CN1181199C - Lichenized bacillus L-25 keratinase and its encoding DNA - Google Patents

Abstract

The present invention relates to L-25 keratinase of bacillus licheniformis, DNA for encoding the keratinase, a carrier derived from the DNA, a host cell transformed by the carrier, an animal feed product containing the keratinase and a skin coating pharmaceutical product containing the keratinase. The keratinase has good hydrolyzation activity, and the conditions for producing the keratinase are flexible; the present invention overcomes the inhibiting effects on keratinase production from catabolic repression agents.

Description

The DNA of Bacillus licheniformis L-25 M-Zyme and this M-Zyme of encoding

The present invention relates to a kind of M-Zyme of Bacillus licheniformis, particularly a kind of Bacillus licheniformis L-25 M-Zyme also relates to the DNA of this M-Zyme of encoding simultaneously.

Chinese patent 951940678.8 discloses a kind of DNA of the Bacillus licheniformis PWD-1 M-Zyme of encoding, this M-Zyme can be used for the hydrolysis feather, but, the hydrolytic activity of this M-Zyme is difficult to satisfactory, the fermentation production process of also finding this M-Zyme in addition is subjected to some conditionalities, cause its industrial output to be affected, for example, carbon sources such as glucose (common catabolic repression agent) are inhibited to the biosynthesizing of PWD-1 bacterial strain extracellular enzyme, thisly check the fermentative production that effect can have a strong impact on this M-Zyme, relevant report referring to, Weickert M.J. and ChamblissG.H., Genetic analysis of the promoter region of the Bacillus subtilis alpha-amylase gene. J.bacteriol, 171,3656-3666 (1989); Fujita Y. and Fujita T., Indentification and nucleotide sequence of the promoter region of theBacillus subtilis gluconate operon.Nucleic Acids Res.14,1237-1252 (1986); And Lin X. etc., Expression of Bacillus licheniformis PWD-1 gene in B. subtilis.J.Industrial Microbiol Biotechnol.19,134-138 (1997).

The objective of the invention is to overcome weak point of the prior art, provide a kind of still can be under the effect of catabolic repression agent by its bacterial strain biosynthesizing and the higher lichenoid form bacillus L-25 M-Zyme of hydrolytic activity in fermentative production, further purpose of the present invention is to provide a kind of DNA of this L-25 M-Zyme of encoding, the present invention also aims to provide a kind of, by this carrier transformed host cells and contain the animal-feed goods of this M-Zyme and skin coating medicament by this DNA deutero-carrier.

The objective of the invention is to realize by following approach.

Bacillus licheniformis L-25 M-Zyme, it is the M-Zyme of the purifying of aminoacid sequence shown in a kind of SEQ of having ID NO:1.

See " industrial microorganism and biotechnology (J.IndustrialMicrobiol ﹠amp for details about Bacillus licheniformis L-25; Biotechnol.) magazine " " bacterium of screening and sign degradation of feather by using from rapeseed cake compost (canolameal compost) " and the ATCC (American Type Culture Collecti) shown in the annex 1 in Augusts in 1999 23 volumes.In this piece paper, L-25 has been described in detail to Bacillus licheniformis, what comprise the evaluation of source, method for screening, feather degraded character of soil and growth and proteolytic activity influences temperature etc., and can know that from annex 1 this L-25 bacterial strain live body is stored in American Type Culture Collecti (ATCC).

From Bacillus licheniformis L-25, can adopt conventional way to separate and the purification M-Zyme.Certainly, also can adopt genetic engineering technique to prepare M-Zyme.

The analysis of Keratin sulfate hydrolytic activity, by the azo Keratin sulfate, a kind of keratin derivatives is measured.The keratic preparation of azo is the method for preparing azoalbumin that adopts Tomarelli etc. to describe, with the feather meal that grinds and sulfuric acid, Sodium Nitrite (NaNO ₂) react and (see: Tomarelli, RM., Charney J., with Harding M.L., The use of azoalbumin as a substrate in thecolormetric determination of peptic and tryptic activity, J.Lab, Clin, Med., 34,428-433 (1949)).Under 50 ℃, analyzing the Keratin sulfate hydrolytic activity with 5 milligrams of azo Keratin sulfate (sees: Lin X., Lee C.-G., Casale E.S., and Shih, J.C.H., Purification andcharacterization of a keratinase from a feather-degrading Bacilluslicheniformis strain.Appl.Environ Microbiol., 58,3271-3275 (1992)).In order to improve comparability, the Keratin sulfate hydrolytic activity of 1 unit is defined as 50 ℃ of following per minutes makes that light absorption value raises 0.01 under the 450nm.

