JPH09201192A - Purification method for polyurethane decomposition esterase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently decompose an ester-based polyurethane with an esterase by providing a method for preparing the esterase capable of decomposing the ester-based polyurethane. SOLUTION: A Comamonas acidovorans TB-35 strain is cultured by using an ester-based polyurethane as a carbon source and a culture solution obtained by the culturing is then centrifuged and separated into a supernatant and a microbial body. An esterase is subsequently separated from the resultant microbial body with a surfactant selected from a solution of sodium cholate, a solution of sodium deoxycholate and a solution of sodium dodecyl sulfate. The prepared solution containing the surfactant and esterase is concentrated by ultrafiltration and the surfactant is then removed to afford the esterase sticking to the microbial body. Furthermore, the resultant polyurethane-decomposing esterase is added in an amount of 1.9-188.0mU to decompose the ester-based polyurethane.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying a polyurethane-degrading esterase derived from a specific microorganism. Polyurethane is used in seat cushions of four-wheeled vehicles and two-wheeled vehicles, textiles of clothing, cushions of shoes, adhesives, paints and the like. Polyurethane is a thermosetting resin and is difficult to recycle, so it is often landfilled. However, polyurethane remains in the soil semipermanently, which also causes a shortage of landfills. Therefore, in recent years, development of technology for treating polyurethane has been studied.

[0002]

2. Description of the Related Art As a processing technology for polyurethane, landfill processing, chemical decomposition processing and incineration processing can be mentioned. In the landfill treatment, used polyurethane or a piece of polyurethane generated in a manufacturing process is directly landfilled. However, it has the following disadvantages. Polyurethane is used for sponges, cushions, etc., and is a resin with a low density. Therefore, if it is buried in the soil, the ground will not solidify and it will not be stable. In addition, the transportation efficiency is poor because the weight is large. Furthermore, since polyurethane is not decomposed by microorganisms and does not rot, it occupies a large proportion in the landfill treatment site, which contributes to the shortage of the treatment site.

The chemical decomposition treatment is a decomposition into gas or petroleum by thermal decomposition. This treatment method has an advantage that the raw material polyol can be regenerated by glycol decomposition. However, since the quality of the polyurethane to be disposed varies, the quality of the decomposition products is not stable, and the cost and labor required for the treatment are enormous, which is a disadvantage of being unprofitable. In the incineration treatment, polyurethane is incinerated in a furnace. However, it has the following disadvantages. First, traditional furnaces cannot withstand the high temperatures generated during polyurethane incineration. Further, in hot-cure polyurethane used for seat cushions of four-wheeled vehicles and two-wheeled vehicles, condensed phosphate ester containing 29% chlorine is used as a flame retardant.
When incinerating polyurethane, hydrogen chloride is generated and corrodes the furnace. Furthermore, carbon dioxide and toxic gases are generated during incineration, which pollutes the atmospheric environment.

In addition to the above, as a treatment technique for polyurethane, treatment with microorganisms can be considered. However, decomposition treatment by microorganisms is at the stage of basic technology as described below, and has not yet been put to practical use. Ether-based polyurethanes made from ether-based polyols are resistant to microorganisms and difficult to decompose. On the other hand, it has been reported that ester polyurethanes made from ester polyols are decomposed by enzymes produced by microorganisms.
For example, “Tokiwa, Y. et al: Agri.
c. Biol. Chem. , 52, p1937 (198
8) ”includes ester-based polyurethanes such as Rhizopus arrhizus and R. de.
It is described to be hydrolyzed by the enzyme Lemar ). In addition, "Toshiaki Nakaji
ma-Kambe et al: FEMS Micro
biology Letters 129, 39-42
(1995) ”describes bacteria isolated from soil ( Coma
monas acidovorans TB-35 strain)
Describes the decomposition of ester polyurethane to produce adipic acid and diethylene glycol. Co
mamonas acidovorans TB-35
The method of decomposing the ester polyurethane by the strain will be described based on experiments as follows.

