CN1084349C

CN1084349C - Process for preparing poly-beta-hydroxy-butyrate

Info

Publication number: CN1084349C
Application number: CN98102281A
Authority: CN
Inventors: 尹进; 沈忠耀; 李红旗
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 1998-06-19
Filing date: 1998-06-19
Publication date: 2002-05-08
Anticipated expiration: 2018-06-19
Also published as: CN1201043A

Abstract

The present invention relates to a method for preparing poly-beta-hydroxybutyric acid ester, which comprises: firstly, genetic engineering bacteria containing both PHB synthetic genes and lambda bacteriophage cracking genes S(-)RRz are constructed; when the bacteria are cultured in a culture medium containing glucose, the content of PHB in thalli can occupy more than 60% of the dry weight of the thalli; cells accumulating PHB are induced by buffer liquid containing chelating agents, the cells are cracked within 20 minutes, and the cracking rate is close to 100%; cell deposits after cracking are washed with a surface active agent SDS, which can obtain PHB whose purity is higher than 99%.

Description

A kind of method for preparing poly-beta-hydroxy-butyrate

The present invention relates to a kind of method for preparing poly-beta-hydroxy-butyrate, belong to technical field of biochemical industry.

Poly-beta-hydroxy-butyrate (PHB) is a kind of high molecular polymer of microorganism synthetic, because the characteristic etc. that has biodegradability, biocompatibility, piezoelectricity, optical activity and can utilize regenerative raw materials in biosynthetic process has unique and wide application prospect in fields such as medical science, pharmacy, agricultural, electronics.But scale operation and the widespread use of PHB still are unrealized at present, one of them most important reason is exactly that to produce the cost of PHB much higher than the petrochemical complex plastics: be that raw material synthetic plastics price is about 1$/kg at present with the oil, and the price of PHB is up to 16$/kg.

The method of utilizing microbial fermentation to prepare PHB at present mainly can be divided into for two steps: the first step is a fermentation culture, to obtain to have accumulated in a large number the cell of PHB; Second step was separation and Extraction PHB from the cell that has accumulated PHB.Because PHB a kind ofly is present in intracellular product in the cell with graininess, molecular weight is up to more than 1,000,000, water insoluble, be only soluble in several class organic solvents such as chloroform, and solubleness is low, so separation and Extraction is very difficult, and the cost height, its separation costs accounts in the production cost of PHB mainly, and is about more than 40%.The method that the breaking cell wall that has developed at present separates PHB mainly contains solvent extration, chemical-agent technique, mechanical process and enzyme process:

(1) solvent extration is to utilize organic solvent that the PHB in the cell is dissolved and isolating method, and used organic solvent has chloroform (patent EP 0,015123 A1), dioxane (patent JP63,198,661), tetrahydrofuran (THF) and derivative thereof (patent JP07 79,788), (patent JP 07 79 for the second cyanogen and third cyanogen, 985), (patent DE 4,215 for diacetyl oxide and acetate, 860), 1,2-propylene glycol (patent AT 380,068) etc.The main drawback of solvent extration is that solvent load is big, so the cost height.

(2) chemical-agent technique is to utilize highly basic such as chemical substance such as sodium hydroxide, clorox, hydrogen peroxide (patent WO94/24, oxygenant such as 302), (patent JP 07 79 for sodium oleate, 787), sodium laurylsulfonate tensio-active agents such as (SDS), or the effect of disodium ethylene diamine tetraacetate sequestrants such as (EDTA), the non-PHB impurity in the cell is transformed into the composition of solubility and the method for removing.The shortcoming of this method is that the use meeting of chemical reagent such as clorox produces severely degrade effect (maximum can reach 50%) to the PHB molecule, and contaminate environment.

(3) enzyme process is the effect that utilizes various lyase (as protein lyase), the non-PHB impurity in the cell is degraded into the composition of solubility and the method (patent US 4,910,1445) of removing.This method need add exogenous enzyme, and separating step is many.

