CN109943553A - A kind of low temperature resistant mutant of chitinase and its application - Google Patents

Abstract

The application passes through the low temperature resistant chitinase disclosed in the prior art screened from marine microorganism, the middle screening after PCR is introduced into mutation, obtain a mutant with wild type comparison with the mutation of 5 amino acid sites, by most adaptable temperature detection, the mutant most adaptive temperature reduces 5 DEG C compared with wild type, more conducively hydrolysis of the enzyme applied to chitin.

Description

A kind of low temperature resistant mutant of chitinase and its application

Technical field

The invention belongs to genescreen renovation technique fields, and in particular to a kind of chitinase enzyme (glucosidase) and It is applied, and in particular to a kind of method screening by gene mutation obtain chitinase mutant and chitinase it is resistance to low The application in chitin degrading of warm mutant.

Background technique

Chitin is also known as chitin, chitin, it is widely present in Crustaceans, mollusk and arthropod shell In, such as shrimp, crab, locust, it is furthermore also widely present in algae, shellfish, the cell wall of Mycophyta and higher plant, is mesh It is only second to the second largest living resources of cellulose in preceding nature, but there are more specific functions than cellulose.Chitin is A kind of white or canescence, translucent flat crystal, it is tasteless, it is natural mucopolysaccharide.Chemical name is 1,4-2- acetylaminohydroxyphenylarsonic acid 2- Deoxidation-β-D glucan, basic unit be acetylglucosamine (GlcNAc) be a kind of straight chain polymer chemical structure such as Under, according to the arrangement mode of strand, there are α, tri- kinds of crystal form objects of β, γ for it.Due to having stronger hydrogen between the polysaccharide macromolecular Key effect, so its physicochemical property is highly stable.Maximum α-the chitin of content, is made of, more hydrogen bonds two antiparallel strands It is connected, soaks not swollen；β-chitin is made of two cocurrent and parallel chains, few more, the chance of the smaller chitin of the hydrogen bond of molecule interchain Water can expand；γ-chitin is three chains, and two in the same direction, and one reversed, similar to α-and β-chitin mixing crystal.

Chitin from marine biosphere accounts for the overwhelming majority of chitin total amount, however chitin sea-bottom deposit but very Few, this is mainly related with effectively decomposition of the marine microorganism to chitin.Marine microorganism is decomposed several by secretion chitinase The carbon source and nitrogen source that fourth matter is bred as own growth, have pushed the recycling of chitin.Chitin is a kind of in marine environment Abundant and important nutrition and energy matter, it is sea that marine microorganism degrade and utilize to chitin by chitinase The key of Cycle of nutrients in foreign environment, it is also critically important to the balance for maintaining the marine environment ecosystem.

Nearest chitin causes the great interest of researcher, converts them to oligosaccharide, oligosaccharide is not only dissolved in Water, and possess a variety of biological functions, such as anti-tumor activity and antifungal activity.Currently, chitin oligosaccharide is industrially It is prepared by chemical method, there are many problems for this, generate if any many short chain oligosaccharides, oligosaccharide low output, separation costs Height, and pollution easily is generated to environment.Opposite, enzymatic hydrolysis chitin has many advantages, and environment compatibility is at low cost, repeats. In recent years, the enzymatic hydrolysis of chitin becomes more and more popular.

Compared with the chitinase of land, ocean chitinase has higher pH and salinity tolerance, helps particularly to answer With.The psychrophilic bacteria that Stefanidi etc. is separated to from the sample lived in from 1 200m of the South Pacific Ocean can secrete several several Fourth matter enzyme degradation chitin.Chitinase also found in Alteromonas sp.strain O-7, Vibrio anguillarum、V.parahaemolyticus。

Cold-adapted enzyme mainly has following four Physiology and biochemistry feature: (1) at low temperature have it is high turnover rate (Kcat) and high Catalytic efficiency (Kcat/Km) is the main adaptive character of cold-adapted enzyme.They, which can make up low temperature, causes chemical reaction rate to reduce Bring influences, and mentions enough enough metabolic activities for biology and maintains enough metabolic fluxs.High catalytic efficiency is (including Km value Decline) it is particularly important to cold-adapted enzyme, because she can guarantee the abundant combination of substrate and enzyme active sites under low temperature.(2) most suitable Operative temperature is low or to have high catalytic activity at low temperature be the prerequisite that enzyme is classified as cold-adapted enzyme.The optimal reaction temperature of cold-adapted enzyme It is generally below 40 DEG C of degree, 10 DEG C to 20 DEG C low compared with common medium temperature enzyme.Cold-active chitinase reported at present is most Suitable operative temperature is minimum at 30 DEG C.(3) structure flexibility is another feature of cold-adapted enzyme.Cold-adapted enzyme is submissive, loose and open Structure feature, help to improve conformation change ability of the enzyme molecule in catalytic process.When the temperature drops, with protein structure The reduction of flexibility and cold denaturation effect, the catalytic rate of enzyme decline therewith.And the high-flexibility of cold-adapted enzyme, it is on the one hand bottom The active site that object reaches enzyme provides more preferably channel and accommodated position, and the combination improved between enzyme active sites and substrate is complementary Property, and be conducive to the release of substrate: activation energy can be reduced on the other hand with the low kinetic energy of compensatory reactionBu Changfanying molecule, it is huge to reduce receiving Energy spent by substrate guarantees that the thermal stability of high catalytic efficiency (4) cold-adapted enzyme of enzyme is poor in low energy consumption.Cold-adapted enzyme The ion coordination that the height flexibility of structure may cause albumen is loose, and the two poles knot due to lacking disulfide bond and rigidity Structure causes cold-adapted enzyme sensitive to thermal denaturation and chemical modification, and stability reduces.In addition, natural environment locating for cold-adapted enzyme lacks Heat selective pressure may be also the reason of causing protein structure thermal instability.

