CN105018402A - Bacillus licheniformis capable of producing alkaline protease in large quantity and application of Bacillus licheniformis - Google Patents

Abstract

The invention relates to the technical field of functional microorganism screening, and particularly provides Bacillus licheniformis capable of producing alkaline protease in large quantity. The Bacillus licheniformis is a mutant strain obtained through an ultraviolet mutagenesis method, a collection number of the Bacillus licheniformis is CCTCC NO: M2015449, and flask-shaking fermentation enzyme activity of the Bacillus licheniformis is up to 18000U/ml and is improved by about 54.5% when compared with that of a starting bacterium; after passage for several times, genetic stability can be maintained, unexpected technical effect is realized, the Bacillus licheniformis can be widely applied to production of the alkaline protease, and lowering of production cost and improving of market competitiveness are facilitated.

Description

The Bacillus licheniformis of one strain high yield alkali protein and application thereof

Technical field

The invention belongs to gene engineering technology field, be specifically related to Bacillus licheniformis and the application thereof of a strain high yield alkali protein.

Background technology

Sumizyme MP (alkaline protease) refers to the enzyme of protein hydrolysate peptide bond within the scope of pH value meta-alkalescence, and its main component is a kind of endo-protease, and catalytic site is Serine, and molecular weight is about 27kDa.Mostly the bacterial strain that domestic industry produces proteolytic enzyme is through Bacillus licheniformis2709 seed selection, is by submerged fermentation, extraction and refining a kind of proteolytic ferment.The industries such as Sumizyme MP is widely used in food, medical treatment, brewages, silk, process hides, in industrial enzymes, occupy the maximum enzyme of ratio, account for about 60% (Mala B.R. etc. of the annual total sales volume in the whole world, 1998, Microbiology and Molecular BiologyReviews).

Sumizyme MP is detergent additive salable in the market, can increase substantially the clean effect of washing composition, has unique washing effect especially to protein dirts such as bloodstain, sweat stain, milk stain, oil stains.But at present basic protein enzyme product is generally monopolized by Novi's letter and Jie Neng Ke Liang great zymin international corporation, it has enzyme running water and equals height, dissolution rate soon and the advantage strong with tolerance during washing composition compatibility.Domestic zymin enterprise will expand the market share in detersive enzyme field, except accelerate development is set out except the excellent novel alkali proteinase of zymologic property, also needs the output improving Sumizyme MP, thus reduces production cost.

Summary of the invention

The object of this invention is to provide Bacillus licheniformis and the application thereof of a strain high yield alkali protein.The present invention, first by proceeding in Bacillus licheniformis (Bacillus licheniformis) by the alkaline protease gene deriving from Bacillus clausii (Bacillus clausii), builds the engineering strain obtaining recombinant expressed Sumizyme MP; Then with this project bacterial strain for the bacterium that sets out carries out ultraviolet mutagenesis, and then filter out a strain Sumizyme MP superior strain, for low cost, large-scale production Sumizyme MP are laid a good foundation.

One aspect of the present invention relates to a kind of Bacillus licheniformis engineering bacteria, and it carries the expression vector expressing Sumizyme MP.

The aminoacid sequence of described Sumizyme MP is SEQ ID NO:1, and its coding nucleotide sequence is SEQID NO:2.

The present invention relates to a kind of Bacillus licheniformis mutant strain on the other hand, for Bacillus licheniformis BL1-43 (Bacillus licheniformis BL1-43), be preserved in the China typical culture collection center of Wuhan, China Wuhan University on July 13rd, 2015, deposit number is CCTCC NO:M2015449.

The invention still further relates to the application of above-mentioned Bacillus licheniformis mutant strain.

The present invention obtains the Bacillus licheniformis mutant strain of a strain high yield alkali protein by the method for ultraviolet mutagenesis, its shake-flask fermentation enzyme activity is up to 18000U/ml, the bacterium that comparatively sets out improves about 54.5%, and after repeatedly going down to posterity, the stability in heredity can also be kept, achieve unexpected technique effect, the production of Sumizyme MP can be widely used in, contribute to reducing production cost, improve the market competitiveness.

