequence 2
Amino acid residue sequence by the substitutions of one or several amino acid residues, missing or addition with the amino acid with sequence 2
The identical active protein as derived from sequence 2 of residue sequence.
The protein that the amino acid residue sequence of sequence 2 is made up of 1023 amino acid residues in sequence table.
Polysaccharide degrading enzyme Chond EI encoding genes, entitled Chond EI, are one of following nucleotides：
1) in sequence table sequence 1 DNA sequence dna；
2) in polynucleotide the protein sequence of sequence 2 polynucleotides；
3) DNA sequence dna limited with sequence 1 in sequence table has more than 95% homology, and encodes identical function protein
DNA sequence dna.
DNA sequence dna in sequence 1 by:3072 base compositions.
Expression vector and cell line containing gene of the present invention belong to protection scope of the present invention.
The primer pair of any fragment in the protein coding gene is expanded also within protection scope of the present invention.
Advantages of the present invention is to be prepared with the tarda separated by ocean the clone of the present invention and expression and purification
Polysaccharide degrading enzyme enzyme Chond EI, there is the characteristics of Product Activity is high, stability is good, with short production cycle, cost is low, be easier to realize
Industrialized production.
Brief description of the drawings：
Fig. 1：The clone of Chond EI genes
Fig. 2：Induced expression of the recombinase in Escherichia coli
Fig. 3：Western blot verify albumen accuracy
Fig. 4：The recombinant protein of purifying
Fig. 5：Temperature influences on enzyme activity
Fig. 6：PH influences on enzyme activity
Fig. 7：Influence of the Nacl concentration to enzyme activity
Embodiment：
The invention will be further described with reference to the accompanying drawings and examples.Embodiment is intended to carry out citing to the present invention to retouch
State, rather than limit the invention in any form.
Embodiment 1, the acquisition of Chond EI genes and the acquisition of recombinase
1st, the acquisition of Edwardsiella tarda Chond EI genes and expression plasmid structure
Choose positioned at chond E1 gene base sequences on Edwardsiella tarda genome, and design primer chond
E1-fwd：ACCTGCATATGTTATCCATGCATCGCACC, chond E1-rev：
ATCAGAAGCTTAGGCATTTTTTTCAGCGCAATC.Using Edwardsiella tarda genome as template, PCR amplifications chond
E1 genes.Amplification system is：95 DEG C of pre-degeneration 2min；95 DEG C of denaturation 20s；55 DEG C of annealing 10s；72 DEG C of extension 1min30s, finally
72 DEG C of 10min are extended to, carry out 30 circulations.The PCR primer both ends obtained have HindIII and NdeI restricted type restriction endonucleases
Site, DNA electrophoresis results are as shown in Figure 1., conventionally will be logical using HindIII and NdeI restricted type restriction enzyme sites
The gene order for crossing PCR synthesis is connected into efficient expression plasmid pET30a, obtains recombinant plasmid pET30a-Chond EI.The structure
The recombinant plasmid built up will be expressed since the sequence 2 in sequence table the 1st up to the Chond E1 maturation proteins of sequence end.
2nd, recombinase Chond EI expression
Recombinant plasmid pET30a-Chond EI are imported in escherichia coli DH5a bacterial strain with transformed competence colibacillus cellular modalities,
It is sequenced by conventional method screening.Successful single bacterium colony is sequenced in culture, extracts plasmid, and table is imported with transformed competence colibacillus cellular modalities
Up in carrier e. coli strain bl21, the μ g/ml of anti-kanamycins (Kan) 30 bacterial strain is screened.Obtained monoclonal bacterium is connect
In kind 5ml LB culture mediums, 37 DEG C are activated overnight, and cultivate to OD600=0.4-0.6, add final concentration of 1mM IPTG inductions
5 hours.Culture medium is abandoned in centrifugation.SDS-PAGE electrophoresis detections, Coomassie brilliant blue dyeing.As a result as shown in Fig. 2 expressing protein exists
115KD positions.Western blot verify albumen accuracy and the feasibility using Histag purifying, as a result as shown in Figure 3.This
Show that the bacterial strain under IPTG inductions, accurately expresses the recombinant C hond EI genes of importing.
