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Rapid and simple colorimetric assay for detecting the enzymatic degradation of biodegradable plastic films
Journal of Bioscience and Bioengineering VOL. 115 No. 1, 111e114, 2013 www.elsevier.com/locate/jbiosc
TECHNICAL NOTE
Rapid and simple colorimetric assay for detecting the enzymatic degradation of biodegradable plastic films Yukiko Shinozaki,1 Takashi Watanabe,1 Toshiaki Nakajima-Kambe,2 and Hiroko K. Kitamoto1, * National Institute for Agro-environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaraki 305-8604, Japan1 and Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan2 Received 21 May 2012; accepted 17 August 2012 Available online 14 September 2012
We developed a rapid and simple method for evaluating the degradation of solid biodegradable plastics (BPs). Dye-containing BP films were used as substrates and the release of dye caused by the degradation of BPs was confirmed by a color change in the enzyme solution after a reaction time of 24 h. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Biodegradation; Poly(butylene succinate-co-adipate); Poly(lactic acid); Polyurethane; Pseudozyma antarctica]
The use of biodegradable plastics (BPs), such as poly(lactic acid) (PLA), poly (ε-caprolactone) (PCL), and poly(butylene succinate-coadipate) (PBSA), has received much attention from the viewpoint of environmental protection and solid-waste management (1,2). In the natural environment, microorganisms can degrade and assimilate BPs as a carbon source and release CO2; thus, the international standard for the evaluation of their biodegradability is based on measuring the amount of CO2 released under the specified conditions for 180 days or less (1,3). To date, various microorganisms, including bacteria, fungi, and yeast, have been reported to degrade BPs (4e14). In many cases, the abilities of these microorganisms to degrade BPs have been assayed easily by measuring the decrease in the turbidity of BP dispersions (4e9). This assay can be used as an initial screening for BPsdegrading microorganisms. However, our preliminary results indicated that there are many microorganisms that can degrade BP dispersions but not solid BPs (14). Therefore, a second round of screening is needed to identify the microorganisms or enzymes that can degrade solid BPs. The degradation of solid BPs could be evaluated by measuring the weight loss of BP films following enzymatic degradation, but this method needs a relatively long time (100 days or less) to confirm (11,13). Alternatively, the degradation could be evaluated by measuring the increase in solubilized monomers or oligomers released from the BP films into the reaction solution. In this method, the concentration of total organic carbon (TOC) is analyzed (4,10). The TOC analyzer can detect a low level of carbon (4 mg/L); thus, the degradation of solid BPs can be confirmed after a relatively short treatment time, although the equipment required for this method is costly.
* Corresponding author. Tel.: þ81 29 838 8355; fax: þ81 29 838 8199. E-mail address: [email protected] (H.K. Kitamoto).
In this study, we developed a rapid and simple method for evaluating the degradation of solid BPs. Hand-made dye-containing BP films were used as substrates. After treatment of the film with an enzyme solution for 24 h, the relative amount of dye released was measured using a colorimetric detector (Fig. 1). The BPs used in the degradation experiments were as follows: PBSA, Bionolle 3020 (weight average molecular weight [Mw] ¼ 1.4 105) and PBS, Bionolle 1020 (Mw ¼ 1.4 105) supplied by Showa Denko K. K. (Tokyo, Japan); PCL (Mw ¼ 1 104 and 7e10 104), poly(DL-lactic acid) (PDLLA) PLA-0005 (Mw ¼ 0.5 104), and PLA-0020 (Mw ¼ 2 104) purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan); and poly (L-lactic acid) (PLLA) (Mw ¼ 1.3 105) supplied by Toyota Motor Co. Ltd. (Aichi, Japan). Each BP pellet was dissolved in dichloromethane at 1 wt%, and then 300 mL of the solution was mixed with 10 mL of Nile blue staining reagent (7% w/v; Muto Pure Chemicals Co., Ltd. Tokyo, Japan). Each dye-containing BP solution was cast onto a waterrepellent printing glass slide (Matsunami Glass Ind. Ltd., Osaka, Japan), with 20 mL in each well (f 7 mm), and was air-dried at room temperature for 24 h. The weight of the BP film and the dye it contained were calculated to be 0.265 and 0.04 mg, respectively (based on the dichloromethane density, 1.3266 g/cm3). Water-dispersed polyurethanes (PU) Superflex 150 (ester and ether-type) and Superflex 840 (aromatic isocyanate and ester-type) supplied by Dai-ichi Kogyo Seiyaku Co. Ltd. (Kyoto, Japan) were also used. To prepare PU films, 300 mL of the undiluted PU solution (27e30 wt%) was mixed with 10 mL of Nile blue staining reagent, and then 10 mL of the mixture was spread onto each well of the glass slide and air-dried at room temperature for 24 h. The BPs-degrading enzyme from Pseudozyma antarctica JCM10317 (PaE) was purified as described earlier (14,15). To compare PaE’s BPs-degradation activity with that of other enzymes, the commercially available enzymes, proteinase K (SigmaeAldrich
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.08.010
112
SHINOZAKI ET AL.