Accompanying drawing explanation

Fig. 1 is the genetic map of plasmid pWBA-aprE.

Embodiment

Below in conjunction with example, method of the present invention is described further, the experimental technique of unreceipted actual conditions in embodiment, usually can condition routinely, condition as described in " Molecular Cloning: A Laboratory guide " that J. Pehanorm Brooker (Sambrook) etc. is write, or run according to the condition that manufacturer advises.Those skill in the art related can understand better by embodiment and grasp the present invention.But, realize method of the present invention and should not be limited to concrete grammar step described in the embodiment of the present invention.

Term involved in the present invention and related assays method are explained as follows:

1, proteinase activity measuring method: adopt National Standard of the People's Republic of China's protease preparation measuring method (GB/T 25327-2009).

2, the definition of Mei Huo unit: 1g solid enzyme powder (or 1mL liquid enzymes), under certain temperature and pH value condition, 1min caseinhydrolysate produces 1 μ g tyrosine, is 1 enzyme activity unit, represents with U/g (U/mL).

3, Sumizyme MP uses folin's methods to measure the vigor of proteolytic enzyme, the solution used comprises: forint uses solution, and (a commercially available folin solution mixes with two parts of water, shake up), sodium carbonate solution (42.4g/L), trichoroacetic acid(TCA) (65.4g/L), gradient pH value damping fluid, casein solution (10.0g/L).Reaction process is as follows: add 1mL enzyme liquid in test tube, and 40 DEG C of temperature bath 2min, add casein solution 1mL, shakes up rear 40 DEG C of temperature bath 10min, adds 2mL solution of trichloroacetic acid, shake up (blank first adds trichoroacetic acid(TCA), then adds casein solution).Take out static 10min, qualitative filter paper filters at a slow speed.Get 1mL filtrate, add sodium carbonate solution 5mL, add Folin reagent and use solution 1mL, 40 DEG C of colour developing 20min, in 680nm wavelength, measure absorbancy with 10mm cuvette.

Embodiment 1 Sumizyme MP expression vector establishment

The extraction of 1.1 genomic dnas

The TIANamp Bacteria DNA Kit of TIAN GEN company prepares Bacillus clausii genomic dna, the operational manual of its preparation process reference reagent box.

1.2 gene clone

With the genome DNA extracted in 1.1 for masterplate, utilize primer 1:5 '-TTTTAGTTCATCGATCGCATCGGCTGCTGAAGAAGCAAAAGAAAAATATT-3 ' and primer 2: 5 '-GCTGAAGCTAGCTTGCATGCTTAATTTAGCGTGTTGCCGCTTCTGCATTG-3 ' carries out pcr amplification, obtain aprE gene fragment.Amplification condition is 98 DEG C of 10min; 98 DEG C of 10s, 58 DEG C of 20s, 72 DEG C of 1min, 30 circulations; 72 DEG C of 10min.E.Z.N.A.Gel ExtractionKit is utilized to reclaim pcr amplification product.

The extraction of 1.3 plasmids

The TIANrep Rapid Mini Plasmid Kit of TIAN GEN company prepares carrier pWB980, the operational manual of its preparation process reference reagent box.

1.4 expression vector establishment

With the plasmid DNA extracted in 1.3 for template, utilize primer 1:5 '-CAATGCAGAAGCGGCAACACGCTAAATTAAGCATGCAAGCTAGCTTCAGC-3 ' and primer 2: 5 '-AATATTTTTCTTTTGCTTCTTCAGCAGCCGATGCGATCGATGAACTAAAA-3 ' carries out pcr amplification, obtain vector gene sequence.Amplification condition is 98 DEG C of 10min; 98 DEG C of 10s, 58 DEG C of 20s, 72 DEG C of 1min, 30 circulations; 72 DEG C of 10min.E.Z.N.A.Gel ExtractionKit is utilized to reclaim pcr amplification product.

