CN103382462A - Bacillus licheniformis lysozyme and its production method and application thereof - Google Patents
Bacillus licheniformis lysozyme and its production method and application thereof Download PDFInfo
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Abstract
The invention discloses a lysozyme which has a nucleotide sequence as shown in the SEQ ID NO:1. Correspondingly, the invention provides a production method of the lysozyme having the nucleotide sequence. By the utilization of escherichia coli, yeast or bacillus subtilis, the lysozyme can be highly expressed. The lysozyme provided by the invention can resist high temperature and high concentration of pepsin and trypsin, and can be used as a bacteriostatic agent to be widely applied in industries of forage, food, agriculture and the like.
Description
Technical field
The present invention relates to field of molecular microbiology, specifically a kind of N,O-Diacetylmuramidase (lysowme) and production thereof and application.
Background technology
N,O-Diacetylmuramidase is a kind of enzyme of solubilized bacteria cell wall, the β in energy bacterium for degrading cell walls between N-second phthalein teichoic acid and N-second phthalein glucosamine-Isosorbide-5-Nitrae glycosidic link, thus destroy cell walls, make bacterium dead due to the infiltration dissolving.Because it has bacteriolysis, therefore the called after N,O-Diacetylmuramidase.
N,O-Diacetylmuramidase extensively is present in Various Tissues, juice such as tears, the saliva of humans and animals, also extensively exists in certain plants, microorganism.For example, (human lysozyme is a kind of small molecular basic protein all to contain N,O-Diacetylmuramidase in people's white cell, serum, placenta, saliva, milk, uterus mucus and kidney, it is secreted by monocytes/macrophages, play nonspecific defense reaction in the human organism), and also all contain abundant N,O-Diacetylmuramidase in the bright juice of papaya, pineapple and Fructus Fici, their also antiviral relevant with plant by inference.
Generally N,O-Diacetylmuramidase is divided 6 classes: C type (hen's egg-white lysozyme), g type (goose albumen N,O-Diacetylmuramidase), invertebrates type, phage, bacterium and plant origin N,O-Diacetylmuramidase.The molecular weight of the N,O-Diacetylmuramidase of different sources, amino acid form and the enzyme characteristic is had nothing in common with each other, but the N,O-Diacetylmuramidase in various sources all has inhibitory effect, is all the important natural immunity factor in multiple biology, the process of opposing bacterial invasion cell.
Compare with other antimicrobial factors, that N,O-Diacetylmuramidase has is activity stabilized, can the freezing or advantages such as drying treatment, has a broad antifungal spectrum, therefore be used as anti-infective material and sanitas in medical science and foodstuffs industry, main Application Areas comprises the food preservatives as high security, has certain health-care effect, can be optionally, killing microorganisms and do not act on other materials in food on purpose, guarantee that the original nutritive ingredient of food do not suffer a loss; Being used as in milk-product and adding that in the factor pair enteron aisle, the putridness microorganism has special killing action, promote directly or indirectly the increment of bifidus bacillus in enteron aisle simultaneously, is the antibacterial protein in infant food; In food flexible packing with lysozyme immobilization on packaging material for food, produce the packaging material for food of resisting effect, to reach sterilizing fresh-keeping function; Play preservative activity at wine and beverage; Have anti-infective and strengthen microbiotic effect effect at health functional food, promoting hemopexis and anastalsis, regeneration in a organized way; Be used for feed anticorrosion agent and sterilant in livestock industry, have the effect of control grice diarrhoea, can promote the health of chicken body, promote digestion, the absorption of nutritive substance in feed.
Because N,O-Diacetylmuramidase has above-mentioned multiple use, being in great demand to N,O-Diacetylmuramidase on market.At present, people mainly produce N,O-Diacetylmuramidase from Ovum Gallus domesticus album.It is higher that yet the N,O-Diacetylmuramidase in Ovum Gallus domesticus album source is produced cost, and wasted the egg starting material and have with the mankind's food needs and conflict.As food, fodder additives, cause that cost on market is higher and N,O-Diacetylmuramidase that conflict with human foods is hard to carry on as a large amount of.