(4) mechanical process is a kind of breaking cell wall, release PHB particulate method, can not purifying PHB.It is utilize high pressure homogenizer be used for destroy cell walls (Harrison et al, Bioseparation, 1991,2:155-166).This method needs expensive equipment.

Generally speaking, solvent extration, chemical-agent technique, mechanical process and enzyme process that the breaking cell wall that has developed at present separates PHB all are not a kind of ideal methods, have directly influenced scale operation and the widespread use of PHB.

As the PHB of intracellular product, its isolating matter of utmost importance is the fragmentation of cell walls.Scientists finds that very early phage can lysing cell, studies show that what play a major role during the lambda particles phage lysing cell is the enzyme that its lysis genes produces.The lysis genes of lambda particles phage comprises three kinds: S, R, Rz, wherein the function of R and Rz gene product is the degradation of cell wall, and the effect of S gene product is the permeability that changes cytolemma, so that the enzyme that R and Rz gene produce passes cytolemma, arrive cell walls, thereby act on cell walls.Lysis genes SRRz is cloned in the intestinal bacteria, if three genes are expressed simultaneously, cell is with very fast death (Garrett et al.Mol.Gen.Genet.1981,182:326-331), and induce lysis genes to express at the different growing stage of cell, its lysis efficiency is different: when the logarithmic phase of cell, the lysis efficiency of cell is the highest, later on along with stepping into the stable growth phase, the lysis efficiency of cell is also along with reducing (Kloos et al, J.Bacteriol., 1994,176 (23): 7352-7361).If but the S gene is the defective gene S (-) that has amber mutation, S (-) gene will not expressed in cell, this moment the cell normal growth, and can be in cell the product of additive gene R and Rz.To accumulate cell 2% chloroform of R and Rz product, perhaps freeze thawing treatment then can make lysis (Crabtree et al, J.Bacteriol., 1984.158 (1): 354-356).The effect of chloroform and freeze thawing treatment is equivalent to the function of S gene product, promptly destroys cytolemma, allows the enzyme of R and Rz can arrive the peptidoglycan layer of cell walls, thus lysing cell.But no matter be that clone SRRz or clone S (-) RRz are used for the report that smudge cells there is no the practical application example up to the present.

The objective of the invention is the genetic engineering technique of upstream and the fermentation technique and the post-processing technology in downstream are combined, a kind of novel method for preparing poly-beta-hydroxy-butyrate is proposed, that is: make up the genetic engineering bacterium that not only contains the PHB synthetic gene but also contain the lambda particles phage lysis genes, make this bacterium both have the ability of a large amount of accumulation PHB, have again and can induce the cracked characteristic, thereby the separation method that can simplify PHB is with preparation PHB.Because in recombination bacillus coli, the accumulation of PHB is to follow the growth of cell to carry out, reach maximum during the phase at stable growth.If the lysis genes SRRz with clone's lambda particles phage comes lysing cell, when separating purpose product P HB, just exist the maximum and most effective contradiction of lysis of product accumulation volume.So the present invention selects to use defective type lysis genes S (-) RRz that has amber mutation, rather than original lysis genes SRRz, the synthetic gene of lysis genes S (-) RRz and PHB is cloned in the same host bacterium.This host bacterium of fermentation culture is when the PHB accumulation reaches maximum in the thalline, with the function of the condition simulation S gene product that adds, to change the permeability of cytolemma, make the enzyme of R and Rz can pass the peptidoglycan layer that cytolemma arrives cell walls, thereby lysing cell discharge the PHB particle.Replace original lysis genes SRRz to solve the maximum and most effective contradiction of lysis of PHB accumulation volume in the cell with lysis genes S (-) RRz.