J. BIOSCI. BIOENG.,
Preparation of BP films on a water-repellent printing glass slide by casting (BPs were dissolved in dichloromethane with Nile blue staining reagent, and dried)
A
Enzyme solution (50 µL) BP film containing Nile blue staining reagent Water-repellent printing glass slide Well (
Apply enzyme solution on the BP film
7 mm)
1
B
2
3
4
PBSA Enzyme reaction (30°C for 24 h) (degradation of BP film release the dye and increase OD600 in reaction solution)
End of enzyme reaction
PLA
C
1
PBSA Collect the reaction solution into a 96-well microplate and measure OD600
1: Control 2: Lipase B 3: Proteinase K 4: PaE
PCL
PCL PLA
2
3
4 1: Control 2: Lipase B 3: Proteinase K 4: PaE
FIG. 1. Evaluation of the relative BPs-degradation activity using dye-containing BP films (dye-BP assay). (A) Cross-sectional view of a BP film and enzyme solution on the glass slide. (B) Photograph of the glass slides with BP films and enzyme solutions after the reaction (30 C for 24 h). The black area of the glass slide has been printed with highly water-repellent mark preventing the enzyme solution from spilling out from the well. (C) Reaction solutions collected after the degradation of dye-containing BP films (PBSA, Mw ¼ 1.4 105; PCL, Mw ¼ 1 104; and PLA, Mw ¼ 2 104). The BP film degradation could be evaluated from OD600 values of the reaction solutions after subtracting the values for the control.
Japan, Tokyo, Japan) and lipase B (CALB L; Novozymes Japan Ltd., Chiba, Japan) were used. The protein concentrations of these enzymes were determined with a protein assay kit (Bio-Rad Laboratories, USA) according to the manufacturer’s instructions, using bovine serum albumin (SigmaeAldrich Japan) as standard. Each BP film on the glass slide was washed twice with 80 mL of distilled water to eliminate slight extra Nile blue dye. In general, this dye is used for histochemical staining of lipoids. It is a mixture of the oxazine sulfate (true Nile blue) which reacts with acidic lipoids (fatty acids, phospholipids and some others), and the oxazone (Nile red) which dissolves in neutral lipoids (16). The color of the PCL film containing Nile blue was red, while that of other BP films was blue (Fig. 1B, control). This color difference is presumed to be due to the inherent characteristics of each BP. Then 50 mL of the appropriately diluted enzymes (lipase B, proteinase K, and PaE) in 20 mM TriseHCl (pH 8.8) were separately dropped onto the BP films (Fig. 1A). To avoid drying of the BP films, the glass slide was put into a small container lined with wet paper, sealed and incubated at 30 C for 24 h. The reaction solutions on the BP films were then collected into a 96-well microplate (roundbottom), and the OD600 values were measured using a multispectrophotometer (Dainippon, Osaka, Japan). The OD600 values of the enzyme solutions were obtained after subtracting the values for the controls. For the substrate and enzyme controls, the enzyme and the substrate were omitted from the reaction solutions, respectively. To predict the OD600 in reaction solution after complete degradation of a BP film, dichloromethane without BP was mixed with Nile blue and cast on a well; then after evaporating dichloromethane, the dye was dissolved in buffer solution and its OD600 was measured, as described above. After the enzymatic reactions with the dye-containing BP films, the color changes in the reaction solutions were observed
(Fig. 1B and C). The OD600 values of the solutions were correlated with the enzyme concentrations (Fig. 2A). The concentrations of the dye in PU films were different from that of other BPs because the initial concentrations of PU (dispersed in water at 27e30 wt%) were higher compared to other BPs (dissolved in dichloromethane at 1 wt%). Nevertheless, OD600 values of the enzyme solutions after the PU films degradation were correlated to the enzyme concentrations, similar to other BPs (Fig. 2A). The actual amount of BPs solubilized by the enzymatic reaction was evaluated by measuring the TOC content of the reaction solutions as follows. The same experimental procedure as above, except for the addition of Nile blue staining reagent, was carried out for PBSA, PCL (Mw ¼ 1 104), PLA-0020, and PU (Superflex 840), and the collected reaction solutions were diluted with 20 mL of Milli-Q water. The diluted solutions were then filtered through a 0.45-mm membrane (DISMIC-25, PTFE; Toyo Roshi Kaisha, Ltd., Tokyo, Japan), and the water-soluble TOC level in the solution was measured using a TOC-V CSH analyzer (Shimadzu Co., Kyoto, Japan). The range of the linear correlation between the TOC level and the PaE concentration was slightly different compared to that between the OD600 and the PaE concentration (Fig. 2A and B). These results may be due to the fact that these two methods detect different products. Specifically, TOC analysis measures the watersoluble oligomers and/or monomers released from BP films, while the colorimetric assay using OD600 measurement determines the amount of dye eluted from BP film during the degradation process. Although the linear correlation ranges showed slight differences, these evaluations of the relative BPs degradation based on TOC or OD600 measurement were basically in agreement with each other. This new assay using dye-containing BP films (dye-BP assay) can therefore be considered useful as a rapid and simple method for evaluating the enzymatic degradation of BP films.