Compared with high temperature environment, under low temperature environment, the viscosity of water is significantly increased, and warm-up movement significantly reduces.Such Under environment, due to the reduction of warm-up movement, the combination of enzyme-to-substrate with separate, the change of enzyme texture image all than under high temperature be stranded Difficulty, temperature can significantly affect the combination of enzyme-to-substrate.Enzyme molecule will have higher catalytic rate under such circumstances, it is necessary to make point More compliant (flexible) that minor structure becomes, to increase the speed of conformational change.Compared with medium temperature enzyme, cold-adapted enzyme it is common Be characterized in: the quantity of salt bridge is reduced, and the interaction of aromatic rings weakens in hydrophobic core, and proline subtracts with arginine residues quantity Few, the hydrophobicity of enzyme weakens, and makes the Interaction enhanced of enzyme and solvent.The change of these structures is all conducive to enzyme molecule flexibility Increase.

Due to cold-active chitinase have reaction temperature it is low, to it is thermo-responsive the features such as, so industrialization utilize chitin The superiority for having medium temperature chitinase incomparable during enzyme degradation chitin production chitin oligo saccharide, using low temperature chitin Enzyme can save heating and cooling two steps, not only saved energy but also saved the time.Meanwhile useless using chitinase processing nature When object and sewage treatment, since cold-active chitinase has the optimal reactive temperature close to natural environment temperature, application Effect is more preferable.This is to environmental protection important in inhibiting.

Currently, reported cold-active chitinase is not very much.To solve the above problems, the present invention will be from prior art public affairs On the basis of the known chitinase opened, the method for passing through random mutation and screening obtains low temperature resistant chitinase.

Summary of the invention

The object of the present invention is to provide a kind of its applications of chitinase mutant.The present invention is by largely screening, finally A kind of low temperature resistant stronger chitinase mutant is obtained, and constructs and has obtained the engineering strain of recombinant expression chitinase, To realize that its application is laid a good foundation.

First aspect present invention is related to a kind of low temperature resistant mutant of chitinase, is that amino acid sequence is and SEQ ID NO: Amino acid sequences alignment shown in 1 has the following mutation of any one or more, and the mutation is selected from: the 501st Arg sports Leu, and the 555th Tyr sports Trp, and the 635th Phe sports Pro, and the 673rd Ala is sported Ser, the 689th Glu sport Thr.

Second aspect of the present invention is related to a kind of low temperature resistant mutant of chitinase, be amino acid sequence be SEQ ID NO:3 It is shown.

The third aspect of the present invention provides the nucleic acid sequence for encoding the above-mentioned low temperature resistant mutant code gene of chitinase； In a specific embodiment, the nucleotides sequence is classified as shown in SEQ ID NO:4.

The fourth aspect of the present invention, the present invention provides a kind of carriers, containing more described in the second aspect of the present invention Nucleotide.Carrier herein includes expression vector, for example, bacterial plasmid, clay, phasmid, yeast plasmid, plant cell are sick Poison, animal virus and various other viral vectors.The carrier being applicable in the present invention includes but is not limited to: the expression in bacterium Carrier (prokaryotic expression carrier), in yeast the carrier (such as pichia vector, Hansenula vectors) of expression, in insect Carrier (vaccinia virus vector, retrovirus of the baculovirus vector, expression in mammalian cells expressed in cell Carrier, adenovirus vector, adeno-associated virus carrier etc.), the plant viral vector of expression and in mammalian milk in plant The various carriers of expression in gland.As long as any plasmid and carrier all can be used in short, can stablize duplication in host cell. Preferred expression carrier includes selectable marker gene, and such as the ampicillin resistance gene of bacterium, tetracycline resistance gene, to block that mould Plain resistant gene, streptomycin resistance gene, chloramphenicol resistance gene；Neomycin resistance gene, the Zeocin resistance base of saccharomycete Cause, the defect selection marker of saccharomycete, such as His, Leu, Trp etc.；Neomycin resistance gene, the Zeocin resistance base of eukaryocyte Cause, dihydrofolate reductase gene and fluorescent protein marker gene etc..Carrier of the invention is preferably prokaryotic vector, more preferably Coli expression carrier；More preferably pET series plasmids；More preferably pET-28a (+) plasmid.