First, polyurethane was synthesized from polydiethylene glycol adipate and 2,4-tolylene diisocyanate. Cut the synthesized polyurethane into dice shapes (about 50 mg), and put 5 ml in a test tube with a diameter of 25 mm.
Liquid medium. The liquid medium was inoculated with 1 platinum loop of the bacterium and shake-cultured (120 vibrations / min) at 30 ° C. When polyurethane was the only carbon source, it decomposed completely in 7 days. The degradation metabolite was mainly diethylene glycol, and a small amount of trimethylolpropane was also found. When polyurethane was the only carbon and nitrogen source, it decomposed by 48% by weight in 7 days. The degradation metabolites were adipic acid and diethylene glycol.

However, the above-mentioned method of inoculating microorganisms to decompose polyurethane may be difficult to decompose due to mutation of the preserved strain or contamination with miscellaneous bacteria. Therefore, an object of the present invention is to provide a method for preparing esterase, which is an enzyme that decomposes ester polyurethane, and to achieve efficient and stable decomposition of polyurethane by the esterase obtained by the method.

[0007]

The invention according to claim 1 is characterized in that the ester-based polyurethane is used as a carbon source, and Coma is used.
monas acidovorans TB-35 strain, a step of centrifuging a culture solution obtained by the culturing to separate a supernatant and cells, a sodium cholate solution, a sodium deoxycholate solution, and dodecyl sulfate. A step of releasing esterase from the bacterium by a surfactant selected from a sodium solution, and a solution containing the surfactant and the esterase are concentrated by ultrafiltration and the surfactant is removed to remove the esterase from the bacterium. The method for purifying a polyurethane-decomposing esterase comprising the steps of: The invention according to claim 2 is the method for decomposing an ester polyurethane, which comprises adding 1.9 to 188.0 mU of the polyurethane-decomposing esterase obtained by the method according to claim 1 to an ester polyurethane. It depends on.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The ester polyurethane in the present invention means a polyurethane using polyester as a polyol. Commonas used in the present invention
acidovorans TB-35 strain (FERM
P-15381) is a bacterium isolated from soil in Tsukuba city, Ibaraki prefecture, and is known to decompose polyurethane. The scientific properties of the TB-35 strain are described below.

Properties Cell morphology Bacillus Gram stain Negative respiration Anaerobic motility + Oxidase + Catalase + Nitrate reduction + Indole formation-Arginine dihydrolase-Urease-β-Galactosidase-Fructose O-F + Acid esculin, addition of gelatin- Hydrolysis of Tween 80 + Mannitol utilization + Gluconate utilization + Adipate utilization + Malate utilization + Citrate utilization + Acetate utilization + Glucose utilization − Arabinose utilization − Mannose Availability-Maltose availability-Caprate availability-Phenylacetate availability-N-acetylglucosamine-Utility Quinone type Ubiquinone (Q-8) G + C mol% 68.8%

Centrifugation of the culture medium is usually 8000 rp
m for 10 minutes. As the surfactant, any of sodium cholate solution, sodium deoxycholate solution, and sodium dodecyl sulfate solution is used. Particularly, sodium cholate solution is preferable because it does not inhibit the enzyme and the extraction efficiency is high. The concentration of sodium cholate solution is
0.0045 to 56.8% by weight, preferably 0.
It is 3 to 5% by weight, and particularly preferably 2% by weight.
Polyurethane degrading enzyme (esterase) is 1.9-1
It is added to the polyurethane at a concentration of 88.0 mU, preferably 15.0-100 mU. If the concentration is less than 1.9 mU or more than 188.0 mU, the polyurethane decomposing activity is lowered and the effect of the present invention cannot be obtained. here,
The esterase activity is measured by measuring the amount of p-nitrophenol released from p-nitrophenylacetate by esterase by measuring the absorbance at 405 nm. The amount of the enzyme that liberates 1 μmol of p-nitrophenol in 1 minute is 1 U (unit). When the polyurethane is decomposed, it is preferable to form the polyurethane into a film shape, a granular shape or the like having a large surface area.

