Polylactic Acid (PLA)-Degrading Microorganisms and PLA

Chapter 27

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Polylactic Acid (PLA)-Degrading Microorganisms and PLA Depolymerases Fusako Kawai* R & D Center for Bio-based Materials, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan *[email protected]

This paper summarizes topics on microorganisms able to degrade polylactic acid (PLA) and PLA depolymerases. Although the glass transition temperature for PLA is high (approximately 60 °C), PLA-degrading microbes can degrade solid PLA at far lower temperatures such as 30 or 37 °C. Such degraders are Actinomycetes belonging to family Nocardiaceae: PLA depolymerases were purified and cloned as serine proteases from genus Amycolatopsis. Thermophilic lipases were obtained from thermophilic Bacillus strains able to grow on PLA at 60 °C, although their contribution to degradation of PLA is skeptical at high temperature as PLA is easily hydrolyzable. Commercially available proteases and lipases are known to act as PLA depolymerases. We found that enantioselectivity of protease-type depolymerases is specific to poly(L-lactic acid), but that of lipase-type depolymerases is preferential to poly(D-lactic acid). Thus, proteases and lipases are categorized into two different classes of PLA depolymerases.

Introduction The start of chemical synthesis of poly(lactic acid) (PLA) dates back to 1932 when Carothers first synthesized PLA of approximately 3,000 Da. In the 1960s, PLA found its use in medical fields as a bio-absorbable material. Since the latter half of the 1980s, plastic waste attracted public concern as an environmental issue. Very recently, rising cost and limited resources of crude oil has turned © 2010 American Chemical Society In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


more attention toward alternative sources. This trend shed light on PLA again as a bio-based material capable of replacing oil-based materials. PLA is chemically synthesized from lactic acid, a representative fermentation product from plant resources, and is thereby defined as a biomass plastic. The biodegradability of biomass plastics is not a main focus, since the CO2 released from PLA by combustion is thought to equal to the CO2 absorbed by plants, thereby yielding zero-emission of CO2. Therefore, these plastics are designated “carbon-neutral.” However, the biodegradability of PLA has already been established since the first report on enzymatic hydrolysis of PLA by William that described the feasibility for proteases and unfeasibility for esterases (1). Later, some lipases and esterases were reported to be able to hydrolyze PLA (2, 3), but they seem to be active only for low molecular weights (4) or poly(DL-lactide) (5). Among acid, neutral and alkaline proteases, only alkaline proteases showed appreciable activity (14 positive results among a total of 22) for high molecular weight PLA (6), but all the commercial lipases were negative (7). Lim et al. showed that all the six mammalian and microbial serine proteases hydrolyzed PLA well (8). There are also many reports on the microbial assimilation of PLA, since Pranamuda et al. first isolated the PLA-assimilating Amycolatopsis sp. strain HT-32 (9). Tokiwa and Calabia concluded that most of the PLA-degrading microorganisms phylogenetically belong to the family of Pseudonocardiaceae and related genera such as Amycolatopsis, Lentzea, etc. in which proteinous materials promote the production of the PLA-degrading enzyme (10). PLA-degrading enzymes from PLA-assimilating microorganisms were purified from different strains of Amycolatopsis approximately at the same time by two groups (11, 12) and were characterized as proteases. Later, both groups cloned the genes (13, 14). It is notable that almost all the degradation tests have been carried out using poly(L-lactic acid) (PLLA) and that no information regarding the biodegradability of poly(D-lactic acid) (PDLA) is available, except that proteinase K hydrolyzed PLLA but not PDLA (15) and that Tomita et al. isolated a thermophilic Bacillus stearothermophilus able to grow at 60 °C on PDLA as a sole carbon source, although they did not mention enzyme activity contributing to the degradation of PDLA (16). As PLA is hydrolyzed at a relatively high rate at high temperatures (>50 °C), whether the strain excretes PDLA-degrading enzyme or utilizes hydrolyzed products depends on future characterization of PDLA-degrading enzyme. Thus, no information has been available on the enzymatic hydrolysis of PDLA until now. This paper summarizes research on PLA-degrading microorganisms and PLA-degrading enzymes. In addition, I will introduce research of our group on enantioselectivity of PLA-degrading enzymes, based on which PLA depolymerases are categorized into two different classes.