3rd, the purifying of expressing protein
E. coli bl21 containing target gene chond E1 in picking conservation pipe, it is placed in mistake in 5ml LB culture mediums
Night cultivates (containing Kan), then expands culture and (contains Kan) into 200ml LB culture mediums, determines OD600=0.4-0.6, enters
Row IPTG (final concentration of 0.1mM) induces 1h, then by 4 DEG C of centrifugation (5000rmin of bacterium solution-1, 15min) 3 times, precipitation every time
It is resuspended with 20mM Tris-HCl buffer solutions (pH7.4-7.6), precipitation is resuspended in the non denatured Wash Buffer for being eventually adding 4ml,
And ultrasonic disruption is carried out, until bacterium solution clear.(power 10%；Work 1s, is spaced 3s, working time 30min).Will be broken
Broken 4 DEG C of bacterium, 5000rmin-115min is centrifuged, takes supernatant, and carry out Thermo HisPur Cobalt Resin affinity chromatographys.
Affinity chromatography method (whole process will be carried out in 4 DEG C)：
(1) appropriate resin is added in centrifuge tube, 700g centrifugation 2min, abandons supernatant；
(2) the Wash Buffer of three times of (resin) volumes are added, are fully mixed；
(3) 700g centrifuges 2min, abandons supernatant, repeats secondary；
(4) Supernatant samples after will be broken mix with resin, and at ambient temperature, rotation mixes 2h, 700g centrifugations 2min.
Supernatant discarding, leave resin；
(5) and then with the Wash Buffer of 3 times of volumes clean resin three times, remove impurity protein；
(6) 120mM, the Elution Buffer elution albumen of 200mM imidazoles, divided with 20mM, 40mM, 80mM is contained respectively
The protein sample flow down is not collected, is preserved in 4 DEG C of centrifuge tubes,
Using HisPur Cobalt Resin purify obtain high-purity C hond EI protein solutions after, using super filter tube from
Heart method, with the PBS desalinization of soil by flooding or leaching and small size is concentrated into, then carries out the SDS-PAGE electrophoresis detections of purification of recombinant proteins.Knot
Fruit is as shown in figure 4, show to be purified into accurately recombinant protein.
Embodiment 2, recombinant C hond EI Enzyme assays
1 protein function is probed into
Enzyme activity definition of the present invention is described as follows with measure：The detection of enzyme activity is using 232nm optical absorption method, an enzyme
Unit of activity (U) produces the enzyme amount required for 1 μM of unsaturated double-bond for substrate of being degraded at 30 DEG C per min.By Sodium Hyaluronate
(Sodium hyaluronate), mosanom (Sodium alginate from brown), liquaemin (Heparin sodium
From porcine intestinal), agarose (Agrose), chondroitin sulfate A (CSA) (Chondroitin sulfate A) it is molten
Yu Shui, the mother liquor that concentration is 10mg/ml is configured to, is tested as follows：Cumulative volume is 100ul.Add substrate 20ul, 250mM
NaH2PO4-Na2HPO4(pH 7.0) 20ul, pure water 30ul, the recombinant C hond EI albumen 30ul of purifying.Enter under the conditions of 30 DEG C
Row digestion reaction, reaction time 12h, then boiling water boil 10min terminating reactions, ice-water bath 10min, in 15000g rotating speed bars
15min is centrifuged under part, collects supernatant.Absorbance change of the absorbance survey in the unit interval under 232nm in 232nm is detected,
The unsaturated double-bond molal quantity for reacting and terminating in rear system is calculated according to molar extinction coefficient.Detected with the Bradford methods of routine
Protein concentration, and specific enzyme activity (unit is U/mg albumen) is calculated, it is defined as enzyme activity and protein concentration (unit mg/ml)
Ratio.Experimental result illustrates that recombinant C hond EI enzymes can selective degradation chondroitin sulfate A (CSA) and two kinds of polysaccharide of hyaluronic acid.
2 enzymes decompose the optimum condition of polysaccharide
It is 300pmoL/ μ L chondroitin sulfate polysaccharide substrate, enzyme liquid, buffer solution (the pH scopes of different pH value by mole
For 5.0-10.0) and water, by 2：1：3：After the ratio mixing of 4 (volume ratios), 10min is reacted at 30 DEG C, finally under 232nm
Detect absorbance.As a result as shown in figure 5, display recombinase reaches maximum vigor in pH8.0, the optimal reaction of recombinase is shown
PH is 8.0.