Therefore, this area is badly in need of a kind of method of biological fermentation of utilizing and is produced N,O-Diacetylmuramidase, reduces the cost of N,O-Diacetylmuramidase, and the N,O-Diacetylmuramidase of producing has again good antagonistic property, can be used as the harmless food of pure natural, fodder additives.
Summary of the invention
As mentioned above, for the needs of this area, the invention discloses a kind of Bacillus licheniformis N,O-Diacetylmuramidase of high temperature resistant, strong stress resistance.Disclosed N,O-Diacetylmuramidase can the By Direct Pyrolysis bacterium, and it is strong to have the withstand high temperatures ability, and tolerance pH value of solution scope is large, can resist the higher stomach en-of concentration and trypsinase, the characteristics such as antibacterial spectrum width.
Further, the invention also discloses the production method of described Bacillus licheniformis N,O-Diacetylmuramidase, method disclosed by the invention can make its high efficient expression in intestinal bacteria, pichia spp, subtilis, thereby reduce production costs, improve unit output and production efficiency, be convenient to suitability for industrialized production.
Accordingly, the invention also discloses described Bacillus licheniformis bacteriolyze application of enzymes, the experiment that the applicant carries out shows that this N,O-Diacetylmuramidase has wide spectrum, Wide High-efficient Antibacterial, the effectively multiple Gram-positive of cracking and Gram-negative bacteria, not only can be used for the prevention and control bacteriosis, can also play antibacterial antisepsis and sterilization effect widely in various food, biological products as additive.
For achieving the above object, the present invention is achieved through the following technical solutions:
A kind of Bacillus licheniformis N,O-Diacetylmuramidase has the nucleotide sequence shown in sequence table SEQ IDNO:1.
This N,O-Diacetylmuramidase is that the Bacillus licheniformis separation from natural soils obtains, the applicant identifies by the method for 16s the Bacillus licheniformis that screens from soil used, to compare with the Bacillus licheniformis of genebank lane database through the sequence of the 16SrRNA of PCR lichem bacillus strain out, the sequence of the sequence 99.9% of 16SrRNA is coincide, can judge that the bacterial strain that screens is the Bacillus licheniformis of biologically widely using, the sequence of the 16SrRNA of this bacterial strain is as shown in SEQIDNO:2.
The applicant measures the zymetology performance of the N,O-Diacetylmuramidase of gained, shows that the gained N,O-Diacetylmuramidase has following physico-chemical property:
The molecular weight 35KD of N,O-Diacetylmuramidase;
N,O-Diacetylmuramidase is comprised of 318 amino acid;
The optimal pH of N,O-Diacetylmuramidase is 8;
The optimum temperature of N,O-Diacetylmuramidase is 37 ℃
The iso-electric point of N,O-Diacetylmuramidase) 8;
PH stability and the thermostability of N,O-Diacetylmuramidase: enzyme has good stability in pH2～10, and enzyme can keep stability preferably in the time of 55 ℃.
Accordingly, the invention discloses the production method of described Bacillus licheniformis N,O-Diacetylmuramidase, N,O-Diacetylmuramidase of the present invention can be in multiple bacterial strain carrier high efficient expression, include but not limited to following several:
(1) adopt e. coli strain bl21 as expressive host, as expression vector, produce Bacillus licheniformis N,O-Diacetylmuramidase with IPTG (isopropylthiogalactoside) abduction delivering with pET2l, pET28 series plasmid.
(2) adopt pichia spp GSl15 bacterial strain as expressive host, with the plasmid of pPICgK series as expression vector, with methanol induction Expression product Bacillus licheniformis N,O-Diacetylmuramidase.
(3) adopt subtilis WB800 as expressive host, produce the Bacillus licheniformis N,O-Diacetylmuramidase with autonomously replicationg vector PGJl03 plasmid as expression vector.
When adopting e. coli bl21 as the production method of Bacillus licheniformis N,O-Diacetylmuramidase of the present invention, concrete comprises the steps:
1, seed culture: picking mono-clonal from the flat board is inoculated in the LB substratum 37 ℃ of lower incubated overnight;
2, abduction delivering: get seed culture medium and be inoculated in fermention medium LB, cultivate after OD value to 0.6, add IPTG and induce, cultivate under 20 ℃;
3, protein extraction: centrifugally obtain cell and with its ultrasonication, the centrifugal cell residue of removing, membrane filtration.