VOL. 115, 2013
TECHNICAL NOTE
PBSA
PCL
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60 40
PLA-0020
A
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0.4
0.8
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0 1.0
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TOC (µg/50µL)
0.8
0.5
1.2
PU (Superflex 840) 0.8
PaE concentration (µg/mL)
0.8
0.4
0.8
1.2
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B 500
0.4 0.2
400 300 200 100
0.0 0.0 0.5 1.0 1.5 2.0 2.5 PaE concentration (µg/mL)
0
PaE concentration (µg/mL)
100
0.0
0.4
A
0.2
40
0 0.0
1.2
PaE concentration (µg/mL)
1.0
0.4
60
20
B
0.6
80
0.0
0 0.4 0.8 1.2 PaE concentration (µg/mL)
1.0 0.5
20
0.0
100 TOC (µg/50µL)
0.5
120
1.5
OD600
1.0
B
2.0
80
TOC (µg/50µL)
OD600
1.5
OD600
A
100
2.0
TOC (µg/50 µL)
A
0.0
113
0 0
0.2
0.4
0.6
0.8
PaE concentration (µg/mL)
0
0.2
0.4
0.6
0.8
PaE concentration (µg/mL)
FIG. 2. Comparison of the results for OD600 and TOC measurements after degradation of BP films by PaE. (A) OD600 of the reaction solution after the degradation of dye-containing BP films (30 C for 24 h, at pH 8.8). (B) TOC released into the reaction solution after the degradation of BP films (without dye). Values are expressed as the mean (SD) at n ¼ 3.
OD600
The relative degradation activities of lipase B, proteinase K and PaE for BPs were compared using this dye-BP assay (Fig. 3). Lim et al. reported that several commercially available lipases and proteinase K degraded PCL and PLA, respectively (17). In this study, lipase B also showed degradation activity for PCL, as in many other lipases. Compared to lipase B and proteinase K, PaE showed relatively higher degradation activities for almost all the tested BPs. These results are compatible with our previous report (15). Furthermore, in this study, it was demonstrated for the first time that PaE could also degrade PUs. The maximum OD600 after complete degradation of BP films except for PUs was determined to be 1.78 0.12, and actual OD600 values of reaction solution after degradation of PBSA and PCL (Mw ¼ 1 104) films by PaE were 1.65 0.17 and 1.64 0.044, respectively (Fig. 3). These results indicated that these films were
2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
Lipase B ( 2 µg/mL) Proteinase K ( 100 µg/mL) PaE ( 2 µg/mL)
FIG. 3. Comparison of the relative BPs-degradating activities of commercially available enzymes and PaE. OD600 in the reaction solution after the enzymatic degradation of dye-containing BP films (30 C for 24 h, at pH 8.8) after subtracting the values for the control. The concentration of proteinase K was 100 mg/mL, lipase B and PaE was 2 mg/mL. Values are expressed as the mean (SD) at n ¼ 3.
