CN1902309A - Method of decomposing polyhydroxyalkanoate resin - Google Patents
Method of decomposing polyhydroxyalkanoate resin Download PDFInfo
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- CN1902309A CN1902309A CNA2004800398780A CN200480039878A CN1902309A CN 1902309 A CN1902309 A CN 1902309A CN A2004800398780 A CNA2004800398780 A CN A2004800398780A CN 200480039878 A CN200480039878 A CN 200480039878A CN 1902309 A CN1902309 A CN 1902309A
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Abstract
A novel microorganism which directly biologically decomposes polyhydroxyalkanoate resins and plastics containing these; and a method of decomposing such a resin or plastic. The method, which is for decomposing a polyhydroxyalkanoate resin, is characterized by decomposing the polyhydroxyalkanoate resin with a thermophile belonging to the genus Streptomyces.
Description
Technical field
The present invention relates to a kind of by means of the method for using new biodegradation method decomposing polyhydroxyalkanoateresin.
Background technology
In recent years, Plastic wastes are handled and have been become problem.Plastic wastes are handled mainly and are undertaken by incineration or landfill.Incineration is problematic, because it has promoted global heating, and landfill is because such as reduce former of the floor area that can be used for this thereby also be problematic.Therefore, biodegradation method has caused people's attention.Polyhydroxyalkanoateresin resin has biological degradability, makes to have developed the various application of this resin as plastics of future generation.In the near future, be similar to the situation of the plastics of current use, the refuse problem of this type of polyhydroxyalkanoateresin resin will occur relating to.
Polyhydroxyalkanoatefrom is a kind of polymkeric substance of hydrolysis in soil or in water system.Polyhydroxyalkanoatefrom is as the substituting bio-degradable plastics material of plastics of the general purpose that is difficult to environment degradable and well-known.Depend on its formation monomer type, the example of this type of polyhydroxyalkanoateresin resin comprises: poly butyric ester, poly-hydroxyl valerate, poly-hydroxycaproic ester, poly-hydroxyl heptanoate, poly-Hydroxyoctanoic acid ester, poly-hydroxyl pelargonate, poly-hydroxydecanoic acid ester and its multipolymer.
Poly butyric ester is a kind of (referring to the non-patent literature 1 and 2) of polyhydroxyalkanoatefrom of the energy that is known as in cell the various microorganisms of conduct of synthetic and storage.In addition, also known poly butyric ester is degraded by aerobic microorganism (non-patent literature 3 to 5) or anaerobion (patent documentation 1 and non-patent literature 6 and 7).
Patent documentation 1: Japanese Patent No.2889953
Non-patent literature 1: " Journal of Bacteriology, " be 1965,90 volumes (U.S.A.), the 1455-1466 page or leaf
Non-patent literature 2: " Macromolecule Bioscience, " 2001,1 volumes, 1-24 page or leaf
Non-patent literature 3: " Journal of Environmental Polymer Degradation, " be 1993,1 volumes (U.S.A.), the 53-63 page or leaf
Non-patent literature 4: " Journal of Environmental Polymer Degradation, " be 1993,1 volumes (U.S.A.), the 227-233 page or leaf
Non-patent literature 5: " Archives of Microbiology, " be 1996,154 volumes (Germany), the 253-259 page or leaf
Non-patent literature 6: " Polymer Symposium " 1993,50 volumes, 745-746 page or leaf
Summary of the invention
The purpose that the present invention will realize
The DeR of the polyhydroxyalkanoatefrom that great majority have been reported so far is limited under 25 ℃ to 30 ℃ temperature carries out.It is believed that also that research fully is used for the technology of the active degraded of this type of polyhydroxyalkanoateresin resin under hot conditions, such as belonging to those of fertilizer.Therefore, the purpose of this invention is to provide a kind of can be at about 40 ℃ to 60 ℃ following these type of polyhydroxyalkanoateresin resins of degraded and comprise the new microorganism of the plastics of this resin, and provide a kind of method that is used for this.
The mode of dealing with problems
Because screening and thorough research widely is realizing above-mentioned purpose, so the inventor has had been found that actinomycetes (actinomycete) with active streptomyces of good decomposing polyhydroxyalkanoateresin (Streptomyces) and the enzyme (having identical activity) for preparing by means of actinomycetes by microbial process.Thereby the inventor has finished the present invention.
Specifically, the invention provides following (1) to (9):
(1) a kind of actinomycetic enzyme derived from streptomyces, it can decomposing polyhydroxyalkanoateresin resin, has at the molecular weight between about 47,000 to 56,000, in best pH between 4 to 10 and the optimum temps between 40 ℃ to 55 ℃;
(2) according to the enzyme of top (1), it induces preparation by polyhydroxyalkanoatefrom, hydroxybutyric acid, poly butyric ester and/or hydroxy butyrate;
(3) according to the enzyme of top (1) or (2), wherein the actinomycetes of streptomyces are hot common streptomycete (Streptomyces thermovulgaris), hot olive streptomycete (Streptomycesthermoolivaceus), heat absorbing water streptomycete (Streptomyces thermohygroscopicus) or Streptomyces thermocarboxydovorans;
(4) according to the enzyme of top (1) or (2), wherein the actinomycetes of streptomyces are the microorganisms with preservation registration number FERM P-19578 preservation.
