JP3470109B2 - Surface treatment method of aliphatic polyester fiber using enzyme - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for decomposing a biodegradable polymer and a method for treating the surface of the polymer. More specifically, the present invention relates to a method for decomposing an aliphatic polyester using an enzyme and a method for treating a surface of an aliphatic polyester fiber or the like using an immobilized enzyme. [0002] High molecular compounds represented by plastics, together with metals and ceramics, are materials closely related to daily life, and plastic products are widely used not only in clothing, food and living but also in various industries and medical treatments. Have been. However, conventional plastics, which have been developed with an emphasis on long-term stability, are not decomposed in the natural environment, and a large amount of unnecessary waste destroys the environment on a global scale. Has become. [0004] In recent years, with increasing public interest in the above-mentioned problems, studies on so-called biodegradable polymers that are degraded by microorganisms in nature have been actively conducted and are being put to practical use. [0005] At present, the subject of research includes polymers produced by microorganisms, natural polymers and synthetic polymers derived from plants and animals. It is difficult to obtain a product that is satisfactory in terms of productivity and the like, and is typified by polyvinyl alcohol, polyethylene glycol, and polyester, but is expected as a biodegradable polymer. [0006] It is known that some of these synthetic polymers are decomposed by microorganisms or enzymes. For example, polyvinyl alcohol, which is a water-soluble polymer, is decomposed by bacteria in the soil [(Agric. Biol. Chem., 37, 74).
7 (1973)]. Microorganisms that degrade polyethylene glycol, which is also a water-soluble polymer, are known, but as the degree of polymerization increases, it becomes difficult for bacteria to degrade, especially polyethylene glycol with a molecular weight of 6,000 or more can be degraded by a single bacterium. Is difficult [J. ferment. Techno
l., 53, 757 (1977)]. [0007] Recently, polyester, which is a water-insoluble polymer, is most expected as a biodegradable synthetic polymer because of its ease of processing. In particular, it has been reported that polycaprolactone (PCL), which is an aliphatic polyester, undergoes hydrolysis in soil [Polym. Preprin].
ts, 13, 629 (1972)], and the degradation of PCL by various lipases has been studied in detail [Agric.
Biol. Chem., 52, 265 (1977)]. As described above, aliphatic polyester is considered to be a biodegradable polymer having more preferable properties in that biodegradation can be performed not only by using microorganisms but also by using enzymes. [0008] A method for decomposing polyester using lipase is known (JP-A-52-827).
74). In the above method, for PCL, for example, Aspergillus, Niger, Geotrichum, Candidum, Rhizopus
・ Delemar, Rhizopus Arizus, Candid
a). It is known that lipases derived from syrindrasse, wheat germ, and pig pancreas act, and it has been revealed that lipases derived from the genus Rhizopus have a particularly strong decomposing ability. However, its decomposing power is not satisfactory, and there has been a strong demand for the development of an enzyme having a stronger decomposing power. [0010] In view of such a situation, the present inventors have found that a lipase produced by a microorganism belonging to the genus Pseudomonas has a very strong ability to degrade an aliphatic polyester as compared with a conventional lipase derived from the genus Rhizopus. The present invention has been completed for the first time by finding out. [0011] Further, the present invention improves the texture while maintaining the strength of fibers and the like by treating the surface of fibers and the like made of aliphatic polyester by immobilizing and using lipase having the ability to degrade aliphatic polyester. A method that can be done has also been invented. The aliphatic polyester to be decomposed in the present invention includes polyglycoside, polylactic acid, polyhydroxybutyrate, PCL, hydroxyvalerate and copolymers thereof. Can be Among them, PCL and the like are materials excellent in enzymatic degradation. The first invention of the present invention relates to a method for decomposing an aliphatic polyester using a lipase produced by a microorganism belonging to the genus Pseudomonas. As