Under optimal pH, by chondroitin sulfate polysaccharide substrate, enzyme liquid, the 150mM that mole is 300pmoL/ μ L
NaH2PO4-Na2HPO4 buffer solutions (pH8.0) and water press 2：1：3：The ratio mixing of 4 (volume ratios), respectively in different temperatures (0
DEG C -70 DEG C) reaction 10min, finally detects absorbance under 232nm.As a result as shown in fig. 6, display recombinase reaches at 30 DEG C
To maximum vigor, the optimal reactive temperature for showing recombinase is 30 DEG C.
Under optimal pH, by chondroitin sulfate polysaccharide substrate, enzyme liquid, the 150mM that mole is 300pmoL/ μ L
NaH2PO4-Na2The NaCl of HPO4 buffer solutions (pH8.0) and different final concentrations (0M, 0.2M, 0.4M, 0.6M, 0.8M, 1M) is molten
Liquid presses 2：1：3：The ratio mixing of 4 (volume ratios), 10min is reacted under optimal pH and optimum temperature, is finally detected under 232nm
Absorbance.As a result as shown in fig. 7, display recombinase has high enzyme vigor in 0.2M NaCl solution.
Enzymatic reaction is carried out using optimum reaction condition, is computed, enzyme activity during using chondroitin sulfate A (CSA) as substrate can reach
To 410 0U/L zymotic fluids, purification of Recombinant zymoprotein specific enzyme activity is up to 25U/mg.
It will be played a significant role herein in the production of polysaccharide degrading enzyme, and the recombinant C hond EI by producing herein
Enzyme can be as chondrosulphatase AC substitute, each field applied to chondrosulphatase AC.For homogeneous or relative
The preparation of homogeneous chondroitin sulfate/HYALURONANS and oligosaccharides, the determination of chondroitin sulfate/hyaluronic acid 26S Proteasome Structure and Function,
And related field, including food, clinic, medicine and scientific research etc..
Sequence table
<110>Qingdao Agricultural University
<120>A kind of polysaccharide degrading enzyme and its encoding gene and application
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cagatctaca gcttcgatca gcccgccgat ctcgctgaca tcaccacgac ccccggctcc 180
catgtcgcgc tgagcgataa acgcgccatc atgggcgaac gcgcactggt ctgggattgg 240
caacgtggcg ccagcctatt gctgcgccac gacatggaga tcccgtcgga tgctcaggtc 300
tccaaagcct ggggacgctc cgccacgccg gtgctctcct tctggatcta caacgagaag 360
ccgatcgacg atgtgcttat cgtcgatctc ggtaacggcc ttaatgctaa caatgaggcc 420
gacgcgggca ctcaggtcaa actgaacttc cagggctggc gcgccgtcgg cgtctcgctc 480
aacaacgatc tggccaatcg ggagatgagc ggcgtcggtg tacgcagcga cgaagatgcc 540
ggcgaaccca gcgtcagccg ctcactcggt aagcatatcg ataccatccg cttccgtgcc 600
ccctccgggg agaagtccgg ccgcctgttc atcgatcgcg tgatgatctc ggtcgatgat 660
gcgcgttatc aatggtcgga ctaccatgtc aagacgcgct acgacgaacc ggagatcgcc 720
tttcatcacg ccgagccaac gctggcgctg acggcggcga accaggctgg cgtcgatctg 780
atccgccaac gactgatcga tgagttcgtc aatggaccac aagacagcaa cctgaaggcc 840
gaagccgatc tcaataagct acgcgccgcc ttccaggcgt tgcagatccg cgtggcggcc 900
gatggcacgc tgagtggacg ccatctgatc accgacaaac agaaggtgct ctatcaaccg 960
gagtatctga gcgcgcaaga taaggcgaaa ttcgatcgtt atgtcatcct gggcgactac 1020
accacactga tgtacaacat cagccatgcc tacctccaga gtcacgacgc agcgacccgg 1080
aaaacgttgg ccgacatgta tctgctgatg acccgtcacc tgctcgatca aggttttgcc 1140
aaaggcagcg gcctggtcac gacccaccac tggggatata gcgcccgctg gtggtacatc 1200