Further, in above-mentioned production expression method, comprise that also the protein extract with step 3 carries out the step of purifying by the nickel post.
Wherein, culture condition, inoculum size, filter membrane specification, nickel post type can be selected according to actual needs in above-mentioned steps.
In aforesaid method, the applicant does not specifically describe N,O-Diacetylmuramidase DNA fragmentation pcr amplification is gone out vector plasmid carrier construction expression plasmids such as being connected to pET21 after the full-length gene fragment and carry out inscribe with Ndel, XhoI endonuclease to obtain monoclonal process, and this process is that those skilled in the art extensively adopt for expressing with the pET21 construction recombination plasmid in intestinal bacteria.
When adopting pichia spp GSl15 bacterial strain as the production method of Bacillus licheniformis N,O-Diacetylmuramidase of the present invention, the Bacillus licheniformis lysozyme gene is incorporated on the karyomit(e) of pichia spp GSl15 bacterial strain with the plasmid of pPICgK series as carrier; On methyl alcohol phenotype used both available methyl alcohol utilize Quick-type (mut+), also available methyl alcohol utilizes type (muts or must-) at a slow speed, at first this production method builds the expression vector pPICgK contain the Bacillus licheniformis gene in bacillus coli DH 5 alpha, adopt method that electricity transforms then Plasmid Transformation to be carried out following process in the pichia spp:
1, seed culture: connect bacterium liquid from the glycerine pipe in the YPD substratum, be inoculated into after cultivation in the BMGY substratum, in 30 ℃ of cultivations;
2, fermentation culture: with bacterium liquid access automatic fermenter in BMGY, control pH as 6.0 take 50% ammoniacal liquor, 30 ℃ of temperature are regulated mixing speed and air flow and are kept dissolved oxygen more than 30%, and stream glycerol adding when glycerine exhausts is until 0D600 arrives 250;
3, abduction delivering: after thalline hunger 0.5～lh, add methyl alcohol to begin abduction delivering.Wherein methanol feeding carries out according to dissolved oxygen, makes whole process dissolved oxygen be controlled at 30～50%.
4, purifying: the pichia spp nutrient solution that will contain the Bacillus licheniformis N,O-Diacetylmuramidase is held back the molecular film ultrafiltration with 10-50 ten thousand and is collected filtrate, with the 5000 molecular weight ultrafiltration collection dopes that dam; Dope adds carboxyl methyl cellulose, and the elution buffer wash-out is collected elutriant, desalination, and frost drying is measured protein content and lysozyme activity.
In aforesaid method, culture condition, inoculum size etc. can be selected according to actual needs.
In aforesaid method, the applicant does not specifically describe N,O-Diacetylmuramidase DNA fragmentation pcr amplification is gone out to be connected to pPIC9K vector plasmid carrier construction expression plasmid and to carry out double digestion with EcoRI, NotI after the full-length gene fragment and obtains monoclonal process, and this process is that those skilled in the art extensively adopt for the pPIC9K construction recombination plasmid.
When adopting subtilis WB800 as the production method of Bacillus licheniformis N,O-Diacetylmuramidase of the present invention, the Bacillus licheniformis lysozyme gene is under the P43 promotor is controlled, with autonomously replicationg vector PGJl03 plasmid as carrier, plasmid is free in the dyeing vivoexpression, and concrete comprises the steps:
1, build the PGJl05 plasmid vector that contains the Bacillus licheniformis gene in bacillus coli DH 5 alpha;
2, adopt method that electricity transforms with the PGJl05 Plasmid Transformation in subtilis;
3, culture expression albumen in the LB substratum, be inoculated in seed culture medium in fermention medium, cultivates under 37 ℃, and with the centrifugal ultrasonication of albumen sample thief of cultivating, check protein expression.
In aforesaid method, culture condition, inoculum size etc. can be selected according to actual needs.