The L-25 M-Zyme can produce in the feather substratum and the hydrolysis feather.Under 37 ℃, at 60-72 hour incubation time, the L-25 M-Zyme produced the highest Keratin sulfate hydrolytic activity, just can be fully with the feather hydrolysis at 48-60 hour.And L-25 can produce the M-Zyme (see figure 4) in rapeseed cake or soybean medium.

With reference to Fig. 4, under 37 ℃, bacillus licheniformis L-25 is at three kinds of substratum---the Keratin sulfate hydrolytic activity that produces in feather, rapeseed cake, the bean powder and the relation of incubation time.Sample is taken from 100 milliliters the flask that contains corresponding substratum.The condition determination of M-Zyme hydrolytic activity: 50 ℃, 15 minutes, 0.2 milliliter of application of sample.

With reference to Fig. 5, compare L-25 and PWD-1 and produced the Keratin sulfate hydrolytic activity, the Keratin sulfate hydrolytic activity of L-25 is higher by 27% than PWD-1's.

With reference to Fig. 6, when adding 1% glucose in the feather substratum, the M-Zyme of PWD-1 does not have proteolytic activity to produce at 37 ℃, and the feather in the substratum is kept perfectly.L-25 is then opposite, has added the feather substratum and comparing of not adding of 1% glucose, produces 67% Keratin sulfate hydrolytic activity, 37 ℃, cultivates after 4 days the feather complete hydrolysis.

The DNA of coding M-Zyme of the purifying of aminoacid sequence shown in SEQ ID NO:1.

Can be specially again

The DNA of coding M-Zyme of the purifying of aminoacid sequence shown in SEQ ID NO:1, it has the nucleotide sequence shown in the SEQ ID NO:2.

From L-25 strains separation M-Zyme gene (kerB)

Under 50 ℃, with the nutrient broth overnight incubation of L-25 bacterial strain with 50 milliliters, use standard method (to see: Ausubel F.M. then, Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A., Struhl K., Preparation and analysis of DNA.In " Currentprotocols in molecular cloning. " vol.1.John Wiley ﹠amp; Sons, Inc., New York, 1987) extract genomic dna.Design two primer (primer 1:5 '-CTCCTGCCAAGCTGAAGC-3 '; Primer 2: 5 '-GATCATGGAACGGTTC-3 '), carry out the PCR reaction as template with the L-25 genomic dna.The M-Zyme gene of amplification is directly cloned a cloning vector PCRII (Invitrogen Co.San Diego.Calif).This genomic dna is checked order.(Promega Co.Madison WI), carries out sequencing reaction, analyzes the nucleotide sequence of kerB for M13 reverse primer (17 bases) that insert use universal primer, side and T7 promoter primer (20 bases).Prepared a pair of inner primer (primer 3:5 '-CTGAATTCAAGCGG-3 ' and primer 4:5 '-CGATGGCAGCGATTCC-3 ') to finish the sequential analysis of kerB.

By common way, above-mentioned sequencing reaction is independently carried out respectively more than three times, with checking result's consistence.

Like this, obtained the sequence of the DNA of coding Bacillus licheniformis L-25 M-Zyme.

L-25 M-Zyme gene and PWD-1 M-Zyme gene are compared:

Above-mentioned sequencing result shows that kerB and Bacillus licheniformis PWD-1 M-Zyme gene (kerA) have high homology.L-25 has two amino different on the aminoacid sequence of inferring with the PWD-1 M-Zyme.And find that the upstream of inferring promotor has several Nucleotide that change has taken place.