almost completely degraded, and that was also confirmed visually (data not shown). The degradation activities of PaE for PCL and PLA seemed to decrease as their molecular weight increased. The OD600 values of controls (simple diffusion) for PBSA and PCL were 0.034 and 0.032 respectively, and those for other BPs were lower than 0.01 (data not shown). On the other hand, proteinase K showed a relatively lower degradation activity at lower enzyme concentrations, e.g., OD600 ¼ 0.018 at 10 mg/mL proteinase K for PLA-0005. To clarify the degradation activities of proteinase K for several BPs, it was used at a higher enzyme concentration (100 mg/mL) than that of PaE and lipase B (2 mg/mL). The results showed that proteinase K has a higher degradation activity for PLLA (Mw ¼ 1.3 105) than PDLLA (PLA-0005, Mw ¼ 0.5 104 and PLA-0020, Mw ¼ 2 104). Kawai et al. demonstrated that proteinase K can hydrolyze PLLA but not poly(D-lactic acid) (PDLA) (18). The lower degradation activities of proteinase K for PLA-0005 and PLA-0020 in this study were therefore considered to be caused by the presence of the D-lactyl unit. In this study, to examine the optimal pH range for the dye-BP assay, we tested the following buffers: 10 mM acetate buffer (pH 4.0e6.0), 10 mM HEPES buffer (pH 6.5e8.5), 10 mM phosphate buffer (pH 5.5e8.0), and 10 mM Tris buffer (pH 7.0e10.0). In the assay for the degradation of PLLA and PU, we observed a direct correlation between the concentrations of PaE and the OD600 values (data not shown). However, the lower pH buffers (10 mM acetate buffer [pH 4.0e5.5], 10 mM HEPES buffer [pH 6.5e7.0], and 10 mM phosphate buffer [pH 5.5e6.5]) could not be used for PBSA and PCL degradation assays because they caused the dye to be eluted from the BP films even in the control treatments. For the PBS and PDLLA degradation assays, the optimal pH range was 5.0e10.0. Although the pH range for the assay was restricted when using some BPs, the results of this study clearly showed that this new dye-BP assay is useful for evaluating the enzymatic degradation of various solid BP films. Since films of BPs such as PBSA and PCL can be removed from the glass slide easily, this assay can also be carried out in a closed tube. However, for BPs such as PBS, PLA and PU which are difficult to recover from glass slides, this assay technique performed on a glass slide as described above would be more suitable. With this method,
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a large number of samples can be analyzed within a short time period, thus, contributing to the efficient screening and characterization of BPs-degradation enzymes. In addition, not only the purified enzyme but also the crude enzyme solution including culture supernatant can be analyzed with this assay. To date, evaluation of the biodegradability of plastics by international standards has been carried out in natural conditions such as in soil and compost (3). For our future work, a comparative study between the BP degradation evaluated by the standard method and by our colorimetric method will be done in order to find correlation between them. We thank Novozymes Japan Ltd., Showa Denko K. K., Toyota Motor Co. Ltd., and Dai-ichi Kogyo Seiyaku Co. Ltd., for generously supplying the lipase B, PBS and PBSA, PLLA pellet, and water-dispersed polyurethanes respectively, used in this study. We also thank Dr. Elvira Suto for proofreading of the manuscript. This research was financially supported by the National Institute for Agro-Environmental Sciences, Japan.
References 1. Song, J. H., Murphy, R. J., Narayan, R., and Davies, G. B. H.: Biodegradable and compostable alternatives to conventional plastics, Philos. Trans. R. Soc. Lond. B Biol. Sci., 364, 2127e2139 (2009). 2. Hopewell, J., Dvorak, R., and Kosior, E.: Plastics recycling: challenges and opportunities, Philos. Trans. R. Soc. Lond. B Biol. Sci., 364, 2115e2126 (2009). 3. Kunioka, M., Ninomiya, F., and Funabashi, M.: Novel evaluation method of biodegradabilities for oil-based polycaprolactone by naturally occurring radiocarbon-14 concentration using accelerator mass spectrometry based on ISO 14855-2 in controlled compost, Polym. Degrad. Stab., 92, 1279e1288 (2007). 4. Pranamuda, H., Tokiwa, Y., and Tanaka, H.: Polylactide degradation by an Amycolatopsis sp. Appl. Environ. Microbiol., 63, 1637e1640 (1997). 5. Nakamura, K., Tomita, T., Abe, N., and Kamio, Y.: Purification and characterization of an extracellular poly(L-lactic acid) depolymerase from a soil isolate, Amycolatopsis sp. strain K104-1, Appl. Environ. Microbiol., 67, 345e353 (2001). 6. Akutsu-Shigeno, Y., Teeraphatpornchai, T., Teamtisong, K., Nomura, N., Uchiyama, H., Nakahara, T., and Nakajima-Kambe, T.: Cloning and sequencing of a poly(DL-lactic acid) depolymerase gene from Paenibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli, Appl. Environ. Microbiol., 69, 2498e2504 (2003).