Target fragment and carrier segments are formed polymer by Overlap extension PCR, amplification system is as follows: 5 × Phusion HF Buffer 10 μ L, 2.5mM dNTPs 8 μ L, Insert gene (aprE fragment) 4 μ L, Linearized vector (pWB980) 6 μ L, Phusion DNA Polymerase 1 μ L, ddH₂o 21 μ L.Amplification condition is 98 DEG C of 10min; 98 DEG C of 10s, 72 DEG C of 3min, 20 circulations; 98 DEG C of 10s, 72 DEG C of 6min, 15 circulations; 72 DEG C of 10min.By polymer Transforming B. subtilis (Bacillus subtilis) 1A751 Host Strains, obtain positive transformant, sequence verification.Nucleotide sequence through this aprE fragment that checks order is SEQ ID NO:2, and the aminoacid sequence of its coding is SEQ ID NO:1.Find through NCBI BLAST comparison, the Sumizyme MP amino acid sequence similarity of SEQ ID NO:1 and Bacillus clausii is 100%.By the plasmid called after pWBA-aprE obtained, plasmid map as shown in Figure 1.

Competence method is utilized pWBA-aprE to be transformed into subtilis 1A751 Host Strains, concrete conversion process is as follows: by the subtilis 1A751 of fresh activation by LB flat board (Tryptones 1%, yeast powder 0.5%, NaCl 1%) (GM I compound method is: 1 × minimum salts solution 95.6ml to be inoculated into 5ml GM I, 20% glucose 2.5ml, 5% caseinhydrolysate 0.4ml, 10% yeast powder juice 1ml; Wherein the compound method of 1 × minimum salts solution is: K₂hPO₄14g/L, KH₂pO₄6g/L, (NH₄)₂sO₄2g/L, trisodium citrate 1g/L, MgSO₄7H₂o 0.2g/L, solvent soln successively in distilled water, 30 DEG C, 125rpm shaking culture spends the night.Within second day, getting 2ml is transferred in 18ml GM I, 37 DEG C, 250rpm cultivates 3.5h.The nutrient solution getting 10ml previous step is again transferred to 90ml GM II, and (GM II compound method is: 1 × minimum salts solution 96.98ml, 20% glucose 2.5ml, 5% caseinhydrolysate 0.08ml, 10% yeast powder juice 0.04ml, 1M MgCl₂0.25ml, 1M CaCl₂in 0.05ml, 37 DEG C, 125rpm cultivates after 90min, 5000g, 10min collected by centrifugation thalline.To suspend gently thalline with 10ml original fluid supernatant liquor, the thalline after suspension is competent cell.Then add in 0.5ml competence appropriate DNA in 37 DEG C, coat resistant panel (kantlex of LB+25 μ g/mL) after 200rpm shaking culture 30min, then 37 DEG C of overnight incubation, secondary daily inspection and checking transformant.

The structure of embodiment 2 Sumizyme MP recombinant bacterial strain

Utilize electric method for transformation that the correct pWBA-aprE Plastid transformation of checking is entered host Bacillus licheniformis A37, obtain positive transformant, extract plasmid and carry out digestion verification.

Concrete conversion process is as follows: by Bacillus licheniformis A37 at flat lining out, incubated overnight, picking list colony inoculation in the growth medium (LB+0.5M sorbitol) of 25ml, 220rpm, 37 DEG C, incubated overnight; Get 2.6ml overnight culture to join in 40ml growth medium (LB+0.5M sorbitol), 220rpm, is cultured to OD600=0.9 ~ 1.0 by 37 DEG C.By bacterium liquid ice bath 5min, then 5000g, 10min, 4 DEG C of collected by centrifugation thalline.Turn substratum (0.5M sorbitol, 0.5Mmannitol, 10%glycerol) with the electricity of 25ml ice-water bath precooling, again overhang thalline, 5000g, 5min, 4 DEG C centrifugal removes supernatant, and rinsing like this 3 times, prepared by electric transformed competence colibacillus.In the competent cell of 60 μ l, add 1 μ g plasmid pWBA-aprE, hatch 5min on ice, add in the electric revolving cup (1mm) of precooling, electric shock once.Electroporation is arranged: 1.0kv, 25 μ F, 200 Ω, time constant=4.5 ~ 5.0ms.The complete taking-up cup that shocks by electricity also adds 1ml recovery media (LB+0.5M sorbitol+0.38M mannitol), 37 DEG C, 150rpm immediately, after recovery 3h, and coated plate.37 DEG C, incubated overnight.Secondary daily inspection and checking transformant.