(5) a kind of method of decomposing polyhydroxyalkanoateresin resin, it comprises makes polyhydroxyalkanoateresin resin contact so that this resin and enzyme reaction with each enzyme according to top (1) to (4);
(6) a kind of method of decomposing polyhydroxyalkanoateresin resin, it comprise polyhydroxyalkanoateresin resin is contacted with the actinomycetes of streptomyces so that this resin and actinomycetes 40 ℃ to 55 ℃ reactions;
(7) according to the method for top (6), wherein the actinomycetes of streptomyces are hot common streptomycete, hot olive streptomycete, heat absorbing water streptomycete or Streptomyces thermocarboxy-dovorans;
(8) according to the method for top (6), wherein the actinomycetes of streptomyces are the microorganisms with preservation registration number FERM P-19578 preservation; With
(9) a kind of actinomycetes of streptomyces, it can decomposing polyhydroxyalkanoateresin resin and is microorganism with preservation registration number FERM P-19578 preservation.
The invention effect
The method of decomposing polyhydroxyalkanoateresin resin of the present invention is a kind of method that is used to handle this type of polyhydroxyalkanoateresin resin and comprises its waste material.This method is based on such technology: it can carry out not generating under any condition such as the waste gas that is generated in traditional incineration process method situation.In addition, compare with landfill disposal, this method is extremely saved time.Thereby this method is very valuable for waste treatment.Specifically, the method that useful being used for of a kind of more environment degraded as the polyhydroxyalkanoateresin resin of bio-degradable plastics is not to relate to simply to wait for resin at the soil natural degradation, but degradative resin by means of the microorganism of using this resinoid of can degrading or enzyme and on one's own initiative.In the equipment of making fertilizer, use degradation method of the present invention can also make polyhydroxyalkanoateresin resin change into useful material, such as organic acid or fertilizer.In addition, use method of the present invention to help the regeneration of polyhydroxyalkanoateresin resin.
This specification sheets has comprised the part or all of content that discloses in the specification sheets of Japanese patent application No.2003-376263 and/or accompanying drawing, this application is the application's a priority document.
The accompanying drawing summary
Fig. 1 represents the variation of the time relevant with passing through MG2 strains for degrading poly butyric ester toner.The residual PHB content (mg) of " ● " and " zero " expression, and the water miscible total organic carbon content of " ■ " and " " expression (TOC, mg), " ◆ " expression stem cell weight (mg), the result of " zero " and " " expression control sample.
Fig. 2 represents the pH stability of enzyme of the present invention.
Fig. 3 represents the temperature stability of enzyme of the present invention.Pre-incubation time under each temperature of every kind of symbolic representation (■: 15 minutes, ●: 30 minutes, ◆: 45 minutes and ▲: 60 minutes).
Implement best mode of the present invention
The invention provides a kind of new actinomycetic enzyme, this endonuclease capable decomposing polyhydroxyalkanoateresin resin derived from streptomyces.Enzyme of the present invention has at the molecular weight between about 47,000 to 56,000, between 4 to 10 and preferred best pH between 7 to 8, and the optimum temps between 40 ℃ to 55 ℃.In addition, have been found that in the presence of polyhydroxyalkanoatefrom, hydroxybutyric acid, poly butyric ester and/or hydroxy butyrate, to induce and prepare enzyme of the present invention.
Term among the present invention " polyhydroxyalkanoateresin resin " is meant polymkeric substance or its multipolymer of polyhydroxybutyrate, poly-hydroxypentanoic acid, poly-hydroxycaproic acid, poly-hydroxyl enanthic acid, poly-Hydroxyoctanoic acid, poly-hydroxyl n-nonanoic acid, poly-hydroxydecanoic acid.In addition, the example of this type of polyhydroxyalkanoateresin resin comprises and uses chemical catalyst by gamma-butyrolactone and another component (for example, beta-propiolactone, beta-butyrolactone, beta-butyrolactone, 6-caprolactone or ω-pentadecalactone) the polyhydroxy-acid multipolymer that obtains of copolymerization, the product of the blend acquisition by above polymkeric substance, with the product that blend by above polymkeric substance and another component polymer obtains, wherein the weight percentage of hydroxy alkane acid ester component is 10% or bigger in each polymkeric substance.In addition, can be used for the number-average molecular weight of polyhydroxyalkanoatefrom of degradation method of the present invention greatly about 10,000 to 10
6Between, and preferably approximately is between 50,000 to 300,000.The present invention is not confined to above-mentioned example especially.The example of this type of polyhydroxyalkanoatefrom of known commercially available acquisition form comprises the multipolymer (by Sigma-Aldrich, Inc. produces) of poly butyric ester and poly butyric ester and poly-hydroxyl valerate.Yet method of the present invention is not limited to their use.