J. BIOSCI. BIOENG., 7. Masaki, K., Kamini, N. R., Ikeda, H., and Iefuji, H.: Cutinase-like enzyme from the yeast Cryptococcus sp. strain S-2 hydrolyzes polylactic acid and other biodegradable plastics, Appl. Environ. Microbiol., 71, 7548e7550 (2005). 8. Maeda, H., Yamagata, Y., Abe, K., Hasegawa, F., Machida, M., Ishioka, R., Gomi, K., and Nakajima, T.: Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae, Appl. Microbiol. Biotechnol., 67, 778e788 (2005). 9. Li, F., Wang, S., Liu, W. F., and Chen, G. J.: Purification and characterization of poly(L-lactic acid)-degrading enzymes from Amycolatopsis orientalis ssp. orientalis, FEMS Microbiol. Lett., 282, 52e58 (2008). 10. Seo, H. S., Um, H. J., Min, J., Rhee, S. K., Cho, T. J., Kim, Y. H., and Lee, J.: Pseudozyma jejuensis sp. nov., a novel cutinolytic ustilaginomycetous yeast species that is able to degrade plastic waste, FEMS Yeast Res., 7, 1035e1045 (2007). 11. Hoshino, A. and Isono, Y.: Degradation of aliphatic polyester films by commercially available lipases with special reference to rapid and complete degradation of poly(L-lactide) film by lipase PL derived from Alcaligenes sp., Biodegradation, 13, 141e147 (2002). 12. Hayase, N., Yano, H., Kudoh, E., Tsutsumi, C., Ushio, K., Miyahara, Y., Tanaka, S., and Nakagawa, K.: Isolation and characterization of poly(butylene succinate-co-butylene adipate)-degrading microorganism, J. Biosci. Bioeng., 97, 131e133 (2004). 13. Nakajima-Kambe, T., Toyoshima, K., Saito, C., Takaguchi, H., AkutsuShigeno, Y., Sato, M., Miyama, K., Nomura, N., and Uchiyama, H.: Rapid monomerization of poly(butylene succinate)-co-(butylene adipate) by Leptothrix sp., J. Biosci. Bioeng., 108, 513e516 (2009). 14. Kitamoto, H. K., Shinozaki, Y., Cao, X. H., Morita, T., Konishi, M., Tago, K., Kajiwara, H., Koitabashi, M., Yoshida, S., Watanabe, T., and other 3 authors: Phyllosphere yeasts rapidly break down biodegradable plastics, AMB Express, 1, 44 (2011). 15. Shinozaki, Y., Morita, T., Cao X.H., Yoshida, S., Koitabashi, M., Watanabe, T., Suzuki, K., Sameshima-Yamashita, Y., Nakajima-Kambe, T., Fujii, T., and. Kitamoto, H.K.: Biodegradable plastic-degrading enzyme from Pseudozyma antarctica: cloning, sequencing, and characterization, Appl. Microbiol. Biotechnol., http://dx.doi.org/10.1007/s00253-012-4188-8 (published online 8 June 2012). 16. Cain, A. J.: The use of Nile Blue in the examination of lipoids, Q. J. Microsc. Sci., 88, 383e392 (1947). 17. Lim, H. A., Raku, T., and Tokiwa, Y.: A new method for the evaluation of biodegradable plastic using coated cellulose paper, Macromol. Biosci., 4, 875e881 (2004). 18. Kawai, F., Nakadai, K., Nishioka, E., Nakajima, H., Ohara, H., Masaki, K., and Iefuji, H.: Different enantioselectivity of two types of poly(lactic acid) depolymerases toward poly(L-lactic acid) and poly(D-lactic acid), Polym. Degrad. Stab., 96, 1342e1348 (2011).
TECHNICAL NOTE
Rapid and simple colorimetric assay for detecting the enzymatic degradation of biodegradable plastic films Yukiko Shinozaki,1 Takashi Watanabe,1 Toshiaki Nakajima-Kambe,2 and Hiroko K. Kitamoto1, * National Institute for Agro-environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaraki 305-8604, Japan1 and Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan2 Received 21 May 2012; accepted 17 August 2012 Available online 14 September 2012
We developed a rapid and simple method for evaluating the degradation of solid biodegradable plastics (BPs). Dye-containing BP films were used as substrates and the release of dye caused by the degradation of BPs was confirmed by a color change in the enzyme solution after a reaction time of 24 h. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Biodegradation; Poly(butylene succinate-co-adipate); Poly(lactic acid); Polyurethane; Pseudozyma antarctica]
The use of biodegradable plastics (BPs), such as poly(lactic acid) (PLA), poly (ε-caprolactone) (PCL), and poly(butylene succinate-coadipate) (PBSA), has received much attention from the viewpoint of environmental protection and solid-waste management (1,2). In the natural environment, microorganisms can degrade and assimilate BPs as a carbon source and release CO2; thus, the international standard for the evaluation of their biodegradability is based on measuring the amount of CO2 released under the specified conditions for 180 days or less (1,3). To date, various microorganisms, including bacteria, fungi, and yeast, have been reported to degrade BPs (4e14). In many cases, the abilities of these microorganisms to degrade BPs have been assayed easily by measuring the decrease in the turbidity of BP dispersions (4e9). This assay can be used as an initial screening for BPsdegrading microorganisms. However, our preliminary results indicated that there are many microorganisms that can degrade BP dispersions but not solid BPs (14). Therefore, a second round of screening is needed to identify the microorganisms or enzymes that can degrade solid BPs. The degradation of solid BPs could be evaluated by measuring the weight loss of BP films following enzymatic degradation, but this method needs a relatively long time (100 days or less) to confirm (11,13). Alternatively, the degradation could be evaluated by measuring the increase in solubilized monomers or oligomers released from the BP films into the reaction solution. In this method, the concentration of total organic carbon (TOC) is analyzed (4,10). The TOC analyzer can detect a low level of carbon (4 mg/L); thus, the degradation of solid BPs can be confirmed after a relatively short treatment time, although the equipment required for this method is costly.