The applicant does not specifically describe N,O-Diacetylmuramidase DNA fragmentation pcr amplification is gone out to be connected to plasmid construction vector expression plasmid and to carry out double digestion with EcoRI, BamHI after the full-length gene fragment and obtains monoclonal process in aforesaid method, and this process is that those skilled in the art extensively adopt.
By aforementioned production method, N,O-Diacetylmuramidase of the present invention is able to high efficient expression in intestinal bacteria, pichia spp, subtilis.
On the basis of the above, the invention also discloses described N,O-Diacetylmuramidase as the application of fungistat, N,O-Diacetylmuramidase of the present invention is inhibited to Gram-positive or Gram-negative bacteria, and especially the fungistat as gram-positive microorganism has significant restraining effect.
Description of drawings
Fig. 1 has shown the impact of pH environment on Bacillus licheniformis lysozyme activity of the present invention;
Fig. 2 has shown the impact of temperature on Bacillus licheniformis lysozyme activity of the present invention;
Fig. 3 has shown that trypsinase and stomach en-are on the impact of Bacillus licheniformis lysozyme activity of the present invention;
Fig. 4 has shown the expression plasmid pET2la-lyso building process that uses in intestinal bacteria;
Fig. 5 is Bacillus licheniformis N,O-Diacetylmuramidase SDS-PAGE figure of the present invention, and swimming lane 1 is maker, and swimming lane 2 is N,O-Diacetylmuramidase Expression in Escherichia colis, and swimming lane 3 is N,O-Diacetylmuramidase expression in pichia spp, and swimming lane 4 is N,O-Diacetylmuramidases of purifying;
Fig. 6 has shown the expression plasmid pPICgK-lyso building process that uses in pichia spp;
Fig. 7 has shown the expression plasmid PGJlO5 building process that uses in subtilis.
Embodiment
The invention will be further described below in conjunction with embodiment, and embodiment is intended to the description of giving an example to the present invention, but not is construed as limiting the invention in any form.
In following concrete enforcement; the applicant provides building process and the primer sequence of N,O-Diacetylmuramidase expression plasmid used in every kind is produced expression method in detail; the process that provides and primer only are used for making those skilled in the art can better understand the present invention, are not particularly limited and protection scope of the present invention is not consisted of.
Embodiment 1
Bacillus licheniformis N,O-Diacetylmuramidase of the present invention has the nucleotide sequence shown in sequence table SEQ IDNO:1, has the aminoacid sequence shown in sequence table SEQ IDNO:3.
Measure N,O-Diacetylmuramidase of the present invention take micrococcus lysodeikticus as substrate in the solution of a series of different pH damping fluids, measure enzyme activity under 37 ℃, result as shown in Figure 1, show that N,O-Diacetylmuramidase optimal pH of the present invention is 8, being 2～10 to have good stability at pH, is to keep 40% relative activity under 2 conditions the time at pH.
Measure the activity of N,O-Diacetylmuramidase of the present invention with micrococcus lysodeikticus as substrate, in pH is 8 damping fluid, process under different temperature condition, result as shown in Figure 2, the optimum temperuture of N,O-Diacetylmuramidase of the present invention is 37 ℃.
Same with reference to shown in Figure 2, in pH is 8 damping fluid, process 4 hours enzyme solution under different temperature condition, measure the bacteriolyze Thermostability, N,O-Diacetylmuramidase of the present invention is processed under 55 ℃ of conditions can also keep 45% enzyme activity in 4 hours.
With N,O-Diacetylmuramidase powder of the present invention respectively the trypsinase and pepsin solution under 0 to 400mg/L different concns gradient, process under 37 ℃ of conditions, measure trypsinase and stomach en-enzyme to the impact of lysozyme activity, estimate with this function that it can keep as feed, foodstuff additive in Digestive tract.As shown in Figure 3, can find out that N,O-Diacetylmuramidase of the present invention can resist certain density trypsinase and pepsic digestion, can also keep nearly 50% vigor under the digestion of 300mg/1 trypsinase and stomach en-enzyme.