Fifth aspect present invention, the present invention provides cells, contain polynucleotides described in third aspect of the present invention. The cell can use carrier conversion or transfection described in the 4th aspect of the present invention and obtain.Cell can be prokaryotic cell, It can be eukaryocyte, e.g., bacterial cell, yeast cells, plant cell, insect cell, mammalian cell etc..Cell is turning After changing or transfecting the gene order containing encoding fusion protein of the present invention, that is, engineering cell or cell strain are constituted, can be used for Fusion protein needed for producing.Suitable conversion or transfection method include but is not limited to: for bacterial cell, such as Calcium Chloride Method, electricity Perforation method；For yeast cells, such as electroporation and protoplast fusion method；For mammalian cell etc., as plastid package, Coprecipitation of calcium phosphate, electro fusion method and microinjection.It is preferred that cell of the invention is Bacillus coli cells.The present invention also relates to And a kind of engineered strain, carry above-mentioned recombinant plasmid.The engineered strain is bacterium, and preferably Gram-negative bacteria is excellent Escherichia coli are selected as, in a specific embodiment, the host is E.coli BL21.

The invention further relates to a kind of compositions containing chitinase high/low temperature mutant, wherein the chitinase is resistance to Low temperature mutant is prepared by the above-mentioned engineering bacteria fermentation containing the expression plasmid.

The invention further relates to the low temperature resistant mutant of above-mentioned chitinase, encoding gene, expression vector, recombinant cells in water Solve the application in chitin.

The beneficial effects of the invention are as follows by genetic engineering means, the mutated library of large sample is constructed, is then therefrom screened Obtain a large amount of screenings and obtain a kind of low temperature resistant mutant of chitinase, the enzyme compare wild type enzyme for, respective Under conditions of most illustrating, absolute enzyme activity has a certain upgrade, while its optimum temperature is 30 DEG C, and is able to maintain at 25 DEG C 70% or more enzyme activity is also able to maintain nearly 30% activity at 10 DEG C, has low temperature tolerance characteristics；Therefore it is more conducive to several Application in fourth matter degradation process.

Detailed description of the invention

Fig. 1: SDS-PAGE electrophoresis detection figure, wherein swimming lane CHI is wild-type protein, and CHI-M5 is mutein, point Son amount size is 127KD.

Fig. 2: chitinase is compareed with respect to enzyme activity-operative temperature curve graph as wild type chitinase.

Specific embodiment

Method of the invention is described further below with reference to example.The experiment of actual conditions is not specified in embodiment Method, " Molecular Cloning:A Laboratory guide " that can be usually write by the routine experiment of molecular biology field, cold spring harbor laboratory etc. Described in condition, or operated according to the specification of the merchandized handlings such as plasmid, bacterial strain manufacturer.The relevant technology in this field Personnel can more fully understand and grasp the present invention by embodiment.

The fallibility PCR that mutation PCR is known to those skilled in the art.

The gene cloning of 1 wild type chitinase of embodiment

The inventors discovered that disclosing one kind in the prior art screened from Pseudoalteromonas bacterium (Pseudoalteromonas sp.) amino acid sequence chitinase as shown in SEQ ID NO:1, coding nucleotide is such as Shown in SEQ ID NO:2, prepared in expression in escherichia coli.In brief, Shanghai JaRa people from Bioisystech Co., Ltd is entrusted Work synthesizes the gene, then connects EcoR I and Hind into pET-28 (+) carrier (New England Biolabs company) Between the restriction enzyme site of III, it is transformed into bacillus coli DH 5 alpha, is screened on the LB plate of the 50mg/L containing ampicillin, is screened The expression vector for including in positive bacterium is named as pET-chi, extracts plasmid, and sequencing identification is correct.

2 screen mutation of embodiment

Applicant in order to further increase the lower temperature resistance of above-mentioned chitinase (amino acid sequence be SEQ ID NO:1), Using the recombinant plasmid pET-chi constructed in embodiment 1 as template, random mutation primer is separately designed, carries out fallibility PCR, Multipoint random mutation is carried out to target gene, as a result, it has been found that some mutation do not influence its lower temperature resistance, some mutation are even Keep lower temperature resistance worse, in addition also some mutation, although heat resistance can be improved, its pH scope of application narrows after mutation, It is undesirable.Finally, applicant, which obtains, can significantly improve low temperature properties and expand the scope of application under acid condition The combination in mutational site: R501L, Y555W, F635P, A673S, E689T；Specially the 501st Arg sports Leu, the 555 Tyr sport Trp, and the 635th Phe sports Pro, and the 673rd Ala sports Ser, the 689th Glu Sport Thr.