Enzyme of the present invention can be obtained by the actinomycetes of streptomyces, and these actinomycetes for example are hot common streptomycete, hot olive streptomycete, heat absorbing water streptomycete or Streptomyces thermocarboxydovorans.
Be used for mechanism that microorganism culturing collects for example physics and chemistry institute (The Institute ofPhysics and Chemical Research, 2-1 Hirosawa, Wako-shi, Saitama, Japan) in, the actinomycetes of above streptomyces are saved and can obtain.In the present invention, use a kind of bacterial strain or the multiple bacterial strain that is preferably selected from hot common streptomycete (JCM4338) [Streptomyces thermovulgaris (JCM4338)], hot olive streptomycete (JCM4921) [Streptomyces thermoolivaceus (JCM4921)], heat absorbing water streptomycete (JCM4917) [Streptomyces thermohygroscopicus (JCM4917)] and Streptomycesthermocarboxydovorans (JCM10367).Yet, be used for bacterial strain of the present invention and be not confined to these bacterial strains especially.
The inventor has also found a kind of actinomycetes of new streptomyces, and it generates above-mentioned enzyme from soil.The following acquisition of actinomycetes of this new streptomyces.(number-average molecular weight (Mn) is 2.1 * 10 with poly butyric ester
5) be dispersed on every kind of nutrient agar.Soil (collecting at Tsukuba-shi) is placed on the egative film that contains this type of nutrient agar, cultivates at 50 ℃ then.From the bacterium that forms the clear zone, obtain new actinomycetes.From be proved to be bacterial strain, in 24 hours of cultivating, form tangible clear zone and have extra high active bacterial strain and be named as the MG2 bacterial strain with degrading activity.In the world, the international monopoly that the MG2 bacterial strain is preserved in Independent Administrative Leged Industrial Technology Complex Inst according to the rule of budapest treaty with preservation registration number FERM P-19578 (FERM ABP-10158) on November 4th, 2003 is organized the preservation center, and [(TsukubaCentral 6 for International Patent Organism Depositary ofNational Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan)].Above-mentioned actinomycetes strain (MG2) can be cultivated under the temperature between 25 ℃ to 60 ℃, and shows good propagation at 50 ℃ especially.As kind being the result of analysis, we find that the MG2 bacterial strain is a kind of new bacterial species, and it shows respectively 97.0% and 96.5% sequence similarity with Streptomyces thermocarboxydovorans and hot streptomyces diastaticus (Streptomycesthermodiastaticus).
The actinomycetes of above-mentioned streptomyces demonstrate and can (in the outside of bacterial body) secrete above-mentioned enzyme of the present invention in substratum.Enzyme of the present invention has as the molecular weight between about 47,000 to 56,000 by the SDS-PAGE electrophoretic analysis.
The present invention also provides a kind of method of decomposing polyhydroxyalkanoateresin resin, and it comprises makes polyhydroxyalkanoateresin resin contact so that this resin and enzyme reaction with above-mentioned enzyme of the present invention.
The present invention also provides a kind of method of decomposing polyhydroxyalkanoateresin resin, it comprise polyhydroxyalkanoateresin resin is contacted with the actinomycetes of streptomyces so that this resin and actinomycetes 40 ℃ to 55 ℃ reactions.
Method of the present invention makes it possible to decomposing polyhydroxyalkanoateresin resin, uses the actinomycetes of streptomyces thus or degrades derived from this actinomycetic enzyme.
The actinomycetic example that preferably is used for the streptomyces of the inventive method comprises hot common streptomycete, hot olive streptomycete, heat absorbing water streptomycete and Streptomyces thermocarboxydovorans.
The actinomycetes that further preferably are used for the streptomyces of the inventive method are to belong to the actinomycetes of depositing in the bacterial strain under the preservation registration number FERM P-19578 in the world.
Particularly preferred actinomycetic example is that the inventor is with the MG2 bacterial strain of preservation registration number FERM P-19578 preservation.
Method of the present invention comprises: in containing the substratum of inorganic salt with the MG2 bacterial strain of polyhydroxyalkanoatefrom and actinomycetes one above-mentioned streptomyces, hot common streptomycete bacterial strain, hot olive streptomycete bacterial strain, heat absorbing water streptomycete bacterial strain or Streptomyces thermocarboxydovorans bacterial strain, perhaps cultivate, thus decomposing polyhydroxyalkanoateresin derived from this type of actinomycetic enzyme.