Except that above-mentioned nucleotide sequence

Obviously, those encode L-25 M-Zyme of the present invention but since the degeneracy of genetic code and on the codon sequence dna sequence dna (or oligonucleotide) different with above-mentioned nucleotide sequence also be one aspect of the present invention, the different nucleotide sequence coded genetic code degeneracy phenomenon with a kind of protein or peptide of this permission extensively sees in the document;

In addition, those encode L-25 M-Zyme of the present invention but since site-directed mutagenesis and on the codon sequence dna sequence dna (or oligonucleotide) different with above-mentioned nucleotide sequence be of the present invention again the one side, equally, the side-directed mutagenesis that is used to improve the M-Zyme characteristic also is well-known.

A kind of duplicating and expression vector, it contains the DNA of M-Zyme of the purifying of the aminoacid sequence of coding shown in SEQ ID NO:1.

This duplicating with expression vector can be specially again

It contains the DNA of M-Zyme of the purifying of the aminoacid sequence of coding shown in SEQ ID NO:1, and this DNA has the nucleotide sequence shown in the SEQ ID NO:2.

Carrier is a kind of reproducible DNA construct.It can be used for the gene of amplification coding L-25 M-Zyme, also can be used for expressing the gene of coding L-25 M-Zyme, also can be used for enlarging and produce and the gene of clone L-25 M-Zyme, but no matter be how to use, the carrier of any L-25 of carrying M-Zyme gene all possesses the essential property of this M-Zyme.

A kind of host cell, it is duplicated and expression vector transforms or transfection by a kind of, and this carrier contains the DNA of M-Zyme of the purifying of the aminoacid sequence of coding shown in SEQ ID NO:1.

This host cell can further be specially

It is duplicated and expression vector transforms or transfection by a kind of, and this carrier contains the DNA of M-Zyme of the purifying of the aminoacid sequence of coding shown in SEQ ID NO:1, and this DNA has the nucleotide sequence shown in the SEQ ID NO:2.

Transformed host cells is normally expressed the L-25 M-Zyme, but in order to clone or the gene of the L-25 M-Zyme that increases and transformed host cells need not the express keratin enzyme.Proper host cell comprises those known host cell concerning those skilled in the art, for example prokaryotic organism host cells.

A kind of animal-feed, it forms main points is that it contains the Bacillus licheniformis L-25 M-Zyme just like aminoacid sequence shown in the SEQ ID NO:1.

The production bacterial strain Bacillus licheniformis L-25 of M-Zyme has following service performance: (1) can produce the M-Zyme of degradation of feather by using under the situation that catabolic repression agent (as: glucose, peptide and amino acid) exists.Have substrate scope and the flexible growth conditions that produces M-Zyme widely.Owing to have these characteristics, this M-Zyme can make an addition in the feed and directly use.(2) because the L-25 bacterial strain can produce described M-Zyme with normal diet (as: rapeseed cake, soyflour etc.) as fermented material.Therefore can prepare M-Zyme product, directly add in the animal-feed, need not further separation in conjunction with specific digestion feed.In addition, feather or other the Keratin sulfate composition of this M-Zyme in can the hydrolysis of animal feed absorbs thereby help animal digestion.

Obviously, the above-mentioned this finished product that the L-25 M-Zyme is directly applied to the method in the animal-feed and obtained have outstanding characteristics and obvious improvement.

A kind of skin coating medicament, its composition main points are: it contains the Bacillus licheniformis L-25 M-Zyme just like aminoacid sequence shown in the SEQ ID NO:1.

The stratum corneum in the skin affected part chronic dermatosis of the scales of skin that peel off (for example based on) is thicker usually, the dermal agent that is coated in epidermis must permeate this stratum corneum could allow skin absorption, thicker stratum corneum will produce very big resistance and directly influence administering effect this infiltration, L-25 M-Zyme of the present invention adds in the skin coating medicament, utilize its good hydrolysis properties attenuate or remove this stratum corneum, thereby ancillary drug is penetrated into the infected area preferably, improves administering effect.And do not contain the Keratin sulfate composition in the general dermal agent, therefore this M-Zyme is advanced in interpolation can not influence the pharmaceutical cpd formation.

In sum, the present invention has following advantage compared to existing technology: have high Keratin sulfate hydrolytic activity, and under the situation that metabolism repressor (as glucose, peptide and amino acid) exists, produce M-Zyme and keep good hydrolytic activity, and these characteristics are that a feather degraded Bacillus licheniformis PWD-1 who is widely known by the people is not available.Certainly because of in this way, L-25 M-Zyme of the present invention is compared the PWD-1 M-Zyme at the hydrolysis feather and further make it change into aspect such as digestible protein to have better industrial application value.