The method for preparing poly-beta-hydroxy-butyrate of the present invention's research, form by following each step:

(1) DNA from λ (sam7 cI857) phage isolates the dna fragmentation that contains lysis genes S (-) RRz, and this fragment is inserted in the plasmid vector, and plasmid in a large number increases;

(2) isolate the dna fragmentation that contains lysis genes S (-) RRz from the plasmid vector that contains lysis genes S (-) RRz, isolate the dna fragmentation that contains the PHB synthetic gene from the plasmid vector that contains the PHB synthetic gene, connect above-mentioned two kinds of dna fragmentations, make up the material that not only contains lysis genes S (-) RRz but also contain the PHB synthetic gene;

(3) plasmid that will not only contain lysis genes S (-) RRz but also contain the PHB synthetic gene changes in the intestinal bacteria, makes up not only to have the ability of a large amount of accumulation PHB but also have the gene engineering colibacillus that can induce crack characteristic;

(4) in containing the substratum of glucose, cultivate this gene engineering colibacillus, make cell not only accumulate PHB but also accumulate the product of lysis genes R and Rz.Culture condition is: pH=5.0-7.5, shake a bottle rotating speed 150-220r/min, temperature 32-40 ℃.When cell grows into stationary phase, when the PHB accumulation volume reaches maximum in the cell, the centrifugal 10-20min of 4000-8000r/min, harvested cell:

(5) with the function treatment cell of the condition simulation S gene product that adds, the inducing cell cracking.The method of simulation S gene product function can be physics method such as pair cell freeze thawing, or chemical method contains the damping fluid re-suspended cell of sequestrant etc. as usefulness, purpose is the permeability that changes cytolemma, makes the enzyme of R and Rz can pass the peptidoglycan layer that cytolemma arrives cell walls, thus lysing cell.

(6) the centrifugal 10-20min of cell pyrolysis liquid 4000-8000r/min, collecting precipitation.Precipitation is washed with surfactant soln, the centrifugal 10-20min of 4000-8000r/min, collecting precipitation, the oven dry, get final product the PHB product, purity can reach more than 99%.

That utilizes that method of the present invention makes up not only contains the PHB synthetic gene but also contains the genetic engineering bacterium of lambda particles phage lysis genes S (-) RRz, has both had the ability of a large amount of accumulation PHB, has again and can induce the cracked characteristic.In containing the substratum of glucose during shake-flask culture in the cell content of PHB can reach and account for more than 60% of dry cell weight, and need not add the expensive inductor isopropylthio-(IPTG) of inducing lysis genes to express in the nutrient solution.Cultivating the cell of back gained can induce with the damping fluid that contains sequestrant at an easy rate, makes lysis, and the cleavage rate of cell is near 100%.After the cell induction cracking, centrifugation precipitation, but the PHB in the preliminary purification cell.Further, the purity of PHB can be brought up to more than 99% with SDS solution washing precipitation.

The method for preparing PHB with traditional microbial fermentation is compared, the present invention is in the same place the fermentation of PHB with separation and combination, have many advantages, the problem of the PHB separation difficulty that can avoid traditional microbial fermentation to prepare bringing in the method for PHB, as: the cost issues that can avoid the required expensive device of mechanical process to bring; Can avoid reagent recovery that chemical-agent technique brings, environmental pollution and to the degradation problem of PHB molecule.Compare with enzyme process, that present method has is simple to operate, lysis control is convenient, need not add advantage such as exogenous enzyme.

In addition, utilization clone lambda particles phage lysis genes S (-) RRz that the present invention created makes up the method that genetic engineering bacterium comes fermentative production and then the inducing cell cracking separates intracellular product with breaking cell wall with the purpose product gene, has wide promotion and application prospect in the preparation of other gene engineering product equally.

Description of drawings:

The gene spectrogram of Fig. 1 lambda particles phage lysis genes S (-) RRz.

The gene spectrogram of Fig. 2 plasmid pUC18.

The gene spectrogram of Fig. 3 plasmid pUC18-S (-) RRz.

The gene spectrogram of Fig. 4 plasmid pTZ18u-PHB.

The gene spectrogram of Fig. 5 plasmid pTU9.