Above-mentioned bacterial strains can liquid form use and be used for the degraded of polyhydroxyalkanoateresin resin.Specifically, for example be supplemented with by alkaline medium and in the minimal medium that contains nitrogenous source of 50ppm to 500ppm yeast extract above-mentioned bacterial strains cultivated and growth prepares this type of nutrient solution in the cultivation that is suitable for them.If necessary, the thalline of above-mentioned bacterial strains can cryodesiccated powder (by the standard method lyophilize) form or be used for the degraded of polyhydroxyalkanoateresin resin with the form of solid preparation, this solid preparation is for example for by following the tablet that the gains compressing tablet is made with thus obtained powder and nutrition source (for example yeast extract, casamino acids or the peptone) blend of various VITAMIN or mineral substance and necessity.
The alkaline medium that is used in the present invention cultivate comprises inorganic salt.The example that is generally used for the cultivation source of this type of substratum comprises nitrogenous source, for example for example potassium primary phosphate, dipotassium hydrogen phosphate, sal epsom, sodium-chlor, ferrous sulfate, Sodium orthomolybdate, sodium wolframate or manganous sulfate of ammonium sulfate, ammonium phosphate or volatile salt and another kind of inorganic salt.Different with the conventional substratum that is used for microorganism, can add a spot of yeast extract, casamino acids, peptone, malt extract etc. effectively.In addition, (DAI-ICHI KOGYO SEIYAKU CO., LTD.), perhaps the octyl group glucosides comes the polyhydroxyalkanoatefrom of dispersed powders shape also can to use Plysurf as tensio-active agent.In addition, in order to induce the preparation of enzyme that can decomposing polyhydroxyalkanoateresin resin, it is effective that polyhydroxyalkanoatefrom, hydroxybutyric acid, poly butyric ester or hydroxy butyrate are added substratum.The pH that is used for substratum is between 4.0 to 10.0, preferably between 5.0 to 8.0.The temperature that is used to cultivate is between 25 ℃ to 70 ℃, preferably between 40 ℃ to 55 ℃.
When by by the actinomycetes of streptomyces for example when the enzyme of MG2 bacterial strain, hot common streptomycete bacterial strain, hot olive streptomycete bacterial strain, heat absorbing water streptomycete bacterial strain or the preparation of Streptomyces thermocarboxydovorans bacterial strain and the method according to this invention decomposing polyhydroxyalkanoateresin resin, this fermentoid can use with the pH regulator agent of suitable interpolation, stablizer, vehicle, tensio-active agent etc.
Preferably by means of with the wherein a kind of of wherein a kind of, the pending polyhydroxyalkanoateresin resin of above-mentioned alkaline medium and above-mentioned bacterial strains or comprise wherein blend and have the powder of this type of bacterial strain, tablet or nutrient solution to add culture vessel and be used for the biodegradation method of polyhydroxyalkanoateresin resin of the present invention.Above-mentioned bacterial strains can be mixed activatory mud or fertilizer.With regard to degradation efficiency, most preferably in advance with the polyhydroxyalkanoateresin resin powdered.Polyhydroxyalkanoateresin resin can be the form of film.In addition, will be added into the amount of the polyhydroxyalkanoatefrom in the alkaline medium preferably between 0.01wt.% to 10wt.%.Microorganism can minimum add.The amount that will be added into the microorganism in the polyhydroxyalkanoateresin resin is preferably 0.01wt.% or more, and this makes the amount that will add to not influence of degradation time.
Degrade that the required time is depended on following factor and different: for example composition of polyhydroxyalkanoateresin resin, form and quantity, the type of the microorganism of use, this quasi-microorganism are with respect to the amount of enzyme or resin and other multiple culture condition.Therefore, can not stipulate fully the required time of degrading.By these conditions are kept several minutes, a few week or some months, successfully decomposing polyhydroxyalkanoateresin resin.
Embodiment
To the present invention further be described particularly by reference embodiment.Yet scope of the present invention is not subjected to the restriction of these embodiment.
The actinomycetic acquisition of streptomyces that (embodiment 1) is new
(number-average molecular weight (Mn) is 2.1 * 10 with poly butyric ester
5, by " BIOGREEN " of MITSUBISHI GASCHEMICAL COMPANY.INC. production) and with above 1% nutrient agar (being supplemented with having of the agar substratum of forming as shown in table 2) that is scattered on the egative film.The soil that Tsukuba-shi is collected is placed on the egative film, cultivates at 50 ℃ then.In the middle of the bacterium that forms the clear zone, obtain new actinomycetes.
The actinomycetic biological characteristic of thus obtained new streptomyces is listed in the table 1.