Above-mentioned five point mutation body is named as Chi-Mut5, amino acid sequence is SEQ ID NO:3, is closed referring to the sequence At a coding nucleotide sequence SEQ ID NO:4.The sequence is the codon bias according to Escherichia coli and optimum synthesis, and And two restriction enzyme sites of BamHI and HindIII are added respectively at the both ends of composition sequence 5 ' and 3 ', by the raw work bioengineering stock in Shanghai Part Co., Ltd completes.

Using recombinant plasmid of operation building described in embodiment 1 containing above-mentioned five point mutation body gene, it is named as pET- chi-mut5。

3 expression and purification of embodiment

Method well known in the art prepares the competent cell of e. coli bl21 (DE3), and with heat shock method by embodiment 1 The expression vector of the wild type expression vector and mutant that construct with embodiment 2 is transformed into competent cell respectively, and screening is positive It is spare after clone PCR and sequence verification.

Transfer needle picking takes positive bacteria to be inoculated in 5mL LB culture medium, with 30 DEG C, 200r/min cultivate 12h, then with It is inoculated in the 400mL LB culture medium of kanamycins containing 50ug/mL by 2% (V/V) inoculum concentration, 8h is cultivated with 30 DEG C, 200r/min. When bacterial density OD600 reaches 0.7, the IPTG that 0.5mM is added carries out inducing expression；16 DEG C of 170rpm shake cultures are stayed overnight.

Thalline were collected by centrifugation, and PBS washing, supernatant is collected by centrifugation in ice domain carrying out ultrasonic bacteria breaking, using AKTA affinity chromatography system, Affinitive layer purification is carried out using two kind albumen (wild type and saltant type) of the Ni2+ chromatographic column to above-mentioned expression, is eluted with imidazoles Afterwards by desalting column desalination, albumen is stored in spare in -20 DEG C of PBS buffer solution, and wild-type protein is named as CHI, saltant type egg White to be named as CHI-M5, purifying obtains albumen and carries out SDS-PAGE detection, the result is shown in Figure 1.

The enzyme activity determination of 4 chitinase of embodiment and its low temperature resistant mutant

1, enzyme activity determination method

1) prepared by tobacco brown spot pathogen: weighing 25g chitin, adds 85% phosphoric acid of 250mL, 30 DEG C of reaction 1.5d add distilled water After colloidal precipitation, washing is until neutral repeatedly.Final concentration is adjusted to 2% with 20mM phosphate buffer (pH7.0).

2) reducing sugar method enzyme activity determination: is determined using 3,5- dinitrosalicylic acid (DNS) colorimetric.Enzyme activity unit is defined as this Enzyme enzyme needed for (i.e. 35 DEG C, the 20mM phosphate buffer of pH7.0 in) generate 1 micromole's reduced sugar per minute under optimum condition Amount.0.5% tobacco brown spot pathogen of 0.25mL is mixed with the appropriate diluted enzyme solution to be measured of 0.25mL, in 35 DEG C of incubation 1hr, centrifugation It goes to precipitate, above resets and add 1.5mL distilled water and 1.5mL DNS solution, solution is mixed, after boiling water educates 5min, is cooled to room temperature, and is mended Add 21.5mL distilled water, mix, surveys OD₅₄₀.Colourimetric number, do standard curve with sugar.

3) determination of protein concentration: the measurement of protein concentration is according to Lowry method, using bovine serum albumin(BSA) as standard.

2, optimum temperature

Respectively in 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, The activity that wild type chitinase and mutant that above-mentioned purifying obtains are measured under the conditions of pH7.0, with highest enzyme activity for 100%, Opposite enzyme activity is calculated, does temperature-with respect to enzyme activity curve.As a result as shown in Fig. 2, the optimum temperature of wild type chitinase is 35 DEG C, at 25 DEG C, residual activity is 50%, and about 25% vigor is still able to maintain at 10 DEG C, and the most suitable action temperature of mutant Degree is 30 DEG C, 30 DEG C, and it is able to maintain at 25 DEG C 70% or more enzyme activity, it is also able to maintain nearly 30% activity at 10 DEG C, has resistance to Low-temperature characteristics.

3, mutant is compared with the enzyme activity of wild type chitinase

Using 20mM phosphate buffer by zymoprotein be diluted to 1mg/ml respectively under optimum (35 DEG C of wild type, PH7, saltant type are 30 DEG C, pH7) hydrocolloid, specifically:

0.5% tobacco brown spot pathogen of 0.25mL is mixed with the appropriate diluted enzyme solution to be measured of 0.25mL, in 35 DEG C of (saltant types It is 30 DEG C) 1hr is incubated, centrifugation goes to precipitate, and above resets and add 1.5mL distilled water and 1.5mL DNS solution, and solution mixes, and boiling water is educated It after 5min, is cooled to room temperature, adds 21.5mL distilled water, mix, survey OD₅₄₀.Colourimetric number, do standard curve with sugar.