The comparison that turbidity changed when e. coli jm109 (pTU9) cell was induced with buffer A after the LBG culture medium culturing of Fig. 6 different IP TG concentration.

Electron microscopic observation before and after Fig. 7 e. coli jm109 (pTU9) cell is induced with buffer A.

(a) induce before (magnification: 13000 *)

(b) induce afterwards (magnification: 2000 *)

The gene spectrogram of Fig. 8 material pTU14.

The LBG of Fig. 9 different IP TG concentration increases and increases the comparison of supporting turbidity variation when afterwards e. coli jm109 (pTU14) cell is induced with buffer A in the foster base.

Electron microscopic observation before and after Figure 10 e. coli jm109 (pTU14) cell is induced with buffer A.

(a) induce before (magnification: 13000 *)

(b) induce afterwards (magnification: 2000 *)

The comparison of Figure 11 large intestine bar figure JM109 (pTZ18u-PHB), e. coli jm109 (pTU9), turbidity variation when e. coli jm109 (pTU14) cell is induced with buffer A.

Electron microscopic observation before and after Figure 12 e. coli jm109 (pTZ18u-PHB) is induced with buffer A.

(a) induce before (magnification: 13000 *)

(b) induce afterwards (magnification: 13000 *)

E. coli jm109 after Figure 13 buffer A is induced (pTU14) cell precipitation is again with the SDS effect variation of PHB purity afterwards.

Introduce embodiments of the invention below:

Embodiment 1:

(1) λ (sam7 c1857) phage DNA length is 48502bp, at first cuts λ (sam7 c1857) DNA with restriction enzyme EcoRI enzyme, is divided into length and is respectively 21226,7421,5804,5643,4878, six parts of 3530bp.

The endonuclease reaction condition is: 37 ℃ of temperature, and time 1.5h, the amount ratio of enzyme and DNA is:

Plasmid or DNA 5 μ g

Damping fluid (10 *) 5 μ l

Restriction enzyme 10U

All the other volumes spend ionized water and mend fair

Cumulative volume is 50 μ l

Agarose gel electrophoresis method (work such as J. Sa nurse Brooker, molecular cloning experiment guide with DNA.Science Press, 1996,304-324) isolate the dna fragmentation that length is 3530bp.Cut the dna fragmentation that length is 3530bp (the endonuclease reaction condition is the same) with restriction enzyme Cla I enzyme then, be divided into length and be 1466bp and 2064bp two sections, lysis genes S (-) RRz is positioned on the dna fragmentation that length is 1466bp (Fig. 1), separates this fragment with the agarose gel electrophoresis method of DNA.

Cut material pUC18 (the endonuclease reaction condition is the same) with restriction enzyme A ccI and EcoR I enzyme, the restriction enzyme site of Acc I and EcoR I is positioned on the multiple clone site of plasmid pUC18 (Fig. 2).The DNA that separates the pUC18 after enzyme is cut then with the agarose gel electrophoresis method of DNA.The DNA that connects the pUC18 after the dna fragmentation that contains lysis genes S (-) RRz is cut to enzyme with the T4 dna ligase goes up (work such as J. Sa nurse Brooker, molecular cloning experiment guide.Science Press, 1996,34-49), obtain a new plasmid, called after pUC18-S (-) RRz size is 4119bp (Fig. 3).

Change this plasmid in the e. coli jm109 (work such as J. Sa nurse Brooker, molecular cloning experiment guide.Science Press, 1996,49-56), make up gene engineering colibacillus JM109 (pUC18-S (-) RRz).The LB substratum (yeast extract powder 5g/L, NaCl 10g/L, the peptone 10g/L that contain 100 μ g/ml ammonia benzyls at 5ml; PH=7.0) insert a single bacterium colony of e. coli jm109 (pUC18-S (-) RRz) in, 220r/min cultivates 10h for 37 ℃, is inoculated in 100ml with 1% inoculum size then and contains in the LB substratum of 100 μ g/ml ammonia benzyls, and the volume that shakes bottle is 250ml.37 ℃, 220r/min cultivates 6h, prepares plasmid pUC18-S (-) RRz (work such as J. Sa nurse Brooker, molecular cloning experiment guide in a large number.Science Press, 1996,24-28).