The applicant tests the bacteriostasis of N,O-Diacetylmuramidase of the present invention, selection comprises the totally 16 strain bacterial strain antimicrobial spectrum experiments such as Gram-positive, Gram-negative, adopt the MIC method to measure N,O-Diacetylmuramidase to the bacteriostatic action of bacterial strain, the results are shown in shown in following table 1, N,O-Diacetylmuramidase of the present invention has bacteriostatic action more widely, the restraining effect that 16 kinds of bacterial strains is had degree varies especially has significant restraining effect to the thicker gram-positive microorganism of cell walls.
Table 1: the restraining effect of N,O-Diacetylmuramidase of the present invention to different mushrooms
Embodiment 2: the expression of Bacillus licheniformis N,O-Diacetylmuramidase of the present invention in e. coli bl21
At first, the structure of expression plasmid: amplify the sequence of N,O-Diacetylmuramidase from Bacillus licheniformis DNA, the PCR process adopts following primer:
Primers F: actgcaCATATGatgggaatcaaaggaatcgac;
Primer R:actgcaCTCGAGttatctaacacgaattttctgacca.
PCR product and carrier are all used Ndel and Xhol double digestion, and link is built into expression plasmid pET2la-lyso.As shown in Figure 4, build the expression plasmid that contains the Bacillus licheniformis lysozyme gene, the plasmid that conversion builds is to the expressive host recipient cell, picking mono-clonal contains to 5m1 in the LB substratum of corresponding resistant, and 37 ℃, the 20Orpm incubated overnight, be cultured to the OD600=0.6 left and right, add IPTG to final concentration lmM, be transferred to 20 ℃, 200rpm continues inducing culture, in this process, and can be at 2h, 4h, 6h, 8h, lOh take a sample respectively to analyze the expression situation.
With the centrifugal lmin of lml bacterium liquid 12000rpm that takes out, collect thalline, add the resuspended precipitation of lmlPBS damping fluid, same centrifugal lmin.Add again 300～5OOmlPBS re-suspended cell, the ultrasonication cell, with 4 ℃ of broken liquid, the centrifugal lOmin of l2000rpm, cleer and peaceful precipitation in separation.
Further, also comprise on the basis of the above the step of carrying out purifying to extracting product, comprising:
(1) irrigate Ni
2+-NTA affinity column carries out slow wash-out with deionized water, avoids introducing bubble in the post bed;
(2) startingbuffer with 10 times of column volumes carries out pre-equilibration, and loading is injected the Ni2+-NTA affinity column with the supernatant liquor after cytoclasis;
(3) startingbuffer with 1O times of ml carries out rinsing, collects filtered solution;
(4) begin to carry out wash-out with the staningbuffer that contains 20 ware M miaows of 5 times of column volumes, and elutriant is collected;
(5) successively with the 40mM that contains of 5 times of column volumes, 60mM, 80mM, l00 ware M, 200mM, the startingbuffer of 300mM and 500mM imidazoles carries out wash-out, respectively elutriant is collected;
(6) detect by SDS-PAGE the effect that reclaims, determine the most suitable wash-out concentration of miaow;
The filtrate of (7) collecting from every pipe is taken out the sample of lOu, adds the 2XSDS gel loading buffer of lOul, shakes up;
(8) boiling water bath is processed 3min;
(9) take out sample, lO μ 1 sample all is splined on electrophoresis on the SDS polyacrylamine gel of proper concn, electrophoresis result as shown in Figure 5.
Embodiment 3: the expression of Bacillus licheniformis N,O-Diacetylmuramidase in pichia spp
At first, as shown in Figure 6, build the expression vector pPICgK that contains the Bacillus licheniformis gene in bacillus coli DH 5 alpha, the structure of expression plasmid: amplify the sequence of N,O-Diacetylmuramidase from Bacillus licheniformis DNA, the PCR process adopts following primer:
The primer F:actgcaGAATTCatgggaatcaaaggaatcgac;
Primer R:actgcaGCGGCCGCttatctaacacgaanttctgacca.