Accompanying drawing 1 is the dna sequence dna figure of coding L-25 M-Zyme.

Accompanying drawing 2 is comparison diagrams of L-25 M-Zyme gene order and PWD-1 M-Zyme gene order.

Accompanying drawing 3 is that KerB clones the into synoptic diagram of plasmid.

Accompanying drawing 4 is that Bacillus licheniformis L-25 is in three kinds of substratum----feather, rapeseed cake, the Keratin sulfate hydrolytic activity of soyflour and the relation of incubation time.

Accompanying drawing 5 is Keratin sulfate hydrolytic activity and time relation that Bacillus licheniformis L-25 and PWD-1 produce in simple feather substratum.

Accompanying drawing 6 is Keratin sulfate hydrolytic activity and time relation that lichenoid form bud sub bar bacterium L-25 and PWD-1 produce in containing the feather substratum of 1% glucose.

Below in conjunction with accompanying drawing the present invention is carried out more detailed description.

Most preferred embodiment:

The acquisition of L-25 M-Zyme:

From Bacillus licheniformis L-25 bacterial strain with separation, the method for purification purification L-25 M-Zyme of routine.

Certainly prepare by under the condition that allows coded M-Zyme to express, cultivating host cell and collecting expressed M-Zyme.M-Zyme can be fused on the suitable secretion leader sequence and express and enter substratum and collect from substratum, or M-Zyme also can express in the born of the same parents, dissolves cell then and collection angle proteolytic enzyme from the molten product of born of the same parents.

From L-25 strains separation M-Zyme gene (kerB):

Genomic dna adopts the standard procedure extracting

The L-25 bacterial strain is cultivated in 100 milliliters of nutritious soup, and 37 ℃, shake training (200rpm), spend the night.Bacterial cell passes through 4000 * g, and 10 minutes, centrifugal collection, resuspending is in 9.5 milliliters of TE damping fluids that contain 30 lysozyme ml solution, ice bath 10 minutes.Then, with Proteinase K (20mg/ml) the adding solution of 0.5 milliliter of 10%SDS and 50 microlitres, 37 ℃ are incubated 1 hour.Subsequently, 1.8 milliliters 5M NaCl and 1.5 milliliters CTAB/NaCl solution (the 0.7M NaCl solution of 10%CTAB) are added sample, and thoroughly mixed.65C is incubated 20 minutes, adds the thorough extracting of equal-volume chloroform/Virahol, centrifugal then 10 minutes.Above water transfer to a new centrifuge tube, and with twice of isopyknic phenol/chloroform/Virahol extracting.The isopropanol precipitating DNA that adds 0.6 volume, 4C, centrifugal 15 minutes.Separated DNA is dissolved in TE or deionized water, is stored in-70 ℃.Final DNA concentration 260nm spectrophotometric determination.

Pcr amplification

Prepare two primers:

Primer 1:5 '-CTCCTGCCAAGCTGAAGC-3 ';

Primer 2: 5 '-GATCATGGAACGGTTC-3 '.

1.46kb M-Zyme gene (kerB) fragment excite with primer 1 and primer 2, carry out pcr amplification reaction with the L-25 genomic dna as template.

KerB is checked order

With the M-Zyme gene of amplification directly the clone advance a cloning vector PCRII (InvitrogenCo.San Diego, Calif).(Promega Co.Madison WI), is used to analyze the kerB sequence for side insertion M13 reverse primer (17 bases) and T7 promoter primer (20 bases).The nucleotide sequence analysis of KerB adopts universal primer, M13 reverse primer and T7 promotor to carry out sequencing reaction.Prepared a pair of inner primer:

Primer 3:5 '-CTGAATTCAAGCGG-3 ';

Primer 4:5 '-CGATGGCAGCGATTCC-3 '

To finish the sequential analysis of kerB.

Determining of final kerB sequence by sequencing reaction independently more than 3.