(2) cut the plasmid pTZ18u-PHB (Fig. 4) that contains the PHB synthetic gene with restriction enzyme Hind III and Ssp I enzyme, isolate the dna fragmentation that contains the PHB synthetic gene with the agarose gel electrophoresis method of DNA then; With restriction enzyme Sca I and Hind III digested plasmid pUC18-S (-) RRz, isolate the dna fragmentation that contains lysis genes S (-) RRz with the agarose gel electrophoresis method of DNA then; Connect above-mentioned two kinds of dna fragmentations with the T4 dna ligase, not only contained lysis genes S (-) RRz but also contained the plasmid of PHB synthetic gene, called after pTU9, size is 10.5kb (Fig. 5).

(3) plasmid pTU9 is changed over to (work such as J. Sa nurse Brooker, molecular cloning experiment guide in the e. coli jm109.Science Press, 1996,49-56), structure not only has the ability of a large amount of accumulation PHB but also have the gene engineering colibacillus JM109 (pTU9) that can induce crack characteristic.

(4) in containing the LB substratum of 100 μ g/ml ammonia benzyls, 5ml inserts a single bacterium colony of e. coli jm109 (pTU9), 220r/min, 37 ℃ increase foster 10h, be inoculated in 1% inoculum size then that 100ml contains 100 μ g/ml ammonia benzyls and IPTG concentration is respectively (0,0.1,0.3 in LBG substratum 0.5mmol/L) (adding 20g/L glucose in the LB substratum), the volume that shakes bottle is 250ml.37 ℃, after 220r/min cultivates 12h, 6000r/min, centrifugal 10min collecting cell, the content of PHB can reach and account for more than 40% of dry cell weight in the cell.

(5) cell is resuspended in the buffer A that contains sequestrant EDTA (2mmol/L EDTA.50mmol/L three (methylol) aminomethane (Tris), pH=8.0) in, make initial O.D.600nm value be about 7,220r/min, 37 ℃ of inducing cell cracking.The result shows, induce 20min with buffer A after, the whole cracking of cell.

Fig. 6 is resuspended in the time curve that back bacteria suspension turbidity changes in the buffer A for cell.As can be seen from Figure, IPTG concentration does not almost influence the cracking of inducing of cultivating the back cell in the substratum.The form of cell before and after inducing with buffer A with scanning electron microscopic observation, result also show, even support cell after cultivating in the base not adding increasing of IPTG, after inducing cracking 20min, do not have complete cell to exist in the solution, the cleavage rate of cell is near 100%, as shown in Figure 7.Hickie among Fig. 7 is the PHB particle, and Fig. 7 (a) induces preceding situation for buffer A: the cellular form that cell is kept perfectly, and the PHB particle is retained in the cell; Fig. 7 (b) is the situation of buffer A after inducing: do not have complete cell to exist, the PHB particle discharges from cell.IPTG is a kind of reagent of costliness, does not add IPTG, is favourable to reducing cost.

Embodiment 2

(1) with (1) of embodiment 1.

(2) with (2) of embodiment 1, just replace Sca I digested plasmid pUC18-S (-) RRz with restriction enzyme Pvu II, the resulting plasmid that had not only contained lysis genes S (-) RRz but also contained the PHB synthetic gene, called after pTU14, size is 9.0kb (Fig. 8).

(3) plasmid pTU14 is changed over to (work such as J. Sa nurse Brooker, molecular cloning experiment guide in the e. coli jm109.Science Press, 1996,49-56), structure not only has the ability of a large amount of accumulation PHB but also have the gene engineering colibacillus JM109 (pTU14) that can induce crack characteristic.