By one of them positive transformant called after Bacillus licheniformis BL1 (Bacilluslicheniformis BL1) of above-mentioned acquisition.

Embodiment 3 Bacillus licheniformis BL1 ultraviolet mutagenesis and screening

The preparation of 3.1 bacteria suspensions

By the beef extract-peptone inclined-plane (extractum carnis 0.5% of Bacillus licheniformis BL1 at the kantlex containing 25 μ g/mL, peptone 1%, NaCl 0.5%, agar 1.5%, pH 7.5) upper streak inoculation, cultivate after 24 hours for 37 DEG C, inoculate the beef extract-peptone liquid nutrient medium (meat extract 0.5% of a single bacterium colony to the kantlex containing 25 μ g/mL, peptone 1%, NaCl 0.5%, pH 7.5), incubated overnight, next day, the beef extract-peptone liquid nutrient medium of the fresh kantlex containing 25 μ g/mL is forwarded to by 1% inoculum size, grow to mid-log phase, 3900rpm collected by centrifugation thalline, outwell supernatant, with sterilized brine once, with granulated glass sphere concussion about 15min, pour centrifuge tube into collect, resuspended with physiological saline, finally adjust cell concn to 10⁸/ mL.

3.2 ultraviolet mutagenesis process and Induced dosage are determined

Open 9W ultraviolet violet light switch, preheating is about 30min.Cut-off footpath 9cm sterilized petri dishes, adding above-mentioned cell concn is 10⁸the bacteria suspension 10mL of/mL, puts into a sterilized magnetic agitator; Opening magnetic stirring apparatus, then open ware lid, is 15cm place in vertical range, stirs respectively and irradiates 30s, 60s, 120s, 180s, 240s; Cover ware lid, close ultraviolet lamp, in dark, hatch 30min.

Postradiation bacteria suspension 0.85% physiological saline, 10 times of dilution method gradient dilutions are become 10^-1~ 10^-6; Get 10^-4, 10^-5, 10^-6three each 100 μ L of dilution bacteria suspension, coating is dull and stereotyped containing the beef extract-peptone of the kantlex of 25 μ g/mL, and each extent of dilution is coated with three flat boards, evenly fills whole planar surface with sterile glass rod; With same operation, the bacterium liquid dilution of getting non-irradiated with ultraviolet radiation is coated with flat board and compares.Above-mentioned coating is dull and stereotyped uniformly, after wrapping with black cloth or newspaper, put 37 DEG C of incubated overnight.

The single colony number under each extent of dilution, flat board grown when adding up different irradiation time, if the single colony number under certain extent of dilution, flat board grown is between 30 ~ 300, then thinks that this extent of dilution is suitable.Single colony number that lower for this extent of dilution three flat boards grow is averaged, by following formulae discovery bacteria suspension concentration:

Bacterium colony mean number × extension rate × 10 under bacteria suspension concentration (CFU/mL)=certain extent of dilution

Lethality rate by under certain ultraviolet treatment dosage of following formulae discovery:

As calculated, under different ultraviolet mutagenesis dosage, the lethality rate of Bacillus licheniformis BL1 is as shown in table 1.

Table 1: ultraviolet mutagenesis lethality rate

As can be seen from the data of table 1, bacteria suspension lethality rate after uv irradiating 30s reaches more than 95%, therefore finally determines that mutation time is 30s.

3.3 caseins dull and stereotyped proteolysis circle primary dcreening operation

After uv irradiating process 30s, viable cell concentrations about 10 in bacteria suspension⁶/ mL; Bacteria suspension 100 μ L after the upper even spread gradient dilution of each beef extract-peptone flat board (containing kantlex 25 μ g/mL); Wrap with black cloth or newspaper, cultivate 24h for 37 DEG C.