As a result the enzyme activity of wild type is in 11.56U/mg, and saltant type in 12.14U/mg, i.e., absolute enzyme activity has certain It is promoted.

The screening of the mutation, which obtains, can be more adaptive to the chitinase of industrial chitin hydrolysis.

SEQUENCE LISTING

<110>University Of Dalian

<120>a kind of low temperature resistant mutant of chitinase and its application

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<160> 4

<170> PatentIn version 3.5

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<211> 1054

<212> PRT

<213> Pseudoalteromonas sp.

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Thr Ser Leu Ala Tyr Asn Lys Phe Ala Gln Ala Gly Ser Lys Tyr Asp

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

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Ile Ala Pro Thr Phe Ser Phe Thr Asn Asn Ser Asn Ile Asp Leu Ser

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Lys Ser Asn Trp Asn Ala Gln Glu Lys Leu Gly Met Ala Val Asp Val

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aaaaacctac aaaatggcat tttaccaagt taccttgcgg cttacggctt agatccagca 2160

aatgatccaa ctgataagct agtgggcact tacactcgtc attacgattc agtggcagta 2220

gcgccgtggc tttggaatgc tgataaaaac gtgttcttgt cgattgaaga tgaagagtca 2280

atgggcacta agcttgatta cgtaatcaac aaaggccttg gcggtattat gttctgggag 2340

ctggcaggtg actttgacta cgacagcgcg aagaaagagt actttatggg ttctactatg 2400

acctcgcttg cttacaataa gtttgcacaa gcgggttcta agtacgacat caacaaaggt 2460

gaaaaaactc accaagtacc agcagaggca gttgacgtaa gctttgaagt gaaagacttc 2520

ccaattggtg acgacaacta cccaattgcg ccaaccttta gttttactaa caactcaaac 2580

attgatttga gcggtgcaaa agtatcgttt gatgtaccag tggctacatc ggcaattttc 2640

aaatcaaact ggaatgcgca agaaaaactc ggcatggcag ttgacgtaaa tcgttcaaac 2700

gcagctggca acaacattgg tggctttgaa aacgagttcc accgtttctc aattaccttt 2760

gttaacgagt ggggcggcgc tgctaagtcg tttgcgccgg gcgaaaccat taatgcgcaa 2820

gtaatgtact acatgccaat tactggccca gtgaaccttg tatttgaaaa agacggtaag 2880

caatacgccg ctaaatttga atacccatat ctaccagatg caacggccgg taacggtggt 2940

ggcaatgatg gcggcggtaa caccgacaca tgtgaaggtg ttgcagtagc tgatattcca 3000

gtttatccaa acttcccaca aacagattgg gcaggcaacc cgtcacacgc tgcaggcggt 3060

gacttgatgg ttgatggcgc atctactgcg gtttacaaag ccaagtggtg gacatcaact 3120

gagcctggca catccgccga ttgggatagt gtgtgtactt tataa 3165

<210> 3

<211> 1054

<212> PRT

<213> Pseudoalteromonas sp.