the lipase that can be used, any lipase derived from the genus Pseudomonas and having the ability to degrade aliphatic polyesters can be used. Examples thereof include lipase PS (manufactured by Amano Pharmaceutical: derived from Pseudomonas cepacia) and lipase AK (manufactured by Amano Pharmaceutical: derived from Pseudomonas florescens). More preferably, lipase PS can be used. These lipases can be crude or highly purified. It is desirable to disperse the aliphatic polyester to be decomposed as finely as possible in order to increase the contact area with the enzyme. For example, it is desirable to disperse the aliphatic polyester in the form of a fine fiber, a thin film, or a powder. The reaction conditions for decomposing the aliphatic polyester using lipase may be any conditions as long as the lipase used exhibits enzymatic activity, but preferably the temperature is 10 to 60 ° C. and the pH is 4 to 9 Buffer. Of course, the reaction can be carried out in a suitable solvent system, for example, a solid system if suitable water is present. The second invention relates to a method for immobilizing a lipase having the ability to degrade an aliphatic polyester on a suitable carrier and treating the surface of the polyester with the immobilized enzyme. As the lipase usable in the present invention, any enzyme capable of decomposing an aliphatic polyester can be used. For example, enzymes derived from Pseudomonas and Rhizopus can be suitably used. More specifically, for example, lipase PS (manufactured by Amano Pharmaceutical: derived from Pseudomonas cepacia), lipase AK (manufactured by Amano Pharmaceutical: derived from Pseudomonas florescens), and lipase F-AP (manufactured by Amano Pharmaceutical: derived from Rhizopus oryzae). Can be More preferably, lipase PS and lipase F-AP can be used. These lipases can be crude or highly purified. As the carrier to be used, a water-soluble polymer carrier is preferable for immersion treatment of fibers and the like. For example, copoly (methyl vinyl ether / maleic anhydride) [Copoly (m
ethylvinylester-co-maleic anhydride)) (MAMEC), polyethylene glycol, polyacrylic acid, polyamine, polyglutamic acid, polylysine, dextran,
Hydroxyethyl starch, carboxymethyl cellulose and the like can be mentioned. More preferably, MAMEC can be used. As a method for immobilizing lipase on the carrier, there are, for example, an ionic bonding method and a covalent bonding method.
In order to stably immobilize the enzyme, a covalent bonding method is desirable. Reactions often used as a covalent bonding method include an active esterification method, a Schiff base bonding method, a diazonium coupling method, a cyanogen bromide activation bonding method, a diisocyanate method, a condensation reagent (carbodiimide method), and a triazinyl derivative bonding method. , Halogenoacetyl derivative binding method,
There are an acid azide derivative binding method, a halogenoacetyl derivative binding method, and the like. However, in the enzyme immobilization reaction, care must be taken so as not to reduce the activity of the enzyme. The conditions for treating fibers or films made of aliphatic polyester using the lipase immobilized as described above can be used under any conditions as long as the lipase to be used exhibits enzymatic activity. Preferably, however, the temperature is between 10 and 60 ° C. in a buffer of pH 4-9. When aliphatic polyester fibers or the like are treated with such an immobilized lipase, only the surface is uniformly decomposed without impairing the stretching strength of the fibers, as compared with the case where the treatment is performed without immobilizing the lipase. can do. That is, aliphatic polyester fibers having different textures can be obtained by changing the immersion time in consideration of the fiber diameter to be treated, the degree of polymerization, and the like. Of course, since lipase is immobilized on a polymer carrier, it can be reused and the stability is improved. Hereinafter, the present invention will be described in detail with reference to examples. Note that the present invention is not limited to these. Example 1 Decomposition of PCL fibers of various lipases PCL fibers were treated as samples using various lipases under the following conditions. The tensile strength retention of the treated fiber was determined by weight loss during drying and simple tensile strength measurement. Further, the surface properties of the fiber sample were observed with a scanning electron microscope. The enzyme