* Corresponding author. Tel.: þ81 29 838 8355; fax: þ81 29 838 8199. E-mail address: [email protected] (H.K. Kitamoto).
In this study, we developed a rapid and simple method for evaluating the degradation of solid BPs. Hand-made dye-containing BP films were used as substrates. After treatment of the film with an enzyme solution for 24 h, the relative amount of dye released was measured using a colorimetric detector (Fig. 1). The BPs used in the degradation experiments were as follows: PBSA, Bionolle 3020 (weight average molecular weight [Mw] ¼ 1.4 105) and PBS, Bionolle 1020 (Mw ¼ 1.4 105) supplied by Showa Denko K. K. (Tokyo, Japan); PCL (Mw ¼ 1 104 and 7e10 104), poly(DL-lactic acid) (PDLLA) PLA-0005 (Mw ¼ 0.5 104), and PLA-0020 (Mw ¼ 2 104) purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan); and poly (L-lactic acid) (PLLA) (Mw ¼ 1.3 105) supplied by Toyota Motor Co. Ltd. (Aichi, Japan). Each BP pellet was dissolved in dichloromethane at 1 wt%, and then 300 mL of the solution was mixed with 10 mL of Nile blue staining reagent (7% w/v; Muto Pure Chemicals Co., Ltd. Tokyo, Japan). Each dye-containing BP solution was cast onto a waterrepellent printing glass slide (Matsunami Glass Ind. Ltd., Osaka, Japan), with 20 mL in each well (f 7 mm), and was air-dried at room temperature for 24 h. The weight of the BP film and the dye it contained were calculated to be 0.265 and 0.04 mg, respectively (based on the dichloromethane density, 1.3266 g/cm3). Water-dispersed polyurethanes (PU) Superflex 150 (ester and ether-type) and Superflex 840 (aromatic isocyanate and ester-type) supplied by Dai-ichi Kogyo Seiyaku Co. Ltd. (Kyoto, Japan) were also used. To prepare PU films, 300 mL of the undiluted PU solution (27e30 wt%) was mixed with 10 mL of Nile blue staining reagent, and then 10 mL of the mixture was spread onto each well of the glass slide and air-dried at room temperature for 24 h. The BPs-degrading enzyme from Pseudozyma antarctica JCM10317 (PaE) was purified as described earlier (14,15). To compare PaE’s BPs-degradation activity with that of other enzymes, the commercially available enzymes, proteinase K (SigmaeAldrich
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.08.010
112
SHINOZAKI ET AL.
J. BIOSCI. BIOENG.,
Preparation of BP films on a water-repellent printing glass slide by casting (BPs were dissolved in dichloromethane with Nile blue staining reagent, and dried)
A
Enzyme solution (50 µL) BP film containing Nile blue staining reagent Water-repellent printing glass slide Well (
Apply enzyme solution on the BP film
7 mm)
1
B
2
3
4
PBSA Enzyme reaction (30°C for 24 h) (degradation of BP film release the dye and increase OD600 in reaction solution)
End of enzyme reaction
PLA
C
1
PBSA Collect the reaction solution into a 96-well microplate and measure OD600
1: Control 2: Lipase B 3: Proteinase K 4: PaE
PCL
PCL PLA
2
3
4 1: Control 2: Lipase B 3: Proteinase K 4: PaE
FIG. 1. Evaluation of the relative BPs-degradation activity using dye-containing BP films (dye-BP assay). (A) Cross-sectional view of a BP film and enzyme solution on the glass slide. (B) Photograph of the glass slides with BP films and enzyme solutions after the reaction (30 C for 24 h). The black area of the glass slide has been printed with highly water-repellent mark preventing the enzyme solution from spilling out from the well. (C) Reaction solutions collected after the degradation of dye-containing BP films (PBSA, Mw ¼ 1.4 105; PCL, Mw ¼ 1 104; and PLA, Mw ¼ 2 104). The BP film degradation could be evaluated from OD600 values of the reaction solutions after subtracting the values for the control.