tcggcactgt tgatgaatcc ggtgctggat caggccaaat tgacacagca ggtctacgat 1260
gcgctgctgt ggtattcgcg tgagtttaaa gctagcttcg acatgaaggt cggcccgcag 1320
agcagtaacc tagattactt caataccctg tcgcgccagc atctcgccct actgctgctg 1380
gagcctaacc cgcaacagcg tatcgacttg ctgaacacct tcagccatta catcagcgga 1440
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cgccatgagg ggaactaccc aggctactcc ttcccggcct ttaagaacgc cgctcagctc 1560
gcctacctgt tgcgcgatac gcctttcgcc ctacagcgcg cggggctgga caagctcaag 1620
caggcgatgg tcgccgcctg gatctacagc aacccgcaga ccgggatctc gctggccggg 1680
cgccacccct tcaactcgcc gtcgctcgcc agcctgacca acgcctacta ctggctggcc 1740
atggcctacg gtaaaaagcc cgatccgacc ctggcggcca tctacctccg cctggcgggg 1800
aagagcgagg ccgatgccaa ggcgctattc ggtagcgcca tcccgccggc agccttaccg 1860
caaggcttct acgcctttaa tggcggcgcc ttcggcatcc accgttggca cgacaagatg 1920
gtgacgttga aagcctttaa cagcaacgtc tggtcctccg agatctacca gaaagataac 1980
cgctatggtc gctaccagag ccatggcgtg gtgcagatca tcaaccaggg ctcgcaagag 2040
gatcagggct atcgccaggc gggttgggac tggaaccgga tgcccggagc caccaccctg 2100
catctgccgt tggaagcact caacagcccc aatcgtcata ccctgatgca gcgcgggagt 2160
caccccttca gcggcacctc atcgctggat ggccgctatg gcatgctggc cttcgatctg 2220
cgcccaatgc gtgatcagcc cagtttcgat caggcattga gcgcgctgaa aagcgtcctg 2280
gccgtcgatg atcgcctgat catggtcggc agcaatctca agagcagcga cagcaagcac 2340
gacttggaga ccaccctgtt ccaactggcc aatcaaccgg gacgcgacag cgccatctgg 2400
gtcaacggac agcgcattga tgccgcctcc tggcagggta gcctgcacaa cggggactgg 2460
ctgatcgatg ccaacggcaa cggctatctg ctggtgaagg ggccgacggc ggaggtccgc 2520
cgccagttac aacactcggc cgacaataag agcatggcgc cgacggaggg ggaattcagc 2580
gtcgcctggc tcaatcatgg caaggcggtc aagaatggcg cctatcagta tctggtggtg 2640
ctcgacgcca ccccgcaacg catgaaccaa ttggcgcaac agctgaagca gggtaaagcc 2700
cccttcaccg tcctgcgcag taacgacggg gttcatgtca tccgcgacaa cctcagccag 2760
gtgacggggt atgtcttcta tcgcacgcaa cgcctcaacg agggcgaggt gatcggcgtc 2820
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accccggatc tcaacatgac acgtcagaag gcggcgaaac cggtcaccat cgacgtcacc 2940
ctacgaggac gttggcaacc ggccacgccg gagcagggca tcgactgctc aaccagcggc 3000
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Met Leu Ser Met His Arg Thr Leu Leu Ala Ala Ser Ile Ala Ala Leu
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Leu Leu Asn Ala Pro Leu Tyr Ala Ala Pro Asn Asp Ala Ala Ala Lys
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Lys Arg Ala Glu Ile Met Arg Ala Gln Ile Tyr Ser Phe Asp Gln Pro
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Ala Asp Leu Ala Asp Ile Thr Thr Thr Pro Gly Ser His Val Ala Leu
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Ser Asp Lys Arg Ala Ile Met Gly Glu Arg Ala Leu Val Trp Asp Trp
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Gln Arg Gly Ala Ser Leu Leu Leu Arg His Asp Met Glu Ile Pro Ser
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Asp Ala Gln Val Ser Lys Ala Trp Gly Arg Ser Ala Thr Pro Val Leu
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Ser Phe Trp Ile Tyr Asn Glu Lys Pro Ile Asp Asp Val Leu Ile Val
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Asp Leu Gly Asn Gly Leu Asn Ala Asn Asn Glu Ala Asp Ala Gly Thr
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Gln Val Lys Leu Asn Phe Gln Gly Trp Arg Ala Val Gly Val Ser Leu