(4) in containing the LB substratum of 100 μ g/ml ammonia benzyls, 5ml inserts a single bacterium colony of e. coli jm109 (pTU14), 220r/min, cultivate 10h for 37 ℃, be inoculated in 1% inoculum size then that 100ml contains 100 μ g/ml ammonia benzyls and IPTG concentration is respectively (0,0.1,0.3 in LBG substratum 0.5mmol/L), the volume that shakes bottle is 250ml.37 ℃, after 220r/min cultivates 12h, 6000r/min, centrifugal 10min collecting cell, the content of PHB can reach and account for 48% of dry cell weight in the cell.

(5) cell is resuspended in the buffer A, makes initial O.D.600nm value be about 8.3,220r/min, 37 ℃ of inducing cell cracking.The result shows, induce 20min with buffer A after, the whole cracking of cell.

Fig. 9 is resuspended in the time curve that back bacteria suspension turbidity changes in the buffer A for cell.As can be seen from Figure, IPTG concentration does not almost influence the cracking of inducing of cultivating the back cell in the substratum.The form of cell before and after inducing with buffer A with scanning electron microscopic observation, the result also shows, even the cell after cultivating in the substratum that does not add IPTG, after inducing cracking 20min, do not have complete cell to exist in the solution, the cleavage rate of cell is near 100%, as shown in figure 10.Hickie among Figure 10 is the PHB particle, and Figure 10 (a) induces preceding situation for buffer A: the cellular form that cell is kept perfectly, and the PHB particle is retained in the cell; Figure 10 (b) is the situation of buffer A after inducing: do not have complete cell to exist, the PHB particle discharges from cell.

Embodiment 3

Not only has the ability of a large amount of accumulation PHB but also have on the basis of the gene engineering colibacillus JM109 (pTU14) that can induce crack characteristic what embodiment 2 made up, the chemical method that present embodiment research is used with chloroform give is simulated the function of S gene product, with the inducing cell cracking.

In containing the LB substratum of 100 μ g/ml ammonia benzyls, 5ml inserts a single bacterium colony of e. coli jm109 (pTU14), 220r/min, cultivate 10h for 37 ℃, be inoculated in 100ml with 1% inoculum size then and contain in the LBG substratum of 100 μ g/ml ammonia benzyls, the volume that shakes bottle is 250ml.37 ℃, after 220r/min cultivated 12h, cell grew into stationary phase, adds the 2ml chloroform in cell culture fluid, and 37 ℃, 220r/min continues to cultivate, and uses microscopic examination behind the 1h, does not have complete cell to exist, and the cleavage rate of cell is near 100%.

Embodiment 4

Not only has the ability of a large amount of accumulation PHB but also have on the basis of the gene engineering colibacillus JM109 (pTU14) that can induce crack characteristic what embodiment 2 made up, present embodiment is studied the function of simulating the S gene product with the physics method of unfreezing, with the inducing cell cracking.

In containing the LB substratum of 100 μ g/ml ammonia benzyls, 5ml inserts a single bacterium colony of e. coli jm109 (pTU14), 220r/min, cultivate 10h for 37 ℃, be inoculated in 100ml with 1% inoculum size then and contain in the LBG substratum of 100 μ g/ml ammonia benzyls, the volume that shakes bottle is 250ml.37 ℃, after 220r/min cultivated 12h, cell grew into stationary phase, after-20 ℃ refrigerator and cooled was frozen 10h, in 37 ℃, 220r/min made it to melt fully with cell culture fluid, use microscopic examination behind the 1h, do not have complete cell to exist, the cleavage rate of cell is near 100%.

Embodiment 5

Present embodiment to the band lysis genes of embodiment 1 and embodiment 2 with do not induce the crack characteristic of back cell not compare with buffer A with the e. coli jm109 cell of lysis genes, purpose is that the cracking of explanation cell is because accumulated in the cell due to the product of lysis genes R and Rz.

Change the plasmid pTZ18u-PHB that only contains the PHB synthetic gene in the e. coli jm109 (work such as J. Sa nurse Brooker, molecular cloning experiment guide.Science Press, 1996,49-56), make up the gene engineering colibacillus JM109 (pTZ18u-PHB) that produces PHB.