<400> 3

Met Ser Asn Lys Leu Val Gly Lys Arg Leu Phe Lys Met Ser Val Leu

1 5 10 15

Ser Leu Ala Val Thr Gly Trp Ala Ala His Ala Ala Val Asp Cys Thr

20 25 30

Asn Ile Ala Glu Trp Gln Ser Gly Val Ala Tyr Thr Gly Gly Gln Gln

35 40 45

Val Gln Lys Ala Asn Thr Ala Tyr Glu Ala Lys Trp Trp Thr Gln Ala

50 55 60

Asp Pro Val Thr His Ser Gly Gln Tyr Gln Glu Trp Lys Asn Leu Gly

65 70 75 80

Ala Cys Ser Asp Gly Gln Val Asn Glu Ser Pro Ile Ala Glu Val Leu

85 90 95

Ser Pro Met Ser Gly Ser Ser Phe Thr Glu Gly Asp Ser Val Val Ile

100 105 110

Glu Ala Gln Thr Ser Asp Ser Asp Gly Thr Val Val Lys Leu Glu Val

115 120 125

Leu Val Asp Gly Val Ala Ile Ala Thr Leu Ser Ser Ala Pro Tyr Ser

130 135 140

Thr Thr Trp Leu Ala Val Thr Gly Gln His Gln Ile Ser Val Arg Ala

145 150 155 160

Thr Asp Asp Lys Gly Ala Glu Thr Thr Ser Pro Ala Asn Ser Val Thr

165 170 175

Val Ala Glu Val Val Pro Asp Asn Tyr Ala Pro Thr Ala Ser Leu Thr

180 185 190

Ser Asn Ala Ser Ser Asn Thr Leu Asn Leu Asn Asp Asn Val Val Leu

195 200 205

Asn Leu Ser Gly Ser Asp Gln Asp Gly Thr Val Thr Ser Leu Glu Leu

210 215 220

Phe Gln Asp Gly Ala Ser Val Ala Gln Ile Ser Gly Ala Thr Gly Ser

225 230 235 240

Phe Asn Phe Thr Ala Thr Ser Ala Gly Asn Thr Gln Phe Ser Leu Val

245 250 255

Ala Thr Asp Asp Lys Gly Ala Thr Ser Asp Ala Gln Thr Leu Ser Phe

260 265 270

Arg Ile Val Asp Gly Thr Thr Gln Pro Gly Asp Arg Asp Ala Cys Lys

275 280 285

Pro Glu Gly Leu Tyr Gln Thr Pro Gly Val Asn Thr Pro Tyr Cys Thr

290 295 300

Ile Tyr Asp Ala Asp Gly Arg Glu Asp Met Gly Ala Asp His Pro Arg

305 310 315 320

Arg Val Ile Gly Tyr Phe Thr Ser Trp Arg Asn Gly Ala Asn Gly Gln

325 330 335

Pro Ser Tyr Leu Val Asp Asp Ile Pro Trp Asp Lys Ile Thr His Ile

340 345 350

Asn Tyr Ala Phe Ala His Val Asp Ala Asn Asn Lys Val Ser Ile Gly

355 360 365

Asp Pro Ser Ala Ala Gly Asn Pro Ala Thr Asn Met Glu Trp Pro Gly

370 375 380

Val Ala Gly Ala Glu Met Asp Pro Thr Leu Pro Tyr Lys Gly His Phe

385 390 395 400

Asn Leu Leu Asn Lys Tyr Lys Lys Leu His Pro Asp Val Lys Thr Leu

405 410 415

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

420 425 430

Gly Asn Arg Val Ala Ser Gly Gly Phe Tyr Thr Met Thr Thr Asn Ala

435 440 445

Asp Gly Ser Val Asn Thr Ala Gly Ile Asn Ala Phe Ala Lys Ser Ala

450 455 460

Val Glu Phe Ile Glu Thr Tyr Gly Phe Asp Gly Val Asp Ile Asp Tyr

465 470 475 480

Glu Tyr Pro Ser Ser Met Asn Asp Ser Gly His Pro Asp Asp Phe Pro

485 490 495

Ile Ser Asn Ala Leu Arg Ala Gly Leu Asn Ala Ser Tyr Gln Val Leu

500 505 510

Met Lys Thr Leu Arg Glu Glu Leu Asp Lys Ala Gly Glu Leu Ala Gly

515 520 525

Lys His Tyr Met Leu Thr Ile Ala Ser Pro Ser Ser Gly Tyr Leu Leu