The sequence of the DNA of the coding L-25 M-Zyme that obtains compares it and well-known Bacillus licheniformis PWD-1 M-Zyme gene (kerA) as shown in Figure 1, as shown in Figure 2, finds:

L-25 M-Zyme gene (kerB) has high similarity with PWD-1 M-Zyme gene (kerA).The coding region of KerB has only two amino acid different with the residue of kerA, and these two amino acid are (Leu-15 in precursor sequence district, the Val-273 of maturing enzyme).The change of the C-of Phe-15 → Leu-15 and maturing enzyme end Ala-273 → Val-273 does not change the hydrophobicity of this position in the precursor sequence district.Two variations all only are that change has taken place a Nucleotide in the codon: TTC (Phe) → TTA (Leu), GCT (Ala) → GTT (Val).Significant change has taken place in the upstream of inferring promotor in two keratin genes, and 70 Nucleotide have 8 differences (accompanying drawing 2).

Infer that the change of promotor upstream region oligonucleotide can change the binding affinity of some modulin, as: negative regulation albumen Hpr and Sin.Other breadboard result has confirmed that the promotor of subtilis gene (aprE) and neutral protease gene (nprE) and upstream region thereof are the target sequences of Hrp and Sin.

About carrier and host cell:

As shown in Figure 3, with the M-Zyme gene among the XbaI-SpeI digestion cutting-out PCRII, insert the XbaI site of plasmid pUB18-P43, thereby obtain a kind of carrier of DNA of M-Zyme of the purifying that contains the aminoacid sequence of coding shown in SEQ ID NO:1.

Operating process according to Dubnau and Gryezan description, plasmid is as a result transformed into protease-deficient subtilis DB104 competent cell, and this cell is a kind of host cell of DNA of M-Zyme of the purifying that contains the aminoacid sequence of coding shown in SEQID NO:1.(see: Dubnau D. and Davidoff-Abelson R., Fate of transforming DNA followinguptake by competent Bacillus subtilis.J.Mol.Biol., 56,209-221 (1971); Gryezan T.J. etc., Characterization of Staphylococcus aureus plasmidsintroduced by transformation into Bacillus subtilis.J.Bacteriol., 134,318-329 (1978)).

Method is: at first, (tryptose blood agar base, TBAB) the screening transformed bacteria falls on the culture dish, uses the dull and stereotyped cultivation of milk-nutrient agar again, further identifies at the Tryptones blood agar culture-medium that contains kantlex (10 μ g/ml).Selection has the bacterium colony of transparent circle to be used for plasmid separation and pcr analysis.The size of plasmid pLK-8 is 5.5kb as a result.As template, use primer 1 and primer 2 with pLK8, the kerB segment of pcr amplification 1.46kb has verified that kerB has changed new plasmid over to and existed in new carrier.

Transforming as a result the DB104 bacterial strain FD-8 (DB104/pLK-8) of plasmid pLK-8 cultivates at the feather substratum and (contains that 10 μ g potassium/ml), the ramp of FD-8 cell is with the thorough hydrolysis of all feathers in the substratum.And in the unconverted blank DB104 substratum that advances plasmid pLK-8 as a result, do not have the hydrolysis of feather to take place, and to cultivate after 5 days, medium still keeps clarifying.

A kind of animal-feed, it contains the Bacillus licheniformis L-25 M-Zyme just like aminoacid sequence shown in the SEQ ID NO:1.

A kind of skin coating medicament, it contains the Bacillus licheniformis L-25 M-Zyme just like aminoacid sequence shown in the SEQ ID NO:1.

Sequence table

(1) general information

1. applicant: Fujian Fudabaite Sci-Tech Devpt Co., Ltd..

2. denomination of invention: the DNA of Bacillus licheniformis L-25 M-Zyme and this M-Zyme of encoding.

3. sequence quantity: 2.