PLA-Degrading Microorganisms and Their PLA Depolymerases Since Pranamuda et al. first isolated the PLA-degrading Amycolatopsis sp. strain HT32 from soil in 1997 (9), many groups isolated various PLA-degrading 406 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


microorganisms, as shown in Table 1. PLA-degrading Actinomycetes belong to the family of Pseudonocardiaceae and related genera, the representative strain of which is Amycolatopsis sp. (10). Stock culture of Amycolatopsis species (15 strains among total 25 strains) made clear zones on PLA agar plates (11, 17), suggesting that this genus contributes a lot for PLA degradation in nature. As approximately 95% of all the soil Actinomycetes are Streptomyces, limited distribution of PLA-degrading Actinomycetes is in accordance with that PLA film is hardly degraded when buried in soil. Except Actinomycetes, Bacillus, Brevibacillus, and Geobacillus have been reported as thermophilic degraders (18–20). Paenibacillus amylolyticus strain TB-13 could decrease turbidity of DL-PLA emulsion at 37 °C, but its degradability toward PLA film is unknown (21). As PLA film is degraded quickly in compost, thermophilic degraders are probable. However, degradation rates by thermophilic degraders are still under discussion. Since PLA is hydrolyzed non-enzymatically and remarkably at 60 °C, the enzymatic contribution to hydrolysis of PLA at 60 °C has to be evaluated carefully, compared with non-enzymatic hydrolysis (with regards to change in film weight, molecular mass and products), but this kind of comparison is insufficient in reports. Although we cannot deny the contribution of thermophilic Actinomycetes to PLA degradation in compost, this should be evaluated carefully in the future. Mayumi et al. recently cloned three genes encoding PLA depolymerases, based on metagenome from compost (22), one of which coded fo a thermostable esterase homologous to Bacillus lipase and showed the binding ability to DL-PLA powders with molecular masses of lower than 20,000. As the expressed enzyme had no activity on L-PLA with molecular masses of approximately 130,000, the enzyme might be able to degrade depolymerized PLA products. We could guess the same mechanism to other thermophilic enzymes. As DL-PLA has lower melting point and higher hydrolytic rate, compared with those of PLLA or PDLA, we should be careful for evaluating biodegradability of PLA (DL-PLA, PLLA and PDLA) in compost. A fungus, Tritirachium album ATCC22563, also showed degradation ability of PLLA, silk fibroin and elastin, which was inducible with gelatin, suggesting the induction of a protease (23). However, the role of the fungus in nature for degradation of PLA is skeptical, since the enzyme was not induced at all in the absence of gelatin.

Table 1. PLA-degrading microorganisms and their degrading enzymesa Microorganisms

PLAdegrading enzyme

Substrate and evaluation of degradability

°C

Ref.

Amycolatopsis sp. strain HT32

protease

L-PLA, film weight

30

(9)

Amycolatopsis sp. strain 3118

“

L-PLA, film weight

30

(26)

Actinomycetes

Continued on next page.

407 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Table 1. (Continued). PLA-degrading microorganisms and their degrading enzymesa


Microorganisms

PLAdegrading enzyme

Substrate and evaluation of degradability

Amycolatopsis sp. strain KT-s-9

“

Amycolatopsis sp. strain 41

°C

Ref.