Proteolytic enzyme has hydrolysis ability to substrate casein, therefore selects casein flat board to form the primary dcreening operation of method as mutagenic strain of transparent circle.By the single bacterium colony grown after mutagenesis, respectively picking dibbling is at 3 identical casein flat board (casein 0.5%, extractum carnis 0.8%, polyprotein peptone 0.5%, yeast extract 0.2%, NaCl 0.2%, agar 1.5%, K containing 25 μ g/mL kantlex₂hPO₄1.8%, ~ pH 7.5) on, on each flat board, the Bacillus licheniformis of dibbling simultaneously BL1 in contrast, after 37 DEG C of cultivation 24h, in the white background of casein flat board, periphery of bacterial colonies becomes grey, transparent, visible proteolysis circle, measure hydrolysis circle D and lawn d size, the ratio D/d of both calculating size, and get 3 dull and stereotyped mean values.Both are primary dcreening operation bacterial strain by mean ratio D/d the greater, and enrichment isolation is to 60 strain mutagenic mutants altogether, and streak inoculation is in the beef extract-peptone inclined-plane conservation containing 25 μ g/mL kantlex.

3.4 shaking flasks are sieved again

By the 60 strain mutagenic mutants that are enriched to, (Strain Designation is BL1-1, BL1-2, BL1-3 ... BL1-60) (yeast soaks powder 0.5% to be inoculated in 50mL seed culture medium respectively with single bacterium colony of starting strain Bacillus licheniformis BL1, Tryptones 0.5%, glucose 1%, K₂hPO₄1.8%, mycin 25 μ g/mL received by card) in, 34 DEG C, 210rpm shaking culture 8h.Then get 2.5mL fermented liquid respectively and be inoculated into 50mL fermention medium (yeast powder 1 ~ 2%, soybean cake powder 2 ~ 5%, maltodextrin 5 ~ 10%, Trisodium Citrate 0.1 ~ 0.5%, CaCl₂0.1 ~ 0.5%, MgSO₄0.1 ~ 0.5%, K₂hPO₄0.5 ~ 2%) in, 34 DEG C, 250rpm shaking culture 72h; Centrifuging and taking supernatant liquor; The Sumizyme MP enzyme adopting National Standard of the People's Republic of China's protease preparation measuring method (GB/T 25327-2009) to measure above-mentioned bacterial strains fermented supernatant fluid is respectively lived.Result shows, and the fermenting enzyme of starting strain is lived as 11650U/mL, and in mutant strain, enzyme running water is put down the highest is Bacillus licheniformis BL1-43, and its fermenting enzyme work, up to 18000U/mL, improves 54.5% than the bacterium that sets out, and achieves unexpected technique effect.

Bacillus licheniformis BL1-43 is gone down to posterity on beef extract-peptone flat board after 5 times, picking list bacterium colony carries out shake flask fermentation again, enzyme is lived and is still improved more than 50% than the bacterium that sets out, and after illustrating that mutant strain Bacillus licheniformis BL1-43 that the present invention obtains goes down to posterity for many times, can also keep the stability in heredity.

Bacillus licheniformis BL1-43 (Bacillus licheniformisBL1-43) is preserved in the China typical culture collection center of Wuhan, China Wuhan University on July 13rd, 2015 by applicant, and deposit number is CCTCC NO:M2015449.

SEQUENCE LISTING

 

<110> Qingdao Weilan Biology Group Co., Ltd.

 

The Bacillus licheniformis of <120> mono-strain high yield alkali protein and application thereof

 

 

<160> 2

 

<170> PatentIn version 3.5

 

<210> 1

<211> 380

<212> PRT

<213> Bacillus clausii (Bacillus clausii)

 

<400> 1

 

Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu Ile

1 5 10 15

 

 

Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala Ala Glu Glu Ala Lys

20 25 30

 

 

Glu Lys Tyr Leu Ile Gly Phe Asn Glu Gln Glu Ala Val Ser Glu Phe

35 40 45

 

 

Val Glu Gln Val Glu Ala Asn Asp Glu Val Ala Ile Leu Ser Glu Glu

50 55 60

 