Japan, Tokyo, Japan) and lipase B (CALB L; Novozymes Japan Ltd., Chiba, Japan) were used. The protein concentrations of these enzymes were determined with a protein assay kit (Bio-Rad Laboratories, USA) according to the manufacturer’s instructions, using bovine serum albumin (SigmaeAldrich Japan) as standard. Each BP film on the glass slide was washed twice with 80 mL of distilled water to eliminate slight extra Nile blue dye. In general, this dye is used for histochemical staining of lipoids. It is a mixture of the oxazine sulfate (true Nile blue) which reacts with acidic lipoids (fatty acids, phospholipids and some others), and the oxazone (Nile red) which dissolves in neutral lipoids (16). The color of the PCL film containing Nile blue was red, while that of other BP films was blue (Fig. 1B, control). This color difference is presumed to be due to the inherent characteristics of each BP. Then 50 mL of the appropriately diluted enzymes (lipase B, proteinase K, and PaE) in 20 mM TriseHCl (pH 8.8) were separately dropped onto the BP films (Fig. 1A). To avoid drying of the BP films, the glass slide was put into a small container lined with wet paper, sealed and incubated at 30 C for 24 h. The reaction solutions on the BP films were then collected into a 96-well microplate (roundbottom), and the OD600 values were measured using a multispectrophotometer (Dainippon, Osaka, Japan). The OD600 values of the enzyme solutions were obtained after subtracting the values for the controls. For the substrate and enzyme controls, the enzyme and the substrate were omitted from the reaction solutions, respectively. To predict the OD600 in reaction solution after complete degradation of a BP film, dichloromethane without BP was mixed with Nile blue and cast on a well; then after evaporating dichloromethane, the dye was dissolved in buffer solution and its OD600 was measured, as described above. After the enzymatic reactions with the dye-containing BP films, the color changes in the reaction solutions were observed
(Fig. 1B and C). The OD600 values of the solutions were correlated with the enzyme concentrations (Fig. 2A). The concentrations of the dye in PU films were different from that of other BPs because the initial concentrations of PU (dispersed in water at 27e30 wt%) were higher compared to other BPs (dissolved in dichloromethane at 1 wt%). Nevertheless, OD600 values of the enzyme solutions after the PU films degradation were correlated to the enzyme concentrations, similar to other BPs (Fig. 2A). The actual amount of BPs solubilized by the enzymatic reaction was evaluated by measuring the TOC content of the reaction solutions as follows. The same experimental procedure as above, except for the addition of Nile blue staining reagent, was carried out for PBSA, PCL (Mw ¼ 1 104), PLA-0020, and PU (Superflex 840), and the collected reaction solutions were diluted with 20 mL of Milli-Q water. The diluted solutions were then filtered through a 0.45-mm membrane (DISMIC-25, PTFE; Toyo Roshi Kaisha, Ltd., Tokyo, Japan), and the water-soluble TOC level in the solution was measured using a TOC-V CSH analyzer (Shimadzu Co., Kyoto, Japan). The range of the linear correlation between the TOC level and the PaE concentration was slightly different compared to that between the OD600 and the PaE concentration (Fig. 2A and B). These results may be due to the fact that these two methods detect different products. Specifically, TOC analysis measures the watersoluble oligomers and/or monomers released from BP films, while the colorimetric assay using OD600 measurement determines the amount of dye eluted from BP film during the degradation process. Although the linear correlation ranges showed slight differences, these evaluations of the relative BPs degradation based on TOC or OD600 measurement were basically in agreement with each other. This new assay using dye-containing BP films (dye-BP assay) can therefore be considered useful as a rapid and simple method for evaluating the enzymatic degradation of BP films.
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TECHNICAL NOTE
PBSA
PCL
B
60 40
PLA-0020
A
0.0
0.4
0.8
0.6
60 40 20
0.0
0 1.0
1.5
2.0
2.5
OD600
80
TOC (µg/50µL)
0.8
0.5
1.2
PU (Superflex 840) 0.8
PaE concentration (µg/mL)
0.8
0.4
0.8
1.2
PaE concentration (µg/mL)
B 500
0.4 0.2
400 300 200 100
0.0 0.0 0.5 1.0 1.5 2.0 2.5 PaE concentration (µg/mL)
0
PaE concentration (µg/mL)
100
0.0
0.4
A
0.2
40
0 0.0
1.2
PaE concentration (µg/mL)
1.0
0.4
60
20
B
0.6
80
0.0
0 0.4 0.8 1.2 PaE concentration (µg/mL)
1.0 0.5
20
0.0
100 TOC (µg/50µL)
0.5
120
1.5
OD600
1.0
B
2.0
80
TOC (µg/50µL)
OD600
1.5
OD600
A
100
2.0
TOC (µg/50 µL)
A
0.0
113
0 0
0.2
0.4
0.6
0.8
PaE concentration (µg/mL)
0
0.2
0.4
0.6
0.8
PaE concentration (µg/mL)
FIG. 2. Comparison of the results for OD600 and TOC measurements after degradation of BP films by PaE. (A) OD600 of the reaction solution after the degradation of dye-containing BP films (30 C for 24 h, at pH 8.8). (B) TOC released into the reaction solution after the degradation of BP films (without dye). Values are expressed as the mean (SD) at n ¼ 3.