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Asn Asn Asp Leu Ala Asn Arg Glu Met Ser Gly Val Gly Val Arg Ser
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Asp Glu Asp Ala Gly Glu Pro Ser Val Ser Arg Ser Leu Gly Lys His
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Ile Asp Thr Ile Arg Phe Arg Ala Pro Ser Gly Glu Lys Ser Gly Arg
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Leu Phe Ile Asp Arg Val Met Ile Ser Val Asp Asp Ala Arg Tyr Gln
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Trp Ser Asp Tyr His Val Lys Thr Arg Tyr Asp Glu Pro Glu Ile Ala
225 230 235 240
Phe His His Ala Glu Pro Thr Leu Ala Leu Thr Ala Ala Asn Gln Ala
245 250 255
Gly Val Asp Leu Ile Arg Gln Arg Leu Ile Asp Glu Phe Val Asn Gly
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Pro Gln Asp Ser Asn Leu Lys Ala Glu Ala Asp Leu Asn Lys Leu Arg
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Ala Ala Phe Gln Ala Leu Gln Ile Arg Val Ala Ala Asp Gly Thr Leu
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Ser Gly Arg His Leu Ile Thr Asp Lys Gln Lys Val Leu Tyr Gln Pro
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Glu Tyr Leu Ser Ala Gln Asp Lys Ala Lys Phe Asp Arg Tyr Val Ile
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Leu Gly Asp Tyr Thr Thr Leu Met Tyr Asn Ile Ser His Ala Tyr Leu
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Gln Ser His Asp Ala Ala Thr Arg Lys Thr Leu Ala Asp Met Tyr Leu
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Leu Met Thr Arg His Leu Leu Asp Gln Gly Phe Ala Lys Gly Ser Gly
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Leu Val Thr Thr His His Trp Gly Tyr Ser Ala Arg Trp Trp Tyr Ile
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Ser Ala Leu Leu Met Asn Pro Val Leu Asp Gln Ala Lys Leu Thr Gln
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Gln Val Tyr Asp Ala Leu Leu Trp Tyr Ser Arg Glu Phe Lys Ala Ser
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Phe Asp Met Lys Val Gly Pro Gln Ser Ser Asn Leu Asp Tyr Phe Asn
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Thr Leu Ser Arg Gln His Leu Ala Leu Leu Leu Leu Glu Pro Asn Pro
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Gln Gln Arg Ile Asp Leu Leu Asn Thr Phe Ser His Tyr Ile Ser Gly
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Ala Leu Ala Gln Thr Pro Pro Gly Ser Asn Asp Gly Leu Arg Pro Asp
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Gly Thr Ala Trp Arg His Glu Gly Asn Tyr Pro Gly Tyr Ser Phe Pro
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Ala Phe Lys Asn Ala Ala Gln Leu Ala Tyr Leu Leu Arg Asp Thr Pro
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Phe Ala Leu Gln Arg Ala Gly Leu Asp Lys Leu Lys Gln Ala Met Val
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Ala Ala Trp Ile Tyr Ser Asn Pro Gln Thr Gly Ile Ser Leu Ala Gly
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Arg His Pro Phe Asn Ser Pro Ser Leu Ala Ser Leu Thr Asn Ala Tyr
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Tyr Trp Leu Ala Met Ala Tyr Gly Lys Lys Pro Asp Pro Thr Leu Ala
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Ala Ile Tyr Leu Arg Leu Ala Gly Lys Ser Glu Ala Asp Ala Lys Ala