4. contact address: country origin: China

Economize: Fujian

City: Fuzhou City

Address: No. 6, energy lane, Gutian road

Postcode: 350005

5. proxy's information: name: Lin Tiankai

Act on behalf of the testimony of a witness number: 35201003

6. telecom information: phone: 0591-3364424

Fax: 0591-3363304

(2) information of SEQ ID NO:1

1. sequence signature:

(1) length: 274 amino acid

(2) type: amino acid

(3) topology: linearity

2. molecule type: protein

3. false the plan:

4. primary source:

(1) organism: Bacillus licheniformis

(2) bacterial strain: L-25

5. sequence description: SEQ ID NO:1

Met?Met?Arg?Lys?Lys?Ser?Phe?Trp?Leu?Gly?Met?Leu?Thr?Ala?Leu?Met?Leu?Val

1 5 10 15

Phe?Thr?Met?Ala?Phe?Ser?Asp?Ser?Ala?Ser?Ala?Ala?Gln?Pro?Ala?Lys?Asn?Val

20 25 30 35

Glu?Lys?Asp?Tyr?Ile?Val?Gly?Phe?Lys?Ser?Gly?Val?Lys?Thr?Ala?Ser?Val?Lys

40 45 50

Lys?Asp?Val?Ile?Lys?Glu?Ser?Gly?Gly?Lys?Val?Asp?Lys?Gln?Phe?Arg?Ile?Ile

55 60 65 70

Asn?Ala?Ala?Lys?Ala?Lys?Leu?Asp?Lys?Glu?Ala?Leu?Lys?Glu?Val?Lys?Asn?Asp

75 80 85 90

Pro?Asp?Val?Ala?Tyr?Val?Glu?Glu?Asp?His?Val?Ala?His?Ala?Leu?Ala?Gln?Thr

95 100 105

Val?Pro?Tyr?Gly?Ile?Pro?Leu?Ile?Lys?Ala?Asp?Lys?Val?Gln?Ala?Gln?Gly?Phe?Lys

110 115 120 125

Gly?Ala?Asn?Val?Lys?Val?Ala?Val?Leu?Asp?Thr?Gly?Ile?Gln?Ala?Ser?His?Pro?Asp

130 135 140 145

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

150 155 160 165

Asn?Gly?His?Gly?Thr?His?Val?Ala?Gly?Thr?Val?Ala?Ala?Leu?Asp?Asn?Thr?Thr?Gly

170 175 180

Val?Leu?Gly?Val?Ala?Pro?Ser?Val?Ser?Leu?Tyr?Ala?Val?Lys?Val?Leu?Asn?Ser?Ser

185 190 195 200

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

205 210 215 220

Met?Asp?Val?Ile?Asn?Met?Ser?Leu?Gly?Gly?Ala?Ser?Gly?Ser?Thr?Ala?Met?Lys?Gln

225 230 235 240

Ala?Val?Asp?Asn?Ala?Tyr?Ala?Arg?Gly?Val?Val?Val?Val?Ala?Ala?Ala?Gly?Asn?Ser

245 250 255 260

Gly?Ser?Ser?Gly?Asn?Thr?Asn?Thr?Ile?Gly?Tyr?Pro?Val?Lys

265 270 274

(3) information of SEQ ID NO:2

1. sequence signature:

(1) length: 1460 base pairs

(2) type: nucleic acid

(3) chain: strand

(4) topology: linearity

2. molecule type: DNA (genomic)

3. false plan the: do not have

4. antisense: do not have

5. primary source:

(1) organism: Bacillus licheniformis

(2) bacterial strain: L-25

4. sequence description: SEQ ID NO:2

CTCCTGCCAA?GCTGAAGCGG?TCTATTCATA?CTTTCGAACC?GAATATTTTT?CTAAAACAGT 60

TATTAATAAC?CAATAAATTT?AAATTGGCCG?TTCAAAAAAA?TGGGTCTACC?ATATAATTCA 120

TTTTTTTTCT?ATAATAAATT?AACAGAATAA?TTGGAATAGA?TTATATTATC?CTTCTATTTA 180

AATTATTCTG?AATAAAGAGG?AGGAGAATAA?GTAATGATGA?GGAAAAAGAG?TTTTTGGCTT 240

GGGATGCTGA?CGGCCTTAAT?GCTCGTGTTC?ACGATGGCAT?TCAGCGATTC?CGCTTCTGCT 300

GCTCAACCGG?CGAAAAATGT?TGAAAAGGAT?TATATTGTCG?GATTTAAGTC?AGGAGTGAAA 360

ACCGCATCTG?TCAAAAAGGA?CATCATCAAA?GAGAGCGGCG?GAAAAGTGGA?CAAGCAGTTT 420

AGAATCATCA?ACGCGGCAAA?AGCGAAGCTA?GACAAAGAAG?CGCTTAAGGA?AGTCAAAAAT 480

GATCCGGATG?TCGCTTATGT?GGAAGAGGAT?CATGTGGCCC?ATGCCTTGGC?GCAAACCGTT 540

CCTTACGGCA?TTCCTCTCAT?TAAAGCGGAC?AAAGTGCAGG?CTCAAGGCTT?TAAGGGAGCG 600

AATGTAAAAG?TAGCCGTCCT?GGATACAGGA?ATCCAAGCTT?CTCATCCGGA?CTTGAACGTA 660

GTCGGCGGAG?CAAGCTTTGT?GGCTGGCCAA?GCTTATAACA?CCGACGGCAA?CGGACACGGC 720

ACACATGTTG?CCGGTACAGT?AGCTGCGCTT?GACAATACAA?CGGGTGTATT?AGGCGTTGCG 780

CCAAGCGTAT?CCTTGTACGC?GGTTAAAGTA?CTGAATTCAA?GCGGAAGCGG?ATCATACAGC 840

GGCATTGTAA?GCGGAATCGA?GTGGGCGACA?ACAAACGGCA?TGGATGTTAT?CAATATGAGC 900

CTTGGGGGAG?CATCAGGCTC?GACAGCGATG?AAACAGGCAG?TCGACAATGC?ATATGCAAGA 960

GGGGTTGTCG?TTGTAGCTGC?AGCAGGGAAC?AGCGGATCTT?CAGGAAACAC?GAATACAATT 1020

GGCTATCCTG?CGAAATACGA?TTCTGTCATC?GCTGTTGGCG?CGGTAGACTC?TAACAGCAAC 1080

AGAGCTTCAT?TTTCCAGTGT?GGGAGCAGAG?CTTGAAGTCA?TGGCTCCTGG?CGCAGGCGTA 1140

TACAGCACTT?ACCCAACGAA?CACTTATGCA?ACATTGAACG?GAACGTCAAT?GGCTTCTCCT 1200

CATGTAGCGG?GAGCAGCAGC?TTTGATCTTG?TCAAAACATC?CGAACCTTTC?AGCTTCACAA 1260

GTCCGCAACC?GTCTCTCCAG?CACGGCGACT?TATTTGGGAA?GCTCCTTCTA?CTATGGGAAA 1320

GGTCTGATCA?ATGTCGAAGC?TGCCGTTCAA?TAACATATTC?TAACAAATAG?CATATAGAAA 1380

AAGCTAGTGT?TTTTAGCACT?AGCTTTTTCT?TCATTCTGTT?GAAGACTGTT?CAATATTTTG 1440

AATCCGTTTC?CATGATCAAG 1460

Claims (8)

1. Bacillus licheniformis L-25 M-Zyme is characterized in that, it is the M-Zyme of the purifying of aminoacid sequence shown in a kind of SEQID of having NO:1.

2. the DNA of coding Bacillus licheniformis L-25 M-Zyme is characterized in that, the M-Zyme of its coding purifying of aminoacid sequence shown in SEQ ID NO:1.

3. the DNA of coding Bacillus licheniformis L-25 M-Zyme according to claim 2 is characterized in that it has the nucleotide sequence shown in the SEQ ID NO:2.

4. one kind is duplicated and expression vector, it is characterized in that, it contains the DNA of M-Zyme of the purifying of the aminoacid sequence of coding shown in SEQ ID NO:1.

5. a kind of duplicating and expression vector according to claim 4 is characterized in that this DNA has the nucleotide sequence shown in the SEQ ID NO:2.

6. a host cell is characterized in that, it is duplicated and expression vector transforms or transfection by a kind of, and this carrier contains the DNA of M-Zyme of the purifying of the aminoacid sequence of coding shown in SEQ ID NO:1.

7. a kind of host cell according to claim 6 is characterized in that, this DNA has the nucleotide sequence shown in the SEQ ID NO:2.

8. an animal-feed is characterized in that, it contains the Bacillus licheniformis L-25 M-Zyme just like aminoacid sequence shown in the SEQ ID NO:1.

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