The e. coli jm109 (pTU9) and the e. coli jm109 (pTU14) that make up with e. coli jm109 (pTZ18u-PHB) with in

embodiment

1 and 2 are cultivated in the LBG substratum respectively: contain single bacterium colony of access in the LB substratum of 100 μ g/ml ammonia benzyls at 5ml, 220r/min, cultivate 10h for 37 ℃, be inoculated in 100ml with 1% inoculum size then and contain in the LBG substratum of 100 μ g/ml ammonia benzyls, the volume that shakes bottle is 250ml.37 ℃, after 220r/min cultivates 12h, 6000r/min, centrifugal 10min collecting cell.The content of PHB can reach and account for 50% of dry cell weight in e. coli jm109 (pTZ18u-PHB) cell.

6000r/min, centrifugal 10min collect the cell of e. coli jm109 (pTZ18u-PHB), e. coli jm109 (pTU9) and e. coli jm109 (pTU14) respectively.Be resuspended in cell in the buffer A respectively, 220r/min, 37 ℃ of inducing cell cracking, the reduction of inducing back e. coli jm109 (pTZ18u-PHB) cell suspension turbidity is than e. coli jm109 (pTU9) and e. coli jm109 (pTU14) much smaller (Figure 11).The result of scanning electron microscopic observation also shows, after e. coli jm109 (pTZ18u-PHB) cell is handled 20min with buffer A, and the cellular form that cell still is kept perfectly, as shown in figure 12.Hickie among Figure 12 is the PHB particle, and Figure 12 (a) induces preceding situation for buffer A, the cellular form that cell is kept perfectly, and the PHB particle is retained in the cell; Figure 12 (b) is the situation of buffer A after inducing: the cellular form that cell is kept perfectly, the PHB particle is retained in the cell.This illustrates that also the internal cause that causes lysis is that lysis genes S (-) RRz expresses, and has accumulated the product of gene R and Rz in cell, the function of S gene product is just simulated in the effect of buffer A, is the external cause that causes lysis.

Embodiment 6

The purpose of present embodiment is after investigating e. coli jm109 (pTU14) cell that embodiment 2 makes up and cultivating in containing the rich medium of glucose, under the situation that PHB content is higher in the cell, cell induce crack characteristic.

In containing the LB substratum of 100 μ g/ml ammonia benzyls, 5ml inserts a single bacterium colony of e. coli jm109 (pTU14), 220r/min, cultivate 10h for 37 ℃, be inoculated in 100ml with 1% inoculum size then and contain increasing of 100 μ g/ml ammonia benzyls foster basic C (yeast extract powder 1.0g/L, peptone 5.0g/L, extractum carnis 3.0g/L, NaCl5.0g/L, glucose 20g/L) in, 220r/min, behind 37 ℃ of cultivation 16h, the content of PHB can reach and account for more than 60% of dry cell weight in the cell.6000r/min, centrifugal 10min collecting cell.Cell is resuspended in the buffer A, makes initial O.D.600nm value be about 10,220r/min, 37 ℃ of inducing cell cracking behind the 20min, do not have complete cell to exist, and the PHB particle discharges from cell, and the cleavage rate of cell is near 100%.

Embodiment 7

The purpose of present embodiment is to investigate the different PHB content that made up by embodiment 2 at e. coli jm109 (pTU14) cell, induces after crack characteristic and the lysis removal situation of non-PHB impurity in the cell after inducing with buffer A.

When e. coli jm109 (pTU14) was cultivated, the content of PHB and culture condition such as inoculum size in cell concn that can reach in the nutrient solution and the cell, to shake bottle rotating speed etc. relevant.The cell of cultivating the different PHB content of gained under the different condition is induced with buffer A respectively, in all cracking fully of 20min inner cell, the not influence of cracking of the PHB pair cell that accumulates in this explanation cell.