595 600 605
Leu Phe Gly Ser Ala Ile Pro Pro Ala Ala Leu Pro Gln Gly Phe Tyr
610 615 620
Ala Phe Asn Gly Gly Ala Phe Gly Ile His Arg Trp His Asp Lys Met
625 630 635 640
Val Thr Leu Lys Ala Phe Asn Ser Asn Val Trp Ser Ser Glu Ile Tyr
645 650 655
Gln Lys Asp Asn Arg Tyr Gly Arg Tyr Gln Ser His Gly Val Val Gln
660 665 670
Ile Ile Asn Gln Gly Ser Gln Glu Asp Gln Gly Tyr Arg Gln Ala Gly
675 680 685
Trp Asp Trp Asn Arg Met Pro Gly Ala Thr Thr Leu His Leu Pro Leu
690 695 700
Glu Ala Leu Asn Ser Pro Asn Arg His Thr Leu Met Gln Arg Gly Ser
705 710 715 720
His Pro Phe Ser Gly Thr Ser Ser Leu Asp Gly Arg Tyr Gly Met Leu
725 730 735
Ala Phe Asp Leu Arg Pro Met Arg Asp Gln Pro Ser Phe Asp Gln Ala
740 745 750
Leu Ser Ala Leu Lys Ser Val Leu Ala Val Asp Asp Arg Leu Ile Met
755 760 765
Val Gly Ser Asn Leu Lys Ser Ser Asp Ser Lys His Asp Leu Glu Thr
770 775 780
Thr Leu Phe Gln Leu Ala Asn Gln Pro Gly Arg Asp Ser Ala Ile Trp
785 790 795 800
Val Asn Gly Gln Arg Ile Asp Ala Ala Ser Trp Gln Gly Ser Leu His
805 810 815
Asn Gly Asp Trp Leu Ile Asp Ala Asn Gly Asn Gly Tyr Leu Leu Val
820 825 830
Lys Gly Pro Thr Ala Glu Val Arg Arg Gln Leu Gln His Ser Ala Asp
835 840 845
Asn Lys Ser Met Ala Pro Thr Glu Gly Glu Phe Ser Val Ala Trp Leu
850 855 860
Asn His Gly Lys Ala Val Lys Asn Gly Ala Tyr Gln Tyr Leu Val Val
865 870 875 880
Leu Asp Ala Thr Pro Gln Arg Met Asn Gln Leu Ala Gln Gln Leu Lys
885 890 895
Gln Gly Lys Ala Pro Phe Thr Val Leu Arg Ser Asn Asp Gly Val His
900 905 910
Val Ile Arg Asp Asn Leu Ser Gln Val Thr Gly Tyr Val Phe Tyr Arg
915 920 925
Thr Gln Arg Leu Asn Glu Gly Glu Val Ile Gly Val Asn Arg Pro Ala
930 935 940
Ile Val Met Thr Arg Pro Gln Glu Gly Gly Leu Val Leu Ser Ala Val
945 950 955 960
Thr Pro Asp Leu Asn Met Thr Arg Gln Lys Ala Ala Lys Pro Val Thr
965 970 975
Ile Asp Val Thr Leu Arg Gly Arg Trp Gln Pro Ala Thr Pro Glu Gln
980 985 990
Gly Ile Asp Cys Ser Thr Ser Gly Asp Glu Thr Arg Leu Arg Phe Arg
995 1000 1005
Ile Asp Phe Gly Ile Pro Gln Gln Ile Ala Leu Lys Lys Met Pro
1010 1015 1020
Claims (3)
1. a kind of entitled Chond EI of polysaccharide degrading enzyme, encoding gene obtains Edwardsiella tarda (E.tarda) environment separation
Bacterial strain, is the protein with the amino acid residue sequence of sequence 2 in sequence table, or the amino acid residue sequence by sequence 2
There is activity identical with the amino acid residue sequence of sequence 2 by the substitution of one or several amino acid residues, missing or addition
The protein as derived from sequence 2.
2. a kind of polysaccharide degrading enzyme described in claim 1, the amino acid residue sequence of sequence 2 is by 1023 in its sequence table
The protein of amino acid residue composition.
3. a kind of polysaccharide degrading enzyme Chond EI encoding genes, entitled Chond EI, are one of following nucleotides：
1) in sequence table sequence 1 DNA sequence dna；
2) in polynucleotide the protein sequence of sequence 2 polynucleotides；
3) DNA sequence dna limited with sequence 1 in sequence table has more than 95% homology, and encodes identical function protein
DNA sequence dna；
4) DNA sequence dna in sequence 1 is by 3072 base compositions.
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