530 535 540

Arg Gly Met Glu Thr Phe Gln Thr Thr Lys Trp Leu Asp Tyr Val Asn

545 550 555 560

Ile Met Ser Tyr Asp Leu His Gly Ala Trp Asn Asp His Val Gly His

565 570 575

Asn Ala Ala Leu Tyr Asp Thr Gly Leu Asp Ser Glu Leu Ala Gln Trp

580 585 590

Asn Val Tyr Gly Thr Lys Glu Phe Glu Gly Ile Gly Tyr Leu Asn Thr

595 600 605

Asp Trp Ala Val Thr Tyr Phe Arg Gly Gly Leu Ser Ala Gly Arg Ile

610 615 620

Asn Ile Gly Val Pro Tyr Tyr Thr Arg Gly Pro Lys Asp Val Ser Gly

625 630 635 640

Gly Glu Asn Gly Leu Trp Gly Arg Ala Ala Leu Pro Asn Gln Ala Asp

645 650 655

Cys Ala Lys Gly Thr Gly Val Gly Glu Lys Asn Lys Cys Gly Asn Gly

660 665 670

Ser Val Gly Ile Asp Asn Met Trp His Asp Leu Asp Glu Asn Gly Asn

675 680 685

Thr Met Pro Ala Gly Ser Asn Pro Leu Trp His Val Lys Asn Leu Gln

690 695 700

Asn Gly Ile Leu Pro Ser Tyr Leu Ala Ala Tyr Gly Leu Asp Pro Ala

705 710 715 720

Asn Asp Pro Thr Asp Lys Leu Val Gly Thr Tyr Thr Arg His Tyr Asp

725 730 735

Ser Val Ala Val Ala Pro Trp Leu Trp Asn Ala Asp Lys Asn Val Phe

740 745 750

Leu Ser Ile Glu Asp Glu Glu Ser Met Gly Thr Lys Leu Asp Tyr Val

755 760 765

Ile Asn Lys Gly Leu Gly Gly Ile Met Phe Trp Glu Leu Ala Gly Asp

770 775 780

Phe Asp Tyr Asp Ser Ala Lys Lys Glu Tyr Phe Met Gly Ser Thr Met

785 790 795 800

Thr Ser Leu Ala Tyr Asn Lys Phe Ala Gln Ala Gly Ser Lys Tyr Asp

805 810 815

Ile Asn Lys Gly Glu Lys Thr His Gln Val Pro Ala Glu Ala Val Asp

820 825 830

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

835 840 845

Ile Ala Pro Thr Phe Ser Phe Thr Asn Asn Ser Asn Ile Asp Leu Ser

850 855 860

Gly Ala Lys Val Ser Phe Asp Val Pro Val Ala Thr Ser Ala Ile Phe

865 870 875 880

Lys Ser Asn Trp Asn Ala Gln Glu Lys Leu Gly Met Ala Val Asp Val

885 890 895

Asn Arg Ser Asn Ala Ala Gly Asn Asn Ile Gly Gly Phe Glu Asn Glu

900 905 910

Phe His Arg Phe Ser Ile Thr Phe Val Asn Glu Trp Gly Gly Ala Ala

915 920 925

Lys Ser Phe Ala Pro Gly Glu Thr Ile Asn Ala Gln Val Met Tyr Tyr

930 935 940

Met Pro Ile Thr Gly Pro Val Asn Leu Val Phe Glu Lys Asp Gly Lys

945 950 955 960

Gln Tyr Ala Ala Lys Phe Glu Tyr Pro Tyr Leu Pro Asp Ala Thr Ala

965 970 975

Gly Asn Gly Gly Gly Asn Asp Gly Gly Gly Asn Thr Asp Thr Cys Glu

980 985 990

Gly Val Ala Val Ala Asp Ile Pro Val Tyr Pro Asn Phe Pro Gln Thr

995 1000 1005

Asp Trp Ala Gly Asn Pro Ser His Ala Ala Gly Gly Asp Leu Met

1010 1015 1020

Val Asp Gly Ala Ser Thr Ala Val Tyr Lys Ala Lys Trp Trp Thr

1025 1030 1035

Ser Thr Glu Pro Gly Thr Ser Ala Asp Trp Asp Ser Val Cys Thr

1040 1045 1050

Leu

<210> 4

<211> 3165

<212> DNA

<213> Pseudoalteromonas sp.