is dissolved in a 0.1 M phosphate buffer (pH 7.0) at a concentration of 0.5 W / V%, and about 75 mg of a PCL-end drawn fiber sample (fiber diameter 275 μm) is added to the enzyme solution (10 ml). Add and treat at 25 ° C with gentle stirring. The reaction conditions and results are shown in Table 1. [Table 1] In Table 1, L-PS is lipase PS "Amano", L-AK is lipase AK "Amano", LF-AP is lipase F-AP-15, and LA-6 is lipase A "Amano" 6. , LM-10 is lipase M "Amano" 10, L-AY30 is lipase AY "Amano" 30, L-PEG is lipase PGE "Amano", NF is neurolase F, PF is pancreatin F (all of these are Amano Pharmaceutical product name). As is clear from Table 1, the lipase derived from Pseudomonas cepacia, ie, the lipase PS "Amano", has a very strong degrading activity, and the lipase derived from Pseudomonas florescens, also belonging to the genus Pseudomonas, namely lipase AK " Amano also has the activity of degrading PCL well. In addition, lipase derived from Rhizopus oryzae, ie, lipase F-AP-15, also degrades PCL well, but lipase derived from Rhizopus niveus, which is the same genus of Rhizopus, ie, neurase F, shows little degradation activity, It can be seen that the lipase does not show any decomposition activity. Example 2 By lipase PS "Amano"
The reaction was performed using the biodegradable lipase PS "Amano" of PCL-end drawn fiber at an enzyme concentration of 0.1 W / V% in the same manner as in Example 1, the reaction time and the degree of decomposition were measured, and the weight loss rate (Wr / Wo) was measured. ) And tensile strength residual ratio (σB, r / σB, o) with time. The results are shown in Table 2 and FIG. [Table 2] When the reaction time was 0 hour, the results were immersed in a buffer solution containing no enzyme for 1 hour. Further, as a result of observing the treated PCL fiber with a scanning electron microscope, it was observed that the fiber treated for 30 hours was greatly eroded by enzymes and decomposed. Example 3 By lipase AK "Amano"
Using a biodegradable lipase AK “Amano” of drawn fibers of PCL end , the reaction was carried out at an enzyme concentration of 0.5 W / V% in the same manner as in Example 1, the reaction time and the degree of decomposition were measured, and the weight loss rate (Wr / Wo) was measured. ) And tensile strength residual ratio (σB, r / σB, o) with time. The results are shown in Table 3 and FIG. [Table 3] When the reaction time was 0 hour, the results were immersed in a buffer solution containing no enzyme for 1 hour. Example 4 PCL using lipase F-AP-15
Example 1 Biodegradation of undrawn fiber Using lipase F-AP-15 at an enzyme concentration of 0.2 W / V%
React in the same manner as above, measure the reaction time and the degree of decomposition,
Weight loss rate (Wr / Wo) and residual tensile strength ratio (σB,
r / σB, o) was determined over time. Table 4 shows the results.
And FIG. [Table 4] When the reaction time was 0 hour, the result was immersed for 1 hour in a buffer solution containing no enzyme. Example 5 By lipase PS "Amano"
Effect of enzyme concentration on biodegradation of PCL powder drawn fiber Using lipase PS "Amano", the enzyme concentration was 0.065-0.2
The reaction was carried out in the same manner as in Example 2 except that the reaction time was changed to W / V%, the reaction time and the degree of decomposition were measured, and the time-dependent change in the weight loss rate (Wr / Wo) was obtained. Wo)
The enzyme concentration dependence was examined by determining 1/2. The results are shown in Table 5, FIG. 4 and FIG. [Table 5] In each case, good linearity is exhibited at the beginning of the decomposition, and it is considered that the decomposition rate proceeds by a first-order reaction with respect to the surface area of the fiber. Example 6 Lipa immobilized on a water-soluble polymer
Decomposition of PCL fiber using ASE PS “Amano” 5.0 g of MAMEC having an average molecular weight of about 20,000 is dissolved in 90 ml of 0.1 M acetate buffer (pH 5.4). Lipase PS "Amano"