PCR product and carrier are all used EcoRl and Notl double digestion, and link is built into expression plasmid pPICgK-lyso.In pichia spp, method for transformation is as follows with Plasmid Transformation for the method that the employing electricity transforms:
At first carrying out cell prepares
(1) in containing the 5Oml centrifuge tube of 5mlYPD, cultivate pichia spp, 30 ℃ are spent the night;
(2 get the 0.1-0.5m1 overnight culture, and inoculation contains the 2L shaking flask of 5OOml fresh culture, and overnight growth is to OD600=1.3-1.5;
(3) at 4 ℃, the centrifugal 5min collecting cell of l500g is with the aqua sterilisa suspension cell of 5OOml precooling;
As above centrifugal, with the aqua sterilisa suspension cell of 250ml precooling; LM sorbyl alcohol suspension cell with the 2Oml precooling; As above centrifugal, with the lM sorbyl alcohol suspension cell of lml precooling, to the about 15ml of final volume.
Cell transforms after being ready to complete:
(1) get the above-mentioned cell of 80ul and mix with 5-2Oug linearizing DNA (being dissolved in 5-lOulTE), change in the 0.2cm electricity revolving cup of precooling.Place 5min on ice;
(2) shock by electricity according to the pichia spp parameter;
(3) the lM sorbyl alcohol that adds immediately the lml precooling is transferred to content in the sterilization centrifuge tube and is divided into the 200-600ul equal portions to cup, is applied on MD or RDB flat board
(4) hatching dull and stereotyped extremely clone at 30 ℃ produces.
Fermentation culture flow process and purifying process are as follows:
Zymotechnique:
1, seed shake-flask culture: connect 200 microlitre bacterium liquid from the glycerine pipe in 5 milliliters of YPD, cultivate that the inoculum size by 1% is inoculated in 5OmlBMGY after l2h, cultivate 24h in 30 ℃, 22Or/min.
2, high density fermentation is cultivated: bacterium liquid in BMGY by 5% inoculum size access lL automatic fermenter, is controlled pH6.0 with 50% ammoniacal liquor, and 30 ℃ of temperature are regulated mixing speed and air flow and are kept dissolved oxygen more than 30%.When glycerine exhausts (dissolved oxygen rises rapidly), begin to flow glycerol adding, when OD600 arrives 250, stop flowing glycerol adding, after thalline hunger 05～lh, add methyl alcohol to begin abduction delivering.Methanol feeding carries out according to dissolved oxygen, makes whole process dissolved oxygen be controlled at 30～50%.
Purifying process:
The pichia spp nutrient solution that will contain the human lysozyme of gene engineering expression is held back the molecular film ultrafiltration with 10-50 ten thousand, collects filtrate: with the 5000 molecular weight ultrafiltration of damming, collection dope; Carboxyl methyl cellulose on dope, the elution buffer wash-out is collected elutriant, crosses G-50 post desalination, and frost drying is measured protein content and lysozyme activity.
Embodiment 4: the expression of Bacillus licheniformis N,O-Diacetylmuramidase in subtilis
At first, the structure of expression plasmid: amplify respectively promotor p43 and signal skin from subtilis DNA, then with merging PCR method, p43 and signal skin are merged.To contain the PCR product of p43 and signal skin and carrier pGJl03 cuts with Xhol and EcoRl enzyme and is built into carrier pGJl04.Amplify the gene of N,O-Diacetylmuramidase from Bacillus licheniformis DNA, PCR product and plasmid pGJl04 are cut with EcoRl and BamHl enzyme, link is built into expression plasmid pGJlO5.
In above-mentioned, subtilis amplification the primer is:
P43-F:actgcaCTCGAGtgtcgacgtgcatgcaggc;
P43-R:tataatggtaccgctatcacttta;
PsacB-SP-F:taaagtgatagcggtaccattataagttctttaggcccgtagtct;
PsacB-SP-R:actgcaGAATTCttctttcgcaaacgcttgagtt。
Amplify the sequence of N,O-Diacetylmuramidase in Bacillus licheniformis DNA, this process the primer is:
Primer RactgcaGAATTCatgggaatcaaaggaatcgac;
Primer R:actgcaGGATCCttatctaacacgaattttctgacca.