<400> 4

atgagtaaca aattagtagg caagcgttta tttaaaatga gcgtgttgtc gttagctgta 60

actgggtggg cagcccatgc agcagtggac tgtacaaaca ttgccgaatg gcaatcaggc 120

gtggcctaca ctggcgggca gcaagtgcaa aaagccaata ctgcttatga ggcgaagtgg 180

tggacacaag ccgatccagt aacccattca gggcaatatc aagagtggaa aaacttaggt 240

gcgtgtagtg atggtcaagt aaacgaatca cctattgcag aggttttatc acctatgagc 300

ggtagttctt ttactgaagg tgacagcgtt gtcattgagg cgcaaacatc agattctgac 360

ggcaccgttg tgaaactaga agtactggtt gatggtgtgg cgattgcaac actaagctct 420

gcgccatatt caacaacttg gcttgcagtg acagggcagc atcaaatttc ggtacgcgct 480

accgatgata aaggtgctga aacaacatcc cctgcaaata gtgtgactgt ggctgaagta 540

gtgccagata actatgcacc aacggcaagc cttactagca atgcgagttc aaataccttg 600

aacttaaatg acaacgttgt cttaaatttg agcggtagcg atcaagatgg cacagtaaca 660

agtcttgaat tgtttcaaga tggcgcgtct gtagcccaaa tttctggcgc aacaggcagt 720

tttaatttta ccgcaacaag cgcaggtaat acgcaattta gtcttgttgc aaccgacgac 780

aaaggcgcaa catctgatgc acaaacatta agtttccgta ttgttgatgg cactacacaa 840

ccgggtgacc gcgatgcctg taagccggaa ggtttgtacc aaacaccggg tgtaaatacg 900

ccgtattgta ctatttacga tgctgatggc cgtgaagata tgggtgccga tcacccacgt 960

cgtgtcattg gttatttcac cagttggcgc aacggtgcca atggccaacc gtcttaccta 1020

gtagatgata ttccttggga taaaatcacc catattaact acgcgtttgc ccatgttgat 1080

gccaataaca aggtttcaat tggcgatcca agtgcggcgg gtaaccctgc taccaatatg 1140

gaatggccag gtgtagcagg tgctgaaatg gacccaacac tgccgtacaa aggtcatttc 1200

aacttactga ataaatataa aaagctccat ccagacgtta aaacactgat ctctgtaggt 1260

ggttgggcag aaacgggcgg ttactttgac gcaaacggca accgtgttgc aagcggtggt 1320

ttctacacta tgacaaccaa tgctgatggc tcagtaaata ctgcgggcat taatgcgttt 1380

gcaaaaagtg cggttgagtt tattgaaacg tacggtttcg atggggttga tattgattat 1440

gaatacccat catcaatgaa tgattcgggt caccctgatg atttccctat ttcaaatgca 1500

ctccgagcag ggctaaatgc ctcttaccaa gtgttaatga aaaccctacg tgaagagctt 1560

gataaagcgg gtgaattagc tggtaaacac tacatgctta ccattgcatc gccgtcatca 1620

ggttacttat tacgtggtat ggaaactttc caaacaacta agtggctaga ttacgtaaac 1680

attatgtctt acgacttgca cggtgcttgg aacgaccacg tgggtcataa cgcagcgctt 1740

tacgatacag ggttagactc tgagcttgcg cagtggaatg tgtatggcac caaagagttt 1800

gaaggcattg gctatttaaa taccgattgg gcggttacgt atttccgcgg tggtttaagc 1860

gcaggtcgca tcaacattgg tgtgccgtac tatacccgtg gtcctaaaga tgtatcgggt 1920

ggcgaaaacg gtttatgggg gcgcgctgcg ttacctaacc aagctgattg tgccaaaggt 1980

actggcgtag gtgagaaaaa caaatgtggt aatggctcag ttggtatcga caacatgtgg 2040

catgaccttg acgaaaacgg taatacaatg cctgcaggta gcaatccgtt atggcatgtt 2100

aaaaacctac aaaatggcat tttaccaagt taccttgcgg cttacggctt agatccagca 2160

aatgatccaa ctgataagct agtgggcact tacactcgtc attacgattc agtggcagta 2220

gcgccgtggc tttggaatgc tgataaaaac gtgttcttgt cgattgaaga tgaagagtca 2280

atgggcacta agcttgatta cgtaatcaac aaaggccttg gcggtattat gttctgggag 2340

ctggcaggtg actttgacta cgacagcgcg aagaaagagt actttatggg ttctactatg 2400

acctcgcttg cttacaataa gtttgcacaa gcgggttcta agtacgacat caacaaaggt 2460

gaaaaaactc accaagtacc agcagaggca gttgacgtaa gctttgaagt gaaagacttc 2520

ccaattggtg acgacaacta cccaattgcg ccaaccttta gttttactaa caactcaaac 2580

attgatttga gcggtgcaaa agtatcgttt gatgtaccag tggctacatc ggcaattttc 2640

aaatcaaact ggaatgcgca agaaaaactc ggcatggcag ttgacgtaaa tcgttcaaac 2700

gcagctggca acaacattgg tggctttgaa aacgagttcc accgtttctc aattaccttt 2760

gttaacgagt ggggcggcgc tgctaagtcg tttgcgccgg gcgaaaccat taatgcgcaa 2820

gtaatgtact acatgccaat tactggccca gtgaaccttg tatttgaaaa agacggtaag 2880

caatacgccg ctaaatttga atacccatat ctaccagatg caacggccgg taacggtggt 2940

ggcaatgatg gcggcggtaa caccgacaca tgtgaaggtg ttgcagtagc tgatattcca 3000

gtttatccaa acttcccaca aacagattgg gcaggcaacc cgtcacacgc tgcaggcggt 3060

gacttgatgg ttgatggcgc atctactgcg gtttacaaag ccaagtggtg gacatcaact 3120

gagcctggca catccgccga ttgggatagt gtgtgtactt tataa 3165

Claims (7)

1. a kind of composition contains a kind of enzyme, which is characterized in that the enzyme has the activity of chitinase, the chitin The activity of enzyme provides the function of degradation in chitin degrading, and the chitinase has low temperature resistant attribute, and the enzyme is (a) or protein (b) as follows:

(a) shown in the SEQ ID NO:1 compared with amino acid sequence, there is one or more following mutation, described is prominent Change is selected from: the 501st Arg sports Leu, and the 555th Tyr sports Trp, and the 635th Phe sports Pro, the 673 Ala sport Ser, and the 689th Glu sports Thr；

(b) its amino acid sequence is as shown in SEQ ID NO:3.

2. a kind of DNA molecular, which is characterized in that the DNA molecular encodes enzyme as described in the appended claim 1.

3. DNA molecular as claimed in claim 2, it is characterised in that the sequence of the DNA molecular is as shown in SEQ ID NO:4.

4. a kind of recombinant vector, which is characterized in that its contain DNA molecular described in claim 2 or 3 and with the DNA molecular The adjusting sequence for expression of connection.

5. host cell, which is characterized in that the host cell contains DNA molecular described in claim 2 or 3 or claim Recombinant vector described in 4.

6. host cell described in claim 5, wherein host cell is specially bacterial cell, preferably Gram-negative Bacterium；More preferably Escherichia coli.

7. composition described in claim 1, the described in any item DNA moleculars of claim 2-3, load as claimed in claim 4 The application of body and the described in any item host cells of claim 5-6 in hydrolysis chitin.