L-PLA, silk fibroin, halo on PLA-agar plate

30

(27)

“

L-PLA, silk powder, casein, film weight

30

(11)

Amycolatopsis sp. strain K104-1

“

L-PLA, casein, fibroin, turbidity of emusion and film weight

37

(12)

Amycolatopsis orientalis IFO12362

“

L-PLA powder, TOC

30, 40

(24)

Lentzea waywayandensis

“

L-PLA, film weight

30

(28)

Kibdelosporangium aridum

“

L-PLA, film weight

30

(29)

Brevibacillus sp.

unknown

L-PLA, film (TOC, GPC, viscosity)

60

(19)

Bacillus stearothermophilus

unknown

D-PLA, film (TOC, GPC, viscosity)

60

(16)

Geobacillus thermocatenulatus

unknown

L-PLA, film (TOC, GPC,viscosity)

60

(20)

Bacillua sinithii strain PL21

esterase

L-PLA, pellet or powder, GPC

60

(18)

Paenibacillus amylolyicus strain TB-13

lipase

DL-PLA, turbidity of emulsion, TOC

37

(21)

Metagenome from compostb

Lipase (esterase)

DL-PLA powder, TOC, absorption of enzymes

60

(22)

Tritirachiium album ATCC 22563

protease

L-PLA, film weight

30

(23)

Cryptococcus sp. strain S-1

Cutinase AB102945

L-PLA, turbidity of emulsion

30

(25)

Aspergillus oryzae RIB40

cutinase

DL-PLA, turbidity of emulsion

37

(30)

Bacteria

Fungi

a

TOC: total organic carbon, GPC: gel permeation chromatography, Ref.; reference homologous to aBacillus lipase.

b

The inside temperature of compost at the secondary fermentation stage is expected to be approximately 60 °C, where degradation of organic materials is most promoted. As vegetative bacterial cells are killed at 55 °C, mature compost 408 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


is free of general pathogenic bacteria and can be used as safe fertilizers for plants. The reason why PLA is not degraded in soil, but well degraded in compost is most probably due to the low density of Pseudonocardiaceae actinomycetes in soil and the high hydrolytic rate of PLA over 50 °C, yielding hydrolyzed products, which are more feasible for microbial degradation than the original high molecular weight of PLA. Enzymatic degradation is probable when enzyme potential and rigidity of molecular chain of a substrate were in accordance with each other, but impossible when binding energy of molecular chain of a substrate is over catalytic energy of an enzyme. However, as shown above, the degradation of PLA was possible at 30 °C far lower than glass transition temperature (Tg: approximately 55 °C), which is not understandable by the flexibility of the substrate molecular chain, but understandable by the reason that PLA absorbs water to cause the collapse of a polymer block, which would produce hydrophilic parts either on the polymer surface or inside of the polymer and become feasible with attack by microbes or enzymes. The fact that hydrolysis of PLA is possible at lower temperatures than Tg seems to support this assumption. Low degradation of PLA film in soil is probably due to low water activity in soil in addition to low density of Pseudonocardiaceae. Thus biodegradation of PLA at 30-37 °C is understandable. On the other hand, thermophilic microbes/enzymes are necessary for recycling of PLA, because thermophilic microbes/enzymes are tougher than mesophilic ones and hydrolysis is more efficient at higher temperatures. Actually PLA depolymerase from A. orientalis IFO12362 showed twice activity at 40 °C as much as that of proteinase K at 37 °C (24). Since the first report of Pranamuda et al on PLA-degrading activity by culture supernatant of Amycolatopsis sp. strain HT32 (9), several enzymes from Pseudonocardiacear were considered to be proteases, as shown in Table 1. Nakamura et al. purified PLA depolymerase from Amycolatopsis sp. strain K104-1 and its N-teminal amino acids had homology with serine protease from earthworm able to degrade fibrin and with serine protease from crab able to degrade collagen (12). The same group also cloned the gene for the enzyme, which had homology with serine proteases from eukaryotic cells belonging to chymotrypsin family (endopeptidase) and produced oligolactide and lactic acid from PLA (13). The enzyme was feasible to degrade PLA film at 30-37 °C. On the other hand, the enzyme from Bacillus sinithii strain PL21 was a thermotolerant esterase (18) and that from Paenibacillus amylolyticus strain TB-13 showed 45-50% homology with mesophilic Bacillus family I-4 lipases (21). These enzymes are all from PLA degraders. On the other hand, Masaki et al. isolated Cryptococcus