The biological characteristic of table 1 MG2 bacterial strain
| Feature | MG2 result |
| Form gramstaining motility catalase oxydase glucose OF aIn fermentability | Irregular, folding and grey black+-++-(oxidation) |
| The acid glucose peptone of the 25 ℃ of 30 ℃ of 50 ℃ of 55 ℃ of 60 ℃ of yeast Fructus Hordei Germinatus+50 μ/ml ovobiocin agar pH10 growth oatmeal agar pH10 melanocyte pigment tyrosine xanthine elastin laminin starch casein yolk agar aesculin gelatin of growing | -+++-+++---+++do not grow++ |
OF
a: oxidative fermentation solution
From thus obtained bacterial strain, obtain 16S-rRNA according to standard method, then specified sequence.Sequence is shown among the SEQ ID NO:1.
As with the sequence of this sequence and the known bacterium of various routines result relatively, find that this bacterium has respectively 97.0% sequence identity and 96.5% sequence identity with Streptomyces thermocarboxydovrans and hot streptomyces diastaticus (Streptomyces thermodiastaticus).Therefore show that this bacterial strain is not conventional known new actinomycetes.The inventor will this new actinomycetes called after MG2 bacterial strain.
The activity of (embodiment 2) degraded poly butyric ester toner
The MG2 bacterial strain that obtains among the embodiment 1 the degraded of upchecking to the PHB powder.
100ml is had shown in the following table 2 alkaline medium formed and 200mg PHB powder, and (number-average molecular weight (Mn) is 2.1 * 10
5" BIOGREEN " by MITSUBISHI GAS CHEMICAL COMPANY.INC. production; Because particle is uniform grading by the genus silk thread screen cloth of 250 μ m) join in the Erlenmeyer flask of 500ml.The MG2 strain cultured solution of 5ml is added flask, and subsequently in gyrate shaker (180rpm) 50 ℃ of cultivations.
Table 2:
| Component | Combined amount (every liter of distilled water) |
| Na 2MoO 4·H 2O Na 2WO 4·2H 2O FeSO 4·7H 2O CaCl 2·2H 2O NaCl MgSO 4·7H 2O (NH 4) 2·SO 4 K 2HPO 4 KH 2PO 4Yeast extract | 0.5mg 0.5mg 10mg 20mg 100mg 200mg 1000mg 1600mg 200mg 100mg |
Used chloroform that substratum is extracted in per 12 hours.Therefore collect undegradable polymkeric substance and use the rotatory evaporator drying then.In addition, also with cell harvesting, centrifugation and dry then.Use TOC-5000 analyzer (Shimadzu Corporation) to measure water miscible organic carbon (TOC).In addition, use 4mM perchloric acid to use the HPLC of sulfonate polystyrene gel post, and analyze degraded products at 40 ℃ as moving phase (0.6 ml/min).Simultaneously, use the bioanalysis instrument (Boehringer Mannheim/R-Biopharm) of enzyme to detect the D-3 hydroxybutyric acid.
The result passes through 24 hours cultivation, also observes 50% PHB powder degraded water miscible total organic carbon (TOC) gathering (Fig. 1) in cell.Detecting D-3 hydroxybutyric acid, hexanodioic acid and succsinic acid in nutrient solution assembles.By 3 days cultivation, the PHB powder was degraded fully.
The degraded of (embodiment 3) poly butyric ester film
The degraded of the PHB film (using the cast film of " BIOGREEN " preparation of producing) of the MG2 bacterial strain that uses scanning electronic microscope to upcheck to obtain among the embodiment 1 by MITSUBISHI GAS CHEMICAL COMPANY.INC..
Cultivate under the condition of those in being similar to embodiment 2.Owing to stick to the effect of the cell on the film, therefore on film, form dome-type hole.Cultivation caudacoria at 6 days is degraded fully.
The various actinomycetic degrading activity of (embodiment 4) streptomyces
(number-average molecular weight (Mn) is 2.1 * 10 with poly butyric ester
5) be scattered on the nutrient agar on the egative film.The actinomycetes strain that on egative film various Streptomycin is belonged to is inoculated and separately then 50 ℃ of cultivations.The degree that the clear zone forms is: +++(in 1 to 2 day, forming), ++ (in 5 to 7 days, forming) ,+(in 8 to 14 days, forming) and-(not formation).
Table 3
| Bacterial strain (JCM registration number) | Temperature of reaction (℃) | The clear zone forms |
| | 50 | +++ |
| S.thermovulgaris(4338) | 50 | +++ |
| S.thermoolivaceus(4921) | 50 | ++ |
| S.thermophilus(4336) | 50 | - |
| S.thermoviolaceous(4437) | 50 | - |
| S.thermohygroscopicus(4917) | 45 | +++ |
| S.thermocarboxydovorans(10367) | 45 | +++ |
| S.thermodiastaticus(4314) | 45 | - |
| S.thermodiastadicus(4840) | 45 | - |
Result from table 3 is clear that, has formed tangible clear zone by MG2 bacterial strain, hot common streptomycete (S.thermovulgaris), hot olive streptomycete (S.thermoolivaceus), heat absorbing water streptomycete (S.thermohygroscopicus) and the Streptomyces thermocarboxydovorans that obtains among the embodiment 1.Confirmed that these bacterial strains have the activity of degraded poly butyric ester.