OD600
The relative degradation activities of lipase B, proteinase K and PaE for BPs were compared using this dye-BP assay (Fig. 3). Lim et al. reported that several commercially available lipases and proteinase K degraded PCL and PLA, respectively (17). In this study, lipase B also showed degradation activity for PCL, as in many other lipases. Compared to lipase B and proteinase K, PaE showed relatively higher degradation activities for almost all the tested BPs. These results are compatible with our previous report (15). Furthermore, in this study, it was demonstrated for the first time that PaE could also degrade PUs. The maximum OD600 after complete degradation of BP films except for PUs was determined to be 1.78 0.12, and actual OD600 values of reaction solution after degradation of PBSA and PCL (Mw ¼ 1 104) films by PaE were 1.65 0.17 and 1.64 0.044, respectively (Fig. 3). These results indicated that these films were
2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
Lipase B ( 2 µg/mL) Proteinase K ( 100 µg/mL) PaE ( 2 µg/mL)
FIG. 3. Comparison of the relative BPs-degradating activities of commercially available enzymes and PaE. OD600 in the reaction solution after the enzymatic degradation of dye-containing BP films (30 C for 24 h, at pH 8.8) after subtracting the values for the control. The concentration of proteinase K was 100 mg/mL, lipase B and PaE was 2 mg/mL. Values are expressed as the mean (SD) at n ¼ 3.
almost completely degraded, and that was also confirmed visually (data not shown). The degradation activities of PaE for PCL and PLA seemed to decrease as their molecular weight increased. The OD600 values of controls (simple diffusion) for PBSA and PCL were 0.034 and 0.032 respectively, and those for other BPs were lower than 0.01 (data not shown). On the other hand, proteinase K showed a relatively lower degradation activity at lower enzyme concentrations, e.g., OD600 ¼ 0.018 at 10 mg/mL proteinase K for PLA-0005. To clarify the degradation activities of proteinase K for several BPs, it was used at a higher enzyme concentration (100 mg/mL) than that of PaE and lipase B (2 mg/mL). The results showed that proteinase K has a higher degradation activity for PLLA (Mw ¼ 1.3 105) than PDLLA (PLA-0005, Mw ¼ 0.5 104 and PLA-0020, Mw ¼ 2 104). Kawai et al. demonstrated that proteinase K can hydrolyze PLLA but not poly(D-lactic acid) (PDLA) (18). The lower degradation activities of proteinase K for PLA-0005 and PLA-0020 in this study were therefore considered to be caused by the presence of the D-lactyl unit. In this study, to examine the optimal pH range for the dye-BP assay, we tested the following buffers: 10 mM acetate buffer (pH 4.0e6.0), 10 mM HEPES buffer (pH 6.5e8.5), 10 mM phosphate buffer (pH 5.5e8.0), and 10 mM Tris buffer (pH 7.0e10.0). In the assay for the degradation of PLLA and PU, we observed a direct correlation between the concentrations of PaE and the OD600 values (data not shown). However, the lower pH buffers (10 mM acetate buffer [pH 4.0e5.5], 10 mM HEPES buffer [pH 6.5e7.0], and 10 mM phosphate buffer [pH 5.5e6.5]) could not be used for PBSA and PCL degradation assays because they caused the dye to be eluted from the BP films even in the control treatments. For the PBS and PDLLA degradation assays, the optimal pH range was 5.0e10.0. Although the pH range for the assay was restricted when using some BPs, the results of this study clearly showed that this new dye-BP assay is useful for evaluating the enzymatic degradation of various solid BP films. Since films of BPs such as PBSA and PCL can be removed from the glass slide easily, this assay can also be carried out in a closed tube. However, for BPs such as PBS, PLA and PU which are difficult to recover from glass slides, this assay technique performed on a glass slide as described above would be more suitable. With this method,
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a large number of samples can be analyzed within a short time period, thus, contributing to the efficient screening and characterization of BPs-degradation enzymes. In addition, not only the purified enzyme but also the crude enzyme solution including culture supernatant can be analyzed with this assay. To date, evaluation of the biodegradability of plastics by international standards has been carried out in natural conditions such as in soil and compost (3). For our future work, a comparative study between the BP degradation evaluated by the standard method and by our colorimetric method will be done in order to find correlation between them. We thank Novozymes Japan Ltd., Showa Denko K. K., Toyota Motor Co. Ltd., and Dai-ichi Kogyo Seiyaku Co. Ltd., for generously supplying the lipase B, PBS and PBSA, PLLA pellet, and water-dispersed polyurethanes respectively, used in this study. We also thank Dr. Elvira Suto for proofreading of the manuscript. This research was financially supported by the National Institute for Agro-Environmental Sciences, Japan.
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