PCR product and carrier are all used EcoRl and Notl double digestion, and link is built into expression plasmid pPICgK-lyso.As shown in Figure 7, build the expression vector PGJlO5 contain the Bacillus licheniformis gene in bacillus coli DH 5 alpha, then adopt method that electricity transforms with Plasmid Transformation in subtilis WB800.
Without in the specified otherwise situation, intestinal bacteria and Bacillus subtillis are all at LB substratum (Trypsin knee: 10g/1 in the present embodiment; Yeast extract: 5g/1; Sodium-chlor: 10g/1; PH7.5) carry out the aerobic shaking culture in; If no special instructions, all growths under 37 ℃ of all bacterial strains.During solid culture, add the agar powder of 1.5g/1 in the LB substratum, add the microbiotic of suitable concn when needing.
At first build the carrier contain the Bacillus licheniformis gene in bacillus coli DH 5 alpha, and adopt method that electricity transforms with Plasmid Transformation in subtilis.
The electricity method for transformation is as follows:
1, raw material is prepared:
40ml (LB+0.5M sorbyl alcohol): peptone 10g/1, yeast powder 5g/1, NaCll0g/1,3.69 sorbyl alcohol pH=7.2; The 2OOml electricity turns substratum (0.5M sorbyl alcohol, 0.5M N.F,USP MANNITOL, 10% glucose): 18g sorbyl alcohol, 18.59 N.F,USP MANNITOL, 20g glucose; L0mlRM (O.5M sorbyl alcohol, 0.38M N.F,USP MANNITOL): 0.9g sorbyl alcohol, 0.7g N.F,USP MANNITOL; The 50ml centrifuge tube of 2 sterilizations.
2, transform
1) inoculate B.subtilis in the 3mlLB substratum, incubated overnight;
2) get in 2.6m1 overnight culture access 40ml (LB+0.5M sorbyl alcohol), 37 ℃, 200rpm is cultured to OD600=0.85-0.95;
3) with bacterium liquid ice-water bath lOmin, 5000g then, 5min, 4 ℃ of centrifugal collection thalline;
4) electricity with the 50ml precooling turns substratum (0.5M sorbyl alcohol, 0.5M N.F,USP MANNITOL, 10% glucose), again blows outstanding thalline, 5000g, and 5min, 4 ℃ of centrifugal supernatants that go, so rinsing is 4 times;
5) thalline after washing blows and is suspended from the lml electricity and turns in substratum, the packing 120 of every EP pipe;
6) with adding 50ngDNA (1～8 μ 1) in 60 μ l competent cells, hatch 2min on ice, add in the electric revolving cup (lmm) of precooling, electric shock once.Electroporation arranges: 2.Okv, lmm shocks by electricity 1 time.(the electric shock result: time constant=4.5～5.0ms, if time constant (4.2, need to increase electricity and turn the rinsing number of times of substratum or improve competent extension rate, obtain higher transformation efficiency);
7) the complete taking-up cup of electric shock also adds lmlRM (LB+0.5M sorbyl alcohol+0.38 N.F,USP MANNITOL) immediately, and 37 ℃, 200rpm, after recovery 3h, coated plate.37 ℃, incubated overnight.
After above-mentioned electricity conversion is completed, with bacterial strain culture expression albumen in the LB substratum, 1% seed culture medium is inoculated in fermention medium, and 37 ℃, 220r/min are cultivated respectively 8 hours, 12 hours, 16 hours and the centrifugal ultrasonication of sample thief in 20 hours, checks protein expression.
The applicant tests embodiment 2,3 product purity, shows that product purity all is not less than 99.6%, illustrates that N,O-Diacetylmuramidase production of the present invention and extracting and purifying method have effectively guaranteed the purity of product.
The applicant has carried out DNA and amino acid sequencing to the product of embodiment 2-4 gained, result shows that its molecular weight of gained N,O-Diacetylmuramidase is consistent with molecular weight in sequence table, and its nucleotide sequence, aminoacid sequence are all consistent with sequence in sequence table SEQ IDNO:l, SEQIDNO:3.
Claims (10)
1. a Bacillus licheniformis N,O-Diacetylmuramidase, is characterized in that having the nucleotide sequence shown in sequence table SEQ IDNO:1.