1.0 g of the enzyme is dissolved in 10 ml of the same buffer solution as described above, and the two are mixed at 20 ° C. and reacted with stirring for 24 hours. After the reaction, unreacted enzyme is removed by fractionation and purification using a 100,000 molecular weight fractionation-like ultrafiltration membrane, and concentrated and freeze-dried to obtain a MAMEC-immobilized enzyme. As a result of quantifying the concentration of the immobilized enzyme by the ninhydrin method, 4.5 wt% was obtained. Using the lipase PS "Amano" immobilized on the water-soluble polymer obtained above, the reaction was carried out at an enzyme concentration of 0.1 W / V% in the same manner as in Example 4, and the reaction time and the degree of decomposition were measured. Then, the time-dependent changes in the weight loss rate (Wr / Wo) and the residual tensile strength ratio (σB, r / σB, o) were determined. The results are shown in Table 6 and FIG. [Table 6] When the reaction time was 0 hour, the results were immersed in a buffer solution containing no enzyme for 1 hour. Further, as a result of observing the treated PCL fiber with a scanning electron microscope, it was found that the surface of the fiber treated for 50 hours was more uniformly decomposed than in Example 2. Further, as is clear from FIG. 6, although the weight reduction rate is not much different from that when the non-immobilized lipase PS “Amano” of Example 2 is used, the rapid decrease in the tensile strength residual ratio is observed. No significant decrease is seen. That is,
The surface of the PCL fiber was decomposed very uniformly, and the surface of the fiber could be treated. Example 7 Lipa immobilized on a water-soluble polymer
Decomposition of PCL fiber using AK AK “Amano” The operation was carried out in the same manner as in Example 6 using lipase AK “Amano” bound to a water-soluble polymer. As a result, Example 6
Similarly, the weight loss rate was not much different from that when the non-immobilized lipase AK “Amano” of Example 3 was used,
There was no sharp drop in the tensile strength residual ratio, and the fiber strength was relatively maintained. Example 8 Lipa immobilized on a water-soluble polymer
Decomposition of PCL fiber using lyase F-AP-15 The same operation as in Example 6 was performed using lipase F-AP-15 bound to a water-soluble polymer. As a result, as in Example 6, the weight loss rate was the same as that of Example 4
Although there was not much difference from when F-AP-15 was used, there was no sharp change in the tensile strength residual ratio. According to the present invention, a biodegradable polymer,
In particular, polycaprolactone, which is an aliphatic polyethylene, can be efficiently decomposed using a lipase derived from Pseudomonas. Further, by using an immobilized enzyme obtained by immobilizing lipase on a water-soluble polymer carrier, expressions such as PCL fiber can be decomposed to improve the texture of the fiber. This has the effect of relatively not impairing the strength.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the reaction time, weight loss rate (Wr / Wo), and residual tensile strength ratio (σB, r / σB, o) when lipase PS “Amano” of Example 2 was used. The change over time is shown. In the figure, ● indicates the weight reduction rate, and ○ indicates the residual tensile strength ratio. FIG. 2 shows the change over time in the reaction time, weight loss rate (Wr / Wo) and residual tensile strength ratio (σB, r / σB, o) when lipase AK “Amano” of Example 3 was used. In the figure, ● indicates the weight reduction rate, and ○ indicates the residual tensile strength ratio. FIG. 3 shows the change over time in the reaction time and the weight loss rate (Wr / Wo) and residual tensile strength ratio (σB, r / σB, o) when lipase F-AP-15 of Example 4 was used. In the figure, ● indicates the weight reduction rate, and ○ indicates the residual tensile strength ratio. FIG. 4 shows the relationship between the reaction time and the weight loss rate when the lipase PS “Amano” of Example 4 was used. In the figure,
, And have enzyme concentrations of 0.065, 0.10, 0.15 and
The result at the time of 0.20 W / V% is shown. FIG. 5 shows the relationship between the reaction time and the square root of the weight loss rate when the lipase PS “Amano” of Example 4 was used. In the figure, and indicate that the enzyme concentrations are 0.065, 0.10, 0.1, respectively.
The results at 5 and 0.20 W / V% are shown. FIG. 6: Immobilized lipase SP “Amano” of Example 6.
2 shows the time-dependent changes in the reaction time, weight reduction rate (Wr / Wo) and residual tensile strength ratio (σB, r / σB, o) when using. In the figure, ● indicates the weight reduction rate, and ○ indicates the residual tensile strength ratio.

Claims (1)

(57) [Claim 1] An immobilized lipase obtained by bonding a lipase capable of decomposing an aliphatic polyester to a water-soluble polymer carrier and contacting the aliphatic polyester fiber. Surface treatment method for aliphatic polyester fibers.