The various actinomycetic degrading activity of (embodiment 5) streptomyces
(powder changes into 180 microns or littler size with the 200mg poly butyric ester; Number-average molecular weight (Mn) is 2.1 * 10
5) have in the substratum (pH7.0) of the composition shown in the table 4 as carbon source adding 100ml.Every kind of bacterial strain on every kind of substratum, listing in the inoculation table 5, and use the 180-rpm gyrate shaker to cultivate 4 days at 50 ℃ subsequently.
After the Powdered poly butyric ester degraded that adds, calculate water miscible total organic carbon (TOC, ppm) content.The results are shown in Table 5.Under the situation that adds according to the bacterium with degradation capability of the present invention, total organic carbon content of Sheng Chenging is 16ppm to 937ppm thus.
Table 4
| Component | Combined amount (every liter of distilled water) |
| Na 2MoO 4·H 2O Na 2WO 4·2H 2O FeSO 4·7H 2O CaCl 2·2H 2O NaCl MgSO 4 (NH4) 2SO 4 K 2HPO 4 KH 2PO 4Yeast extract | 0.5mg 0.5mg 10mg 100mg 200mg 1000mg 1600mg 200mg 100mg 200mg |
Table 5
| Bacterial strain | Temperature (℃) | Total dissolved organic carbon content (ppm) |
| The MG2 | 50 | 937 |
| Streptomyces thermovulgaris bacterial strain (JCM4438) | 50 | 13 |
| Streptomyces thermoolivaceus bacterial strain (JCM4921) | 50 | 16 |
| Streptomyces thermohygroscopicus (JCM4917) | 45 | 123 |
| Streptomyces thermocarboxydovorans (JCM10367) | 45 | 16 |
As mentioned above, having confirmed the MG2 bacterial strain--a kind of actinomycetes of-streptomyces, hot common streptomycete bacterial strain, hot olive streptomycete bacterial strain, heat absorbing water streptomycete bacterial strain and Streptomycesthermocarboxydovorans bacterial strain can be degraded as the poly butyric ester of polymkeric substance.
The pH stability of (embodiment 6) enzymic activity
Use the MG2 bacterial strain check that obtains among the embodiment 1 to make the pH stability of the enzymic activity of polyhydroxyalkanoatefrom degraded.
The MG2 bacterial strain is added 100ml contain 100mg PHB powder and have in the substratum of the composition shown in the table 4, be accompanied by at 50 ℃ then and shake and cultivated 48 hours.After the filtration, filtrate is dialysed to 0.01M phosphate buffered saline buffer (pH 7.0).The damping fluid (for example, the pH3.0 of 3.9ml, 3.5,4.0 or 5.0 citrate buffer (0.1M) and the pH5.5,6.0,6.5,7.0 of 3.9ml, 7.5 or 8.0 phosphate buffered saline buffer (0.1M)) that will have various pH adds in the enzyme solution (1ml respectively does for oneself) of dialysis thus.5 ℃ cultivate 24 hours after, in 50 ℃ of damping fluids that containing 10mg PHB and 0.5% (w/v) octyl group glucopyranoside, reacted 16 hours.Measure water miscible total organic carbon and the content of DL-hydroxybutyric acid sodium salt in each substratum in the mode that is similar to embodiment 2.The result of detection DL-hydroxybutyric acid sodium salt as shown in Figure 2.
Shown in the result among Fig. 2, confirmed in the pH scope of enzyme of the present invention between 4 to 10 stable.Especially, the pH scope endoenzyme between 5 to 8 can keep its high reactivity.
The temperature stability of (embodiment 7) enzymic activity
Check the temperature stability of the enzymic activity of the MG2 bacterial strain that makes the polyhydroxyalkanoatefrom degraded.
Cultivated bacterial strain 48 hours.The substratum of sampling 5ml is accompanied by at 50 ℃, 60 ℃, 70 ℃, 80 ℃ or 90 ℃ then and shakes and cultivated 60 minutes.After 4 hours, measure water miscible total organic carbon content in every kind of substratum 50 ℃ of cultivations.Reaction mixture is made of following material: 0.5% (w/v) the octyl group glucopyranoside of 10mg PHB powder, 0.1M phosphate buffered saline buffer (pH 7.0), 0.1ml and 1ml are the substratum that micropore (Milipore) strainer of 0.2 μ m obtains by means of bacterial body being filtered by the aperture.Fig. 3 has shown the result.
Based on these results, confirmed that enzyme of the present invention is stable in 60 ℃ temperature range at the most.