2. Bacillus licheniformis N,O-Diacetylmuramidase according to claim 1 is characterized in that physico-chemical property is:
The molecular weight 35KD of N,O-Diacetylmuramidase;
N,O-Diacetylmuramidase is comprised of 318 amino acid;
The optimal pH of N,O-Diacetylmuramidase is 8;
The optimum temperature of N,O-Diacetylmuramidase is 37 ℃;
The iso-electric point of N,O-Diacetylmuramidase) 8;
PH stability and the thermostability of N,O-Diacetylmuramidase: enzyme has good stability in pH2～10, and enzyme can keep stability preferably in the time of 55 ℃.
3. the production method of Bacillus licheniformis N,O-Diacetylmuramidase claimed in claim 1, it is characterized in that comprising and adopt e. coli strain bl21 as expressive host, as expression vector, produce the step of Bacillus licheniformis N,O-Diacetylmuramidase with pET2l, pET28 series plasmid with the IPTG abduction delivering.
4. production method according to claim 3, is characterized in that comprising the steps:
(1) seed culture: picking mono-clonal from the flat board is inoculated in the LB substratum 37 ℃ of lower incubated overnight;
(2) abduction delivering: get seed culture medium and be inoculated in fermention medium LB, cultivate after OD value to 0.6, add IPTG to induce, in 20 ℃ of cultivations;
(3) protein extraction: the centrifugal cell that obtains, will remove cell residue after its ultrasonication, adopt membrane filtration, then ni-sepharose purification and get final product.
5. the production method of Bacillus licheniformis N,O-Diacetylmuramidase claimed in claim 1, it is characterized in that comprising and adopt pichia spp GSl15 bacterial strain as expressive host, with the plasmid of pPICgK series as expression, with the step of methanol induction Expression product Bacillus licheniformis N,O-Diacetylmuramidase.
6. production method according to claim 5, is characterized in that comprising the steps:
At first build the expression vector pPICgK that contains the Bacillus licheniformis gene in bacillus coli DH 5 alpha, the method that the employing electricity transforms in pichia spp, is then carried out following process with Plasmid Transformation:
(1) seed culture: be inoculated in the BMGY substratum cultivation under 30 ℃ in the YPD substratum after inoculation bacterium liquid is cultivated from the glycerine pipe;
(2) fermentation culture: with bacterium liquid access automatic fermenter in the BMGY substratum, control pH as 6.0 take 50% ammoniacal liquor, 30 ℃ of temperature are regulated mixing speed and air flow and are kept dissolved oxygen more than 30%; Stream glycerol adding when glycerine exhausts is until OD600 arrives 250;
(3) abduction delivering: after the hungry 0.5-1h of thalline, add methanol induction to express.
(4) protein extraction: the above-mentioned pichia spp nutrient solution that contains the Bacillus licheniformis N,O-Diacetylmuramidase of gene engineering expression is held back the molecular film ultrafiltration with 10-50 ten thousand, then with the 5000 molecular weight ultrafiltration of damming, collect dope; Add carboxyl methyl cellulose in dope, the elution buffer wash-out is collected elutriant, desalination, and frost drying, and get final product.
7. the production method of Bacillus licheniformis N,O-Diacetylmuramidase claimed in claim 1, it is characterized in that comprising and adopt subtilis WB800 as expressive host, produce the step of Bacillus licheniformis N,O-Diacetylmuramidase with autonomously replicationg vector PGJl03 plasmid as expression vector.
8. production method according to claim 7, is characterized in that comprising the steps:
(1) build the PGJl03 plasmid vector that contains the Bacillus licheniformis gene in bacillus coli DH 5 alpha;
(2) adopt method that electricity transforms with Plasmid Transformation in subtilis;
(3) above-mentioned bacterial strains culture expression albumen in the LB substratum, seed culture medium is inoculated in fermention medium, cultivates under 37 ℃, and the then centrifugal ultrasonication of sample thief checks protein expression.
9. Bacillus licheniformis N,O-Diacetylmuramidase claimed in claim 1 is as the application of fungistat.
10. application according to claim 9 is characterized in that described N,O-Diacetylmuramidase can be used as the fungistat of gram-positive microorganism.
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