[0011]

EXAMPLE Polydiethylene glycol adipate (functional group at polyol end is 2.7, weight average molecular weight is 250)
0) and 2,4-tolylene diisocyanate were used to synthesize an ester polyurethane. The synthesized ester polyurethane is cut into dice shapes (about 50 mg) and the diameter is 25
It was placed in 5 ml of liquid medium in a mm test tube. The composition of the medium is shown in Table 1. The final pH of the medium was adjusted to 7.2.

[0012]

[Table 1]

Examples 1 to 3 In liquid medium, Commonas acidovoran
One platinum loop was inoculated with the s TB-35 strain, and shake culture (120 oscillations / minute) was performed at 30 ° C for 7 days. Then, the culture solution was centrifuged to separate into a supernatant and cells. The supernatant was concentrated by ultrafiltration to obtain extracellular esterase. On the other hand, the separated bacterial cells were treated with 0.1M phosphate buffer (pH: 2% by weight sodium cholate).
7) and released the esterase in phosphate buffer. After separating the cells by centrifugation at 8000 rpm for 10 minutes, the supernatant of the phosphate buffer is ultrafiltered using an ultrafilter to concentrate and remove cholic acid.
A bacterial cell-attached esterase was obtained. The microbial cell-attached esterase was adjusted to have a concentration of 1.9 to 188.0 mU to prepare an enzyme solution. 1 mL of enzyme solution was added to 10 mg of polyurethane synthesized into a film having a thickness of 1 mm, and 30
The reaction was carried out at 48 ° C for 48 hours. As a result, the weight of polyurethane was reduced by 0.4 to 1.3 mg. Table 2 shows the results.

[0014]

[Table 2]

Comparative Example 1 An experiment was carried out in the same manner as in Examples 1 to 3 except that the esterase concentration was 1880.0 mU. Table 2 shows the results.

Comparative Examples 2 to 4 For extracellular esterases, experiments were conducted in the same manner as in Examples 1 to 3. Table 2 shows the results. When extracellular esterase was used, polyurethane was hardly decomposed at any concentration. Table 2 shows the results.

Control Example An experiment was conducted in the same manner as in Examples 1 to 3 except that a phosphate buffer solution having a pH of 7.0 was used instead of the enzyme solution. Table 2 shows the results.

[0018]

EFFECTS OF THE INVENTION According to the present invention, the temperature is kept at 30 ° C. without any drawback in treating polyurethane by the conventional method, without requiring a large-scale machine or a great labor.
Ester polyurethane can be decomposed and treated efficiently by a simple operation. In addition, microorganisms are difficult to store and easily cause mutations by subculture, but stable storage of polyurethanes is possible when stored as the enzyme obtained in the present invention. Furthermore, according to the present invention, useful chemical substances such as diethylene glycol can be obtained as a decomposition product of polyurethane.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:01) (72) Inventor Noriko Kanehara 300 Takazuka-cho, Hamamatsu-shi, Shizuoka Suzuki Stock Company ( 72) Inventor Yoko Iwata, 300 Takatsuka-cho, Hamamatsu City, Shizuoka Prefecture Suzuki Stock Company

Claims (2)

[Claims] 1. An ester-based polyurethane is used as a carbon source for Commonas acidovorans T.
A step of culturing the B-35 strain, a step of centrifuging the culture solution obtained by the culturing to separate into a supernatant and cells, a sodium cholate solution, a sodium deoxycholate solution, and a sodium dodecyl sulfate solution. A step of releasing esterase from the microbial cell with a surfactant selected from the following, and a solution containing the surfactant and the esterase is concentrated by ultrafiltration and the surfactant is removed to obtain a microbial cell-attached esterase. A method for purifying a polyurethane-decomposing esterase comprising the steps of: 2. A method for decomposing an ester-based polyurethane, which comprises adding 1.9 to 188.0 mU of the polyurethane-decomposing esterase obtained by the method according to claim 1 to the ester-based polyurethane.