Inducing of the degrading activity of (embodiment 8) use matrix
Whether check observes inducing of enzymic activity in the presence of various response matrix.
In table 6 and table 7 listed various matrix in the presence of detect the growing amount of water miscible total organic carbon (TOC) and the growing amount of hydroxybutyric acid.The composition of reaction mixture is identical with embodiment 6.Reacted 16 hours at 50 ℃.As a result, as shown in table 6 and table 7, detect that soluble T OC content and hydroxybutyric acid content significantly increase in the presence of polyhydroxyalkanoatefrom, hydroxybutyric acid, poly butyric ester and/or hydroxy butyrate.
Table 6
| Matrix | The growing amount of water-soluble TOC (mg/L) | The growing amount of hydroxybutyric acid (mg/L) |
| PHB | 926 | 879 |
| (R)-(-)-3-sodium-3-hydroxybutyric acid | 731 | 820 |
| L-(+)-β-Qiang Dingsuan | 8 | 4.8 |
| DL-3-hydroxyl-butanic acid sodium | 306 | 269 |
| DL-3-hydroxyl-butanic acid ethyl ester | 618 | 536 |
| (R)-(-)-3-hydroxyl-methyl butyl | 1394 | 943 |
| (S)-(+)-3-hydroxyl-methyl butyl | 79 | 69 |
| (S)-(+)-3-hydroxyl-butanic acid ethyl ester | 69 | 66 |
| Beta-hydroxy isovaleric acid (3-hydroxy-3-methyl butyric ester) | - | 2.98 |
| Beta-butyrolactone (Beta-methyl propiolactone) | - | 3.6 |
Table 7
| Matrix | The growing amount of water-soluble TOC (mg/L) |
| PHB | 622 |
| Glucose | 17 |
| Sucrose | 5 |
| Lactose | 6 |
| Maltose | 3 |
| Starch | 20 |
| Glycerine | 12 |
(reference example 1)
Using the MG2 bacterial strain that obtains among the embodiment 1 to check in the mode that is similar to embodiment 4 makes the active of various polymer degradations exist or not exist.
Polymer powder used herein is: poly-(succsinic acid glycol ester) (PES; Number-average molecular weight is 5.92 * 10
4By NIPPON SHOKUBAI CO., LTD. produces), poly-(ester carbonic ether) (PEC; Carbonate content is 18mol%; Number-average molecular weight is 2.4 * 10
5Produce by MITSUBISHI GASCHEMICAL COMPANY.INC.), polycaprolactone (PCL; Trade(brand)name: Tone P-767; Number-average molecular weight is 6.73 * 10
4Produce by Union Carbide Corporation), poly-(butylene succinate) (PBS; Trade(brand)name: Bionolle 1020; Number-average molecular weight is 4.8 * 10
4By SHOWAHIGH POLYMER CO., LTD. produces) and poly-(L-rac-Lactide) (PLA; Trade(brand)name: Lacty1012; Number-average molecular weight is 1.88 * 10
5Produce by Shimadzu Corporation).On 50 ℃ of every kind of nutrient agars that comprising every kind of polymkeric substance, cultivate the MG2 bacterial strain.Table 8 has shown the result.
Table 8
| Polymkeric substance | The clear zone forms |
| Poly-(3-butyric ester) | +++ |
| Poly-(succsinic acid glycol ester) | ++ |
| Poly-(ester carbonic ether) | ++ |
| Polycaprolactone | + |
| Poly-(butylene succinate) | + |
| Poly-(L-rac-Lactide) | - |
Result from table 8 is clear that not only poly butyric ester resin but also polydiethylene glycol succinate are also degraded by contacting with the MG2 bacterial strain with polyestercarbonate.In addition, though it shows a little less than the activity that polycaprolactone and polybutylene succinate are degraded equally.At the nutrient solution that uses the MG2 bacterial strain with measured under the accumulative situation of water-soluble degradation product and observed such trend similarly.
All publications, patent and the patent application of being quoted in this article is hereby incorporated by with their integral body.
Sequence table
＜110〉(the National Institute of Advanced Industrial of Independent Administrative Leged Industrial Technology Complex Inst
Science and Technology)
＜120〉method of decomposing polyhydroxyalkanoateresin resin
<130>PH-2286-PCT
<150>JP2003/376263
<151>2003-11-05
<160>1
<170>PatentIn Ver.2.1
<210>1
<211>1343
<212>DNA
＜213〉streptomycete (Streptomyces sp.)
<400>1
agtttgatcc tggctcagga cgaacgctgg cggcgtgctt aacacatgca agtcgaacga 60
tgaaccactt tcggtgggga ttagtggcga acgggtgagt aacacgtggg caatctgccc 120
tgcactctgg gataaccccg ggaaaccggg gctaataccg gatacgaccc ccgggggcat 180
cctcgggggt ggaaagctcc ggcggtgcag gatgagcccg cggcctatca gctggttggt 240
gaggtaacgg ctcaccaagg cgacgacggg tagccggcct gagagggcga ccggccacac 300
tgggactgag acacggccca gactcctacg ggaggcanca gtggggaata ttgcacaatg 360
ggcgcaagcc tgatgcagcg acgccgcgtg agggatgacg gccttcgggt tgtaaacctc 420
tttcagcagg gaagaagccg cgaggtgacg gtacctgcag aagaagcgcc ggctaactac 480
gtgccagcag ccgcggtaat acgtagggcg cgagcgttgt ccggaattat tgggcgtaaa 540
gagctcgtag gcggcttgtc gcgtcggttg tgaaagcccg gggcttaacc ccgggtctgc 600
agtcgatacg ggcaggctgg agttcggcag gggagatcgg aattcctggt gtagcggtga 660
aatgcgcaga tatcaggagg aacaccggtg gcgaaggcgg atctctgggc cgatactgac 720
gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta 780
aacggtgggc actaggtgtg gggggcattc cacgtcctcc gtgccgcagc taacgcatta 840
agtgccccgc ctggggagta cggccgcaag gctaaaactc aaaggaattg acgggggccc 900
gcacaagcgg cggagcatgt ggcttaattc gacgcaacgc gaagaacctt accaaggctt 960
gacatacacc ggaaacancc agagatggnt gcccccttgt ggtcggtgta caggtggtgc 1020
atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080
ttgntcccgt gttgccagca cgcccctcgg ggtggtgggg actcacggga gaccgccggg 1140
gtcaactcgg aggaaggtgg ggacgacgtc aagtcatcat gccccttatg tcttgggctg 1200
cacacgtgct acaatggccg gtacaatgag ctgcgatacc gcgaggtgga gcgaatctca 1260
aaaagccggt ctcagttcgg attggggtct gcaactcgac cccatgaagt cggagtcgct 1320
agtaatcgcg gatcagcatg cca 1343
Claims (9)
1. actinomycetic enzyme derived from streptomyces, it can decomposing polyhydroxyalkanoateresin resin, has at the molecular weight between about 47,000 to 56,000, in best pH between 4 to 10 and the optimum temps between 40 ℃ to 55 ℃.
2. according to the enzyme of claim 1, it induces preparation by polyhydroxyalkanoatefrom, hydroxybutyric acid, poly butyric ester and/or hydroxy butyrate.
3. according to the enzyme of claim 1 or 2, wherein the actinomycetes of this streptomyces are hot common streptomycete, hot olive streptomycete, heat absorbing water streptomycete or Streptomyces thermocarboxydovorans.
4. according to the enzyme of claim 1 or 2, wherein the actinomycetes of this streptomyces are the microorganisms with preservation registration number FERM P-19578 preservation.
5. the method for a decomposing polyhydroxyalkanoateresin resin, it comprises makes polyhydroxyalkanoateresin resin and contacts so that this resin and enzyme reaction according to each enzyme in the claim 1 to 4.
6. the method for a decomposing polyhydroxyalkanoateresin resin, it comprise polyhydroxyalkanoateresin resin is contacted with the actinomycetes of streptomyces so that this resin and this actinomycetes 40 ℃ to 55 ℃ reactions.
7. according to the method for claim 6, wherein the actinomycetes of this streptomyces are hot common streptomycete, hot olive streptomycete, heat absorbing water streptomycete or Streptomyces thermocarboxydovorans.
8. according to the method for claim 6, wherein the actinomycetes of this streptomyces are the microorganisms with preservation registration number FERM P-19578 preservation.
9. the actinomycetes of a streptomyces, it can decomposing polyhydroxyalkanoateresin resin and is microorganism with preservation registration number FERM P-19578 preservation.
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| CN (1) | CN1902309A (en) |
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| WO2010050482A1 (en) | 2008-10-27 | 2010-05-06 | 東洋製罐株式会社 | Method for producing oligomer and/or monomer by degrading biodegradable resin |
| JP2010138390A (en) * | 2008-11-12 | 2010-06-24 | Toyo Seikan Kaisha Ltd | Solution and method for degrading easily degradable resin composition |
| JP5445756B2 (en) * | 2008-11-12 | 2014-03-19 | 東洋製罐株式会社 | Method for decomposing easily decomposable resin composition |
| WO2017099209A1 (en) | 2015-12-11 | 2017-06-15 | 東レ株式会社 | Method for producing 3-oxoadipic acid |
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| JPH07155180A (en) * | 1993-12-06 | 1995-06-20 | Snow Brand Milk Prod Co Ltd | New phb degrading enzyme, its production and microorganism capable of producing the same enzyme |
| JPH10191980A (en) * | 1997-01-08 | 1998-07-28 | Taisei Corp | Polyhydroxyalkanoate-decomposing enzyme and its production |
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2004
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