 

Glu Glu Val Glu Ile Glu Leu Leu His Glu Phe Glu Thr Ile Pro Val

65 70 75 80

 

 

Leu Ser Val Glu Leu Ser Pro Glu Asp Val Asp Ala Leu Glu Leu Asp

85 90 95

 

 

Pro Ala Ile Ser Tyr Ile Glu Glu Asp Ala Glu Val Thr Thr Met Ala

100 105 110

 

 

Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala His

115 120 125

 

 

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

130 135 140

 

 

Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser Phe

145 150 155 160

 

 

Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr His

165 170 175

 

 

Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly

180 185 190

 

 

Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala Ser

195 200 205

 

 

Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala Gly

210 215 220

 

 

Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser Pro

225 230 235 240

 

 

Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly Val

245 250 255

 

 

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

260 265 270

 

 

Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln Asn

275 280 285

 

 

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

290 295 300

 

 

Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr Ala

305 310 315 320

 

 

Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala Ala

325 330 335

 

 

Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile Arg

340 345 350

 

 

Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu Tyr

355 360 365

 

 

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

370 375 380

 

 

<210> 2

<211> 1143

<212> DNA

<213> Bacillus clausii (Bacillus clausii)

 

<400> 2

atgaagaaac cgttggggaa aattgtcgca agcaccgcac tactcatttc tgttgctttt 60

 

agttcatcga tcgcatcggc tgctgaagaa gcaaaagaaa aatatttaat tggctttaat 120

 

gagcaggaag ctgtcagtga gtttgtagaa caagtagagg caaatgacga ggtcgccatt 180

 

ctctctgagg aagaggaagt cgaaattgaa ttgcttcatg aatttgaaac gattcctgtt 240

 

ttatccgttg agttaagccc agaagatgtg gacgcgcttg aactcgatcc agcgatttct 300

 

tatattgaag aggatgcaga agtaacgaca atggcgcaat cagtgccatg gggaattagc 360

 

cgtgtgcaag ccccagctgc ccataaccgt ggattgacag gttctggtgt aaaagttgct 420

 

gtcctcgata caggtatttc cactcatcca gacttaaata ttcgtggtgg cgctagcttt 480

 

gtaccagggg aaccatccac tcaagatggg aatgggcatg gcacgcatgt ggccgggacg 540

 

attgctgctt taaacaattc gattggcgtt cttggcgtag cgccgagcgc ggaactatac 600

 

gctgttaaag tattaggggc gagcggttca ggttcggtca gctcgattgc ccaaggattg 660

 

gaatgggcag ggaacaatgg catgcacgtt gctaatttga gtttaggaag cccttcgcca 720

 

agtgccacac ttgagcaagc tgttaatagc gcgacttcta gaggcgttct tgttgtagcg 780

 

gcatctggga attcaggtgc aggctcaatc agctatccgg cccgttatgc gaacgcaatg 840

 

gcagtcggag ctactgacca aaacaacaac cgcgccagct tttcacagta tggcgcaggg 900

 

cttgacattg tcgcaccagg tgtaaacgtg cagagcacat acccaggttc aacgtatgcc 960

 

agcttaaacg gtacatcgat ggctactcct catgttgcag gtgcagcagc ccttgttaaa 1020

 

caaaagaacc catcttggtc caatgtacaa atccgcaatc atctaaagaa tacggcaacg 1080

 

agcttaggaa gcacgaactt gtatggaagc ggacttgtca atgcagaagc ggcaacacgc 1140

 

taa 1143

Claims (4)

1. a Bacillus licheniformis engineering bacteria, it carries the expression vector expressing Sumizyme MP.

2. engineering bacteria as claimed in claim 1, it is characterized in that, the aminoacid sequence of described Sumizyme MP is SEQ ID NO:1, and coding nucleotide sequence is SEQ ID NO:2.

3. a Bacillus licheniformis, is characterized in that, the deposit number of described Bacillus licheniformis is CCTCC NO:M2015449.

4. the application of Bacillus licheniformis according to claim 3 in Sumizyme MP is produced.