After cell was induced cracking with buffer A, the non-PHB composition of some in the cell was dissolved in the buffer A in this process, 6000r/min, and after centrifugal 10min removes supernatant liquor, but the PHB in the preliminary purification cell, the result is as shown in table 1.Induce the content of PHB in the preceding cell high more, the content of PHB is also high more in the precipitation that is obtained after inducing.The cell of the different PHB content of table 1 induces before relatively the inducing of back PHB content PHB in the cell to induce behind the postprecipitation content P of non-PHB material in the cell with buffer A _PHBO(%) the content P of PHB _PHBClearance θ (%) (%)

41.10 69.03 68.69

43.23 71.72 69.95

46.29 74.70 70.81

60.61 85.15 73.17

66.07 90.53 79.63

The clearance θ of non-PHB impurity is defined as:

Embodiment 8

The purpose of present embodiment is the method that is further purified PHB on the basis of embodiment 7 cell of research after cracking, to obtain highly purified PHB product.

In containing the LB substratum of 100 μ g/ml ammonia benzyls, 5ml inserts a single bacterium colony of e. coli jm109 (pTU14), 220r/min, cultivate 10h for 37 ℃, being inoculated in 50ml with 5% inoculum size then contains in the culture medium C of 100 μ g/ml ammonia benzyls, 220r/min, behind 37 ℃ of cultivation 16h, cell concentration is 4.25g/L, and the content of PHB can reach and account for 66% of dry cell weight in the cell.6000r/min, centrifugal 10min collecting cell.Cell is suspended with the 25ml buffer A, and making dried bacteria concentration is 8.5g/L, and 220r/min handles 20min, inducing cell cracking for 37 ℃.6000r/min, centrifugal 10min precipitation separation, PHB content can bring up to 90.5% in the precipitation.Precipitation is further used the SDS solution 220r/min of different concns, behind 37 ℃ of effect 10min, and 6000r/min, centrifugal 10min precipitation separation can be further purified PHB, and the result when the SDS consumption is 4g/L, can obtain purity and be 99.66% PHB as shown in figure 13.

Claims

1, a kind of method for preparing poly-beta-hydroxy-butyrate is characterized in that being made up of following each step:

(1) DNA from λ (sam7 cI857) phage isolates the dna fragmentation that contains lysis genes S (-) RRz, this fragment is inserted in the plasmid vector amplification plasmid;

(2) isolate the dna fragmentation that contains lysis genes S (-) RRz from the plasmid vector that contains lysis genes S (-) RRz, isolate the dna fragmentation that contains the PHB synthetic gene from the plasmid vector that contains the PHB synthetic gene, connect above-mentioned two kinds of dna fragmentations, make up the plasmid that not only contains lysis genes S (-) RRz but also contain the PHB synthetic gene;

(3) plasmid that will not only contain lysis genes S (-) RRz but also contain the PHB synthetic gene changes in the intestinal bacteria, makes up not only to have the ability of accumulation PHB but also have the gene engineering colibacillus that can induce crack characteristic;

(4) in containing the substratum of glucose, cultivate this gene engineering colibacillus, make cell not only accumulate PHB but also accumulate the product of lysis genes R and Rz, culture condition is: pH=5.0-7.5, shake a bottle rotating speed 150-220r/min, temperature 32-40 ℃, the centrifugal 10-20min of 4000-8000r/min then, harvested cell;

(5) with the function treatment cell of the condition simulation S gene product that adds, the inducing cell cracking, the method of simulation S gene product function is the cell freeze thawing, perhaps for using the damping fluid re-suspended cell that contains sequestrant, change the permeability of cytolemma, make the enzyme of R and Rz pass the peptidoglycan layer that cytolemma arrives cell walls, thus lysing cell;

(6) the centrifugal 10-20min of cell pyrolysis liquid 4000-8000r/min, collecting precipitation, precipitation is washed with surfactant soln, the centrifugal 10-20min of 4000-8000r/min, collecting precipitation, oven dry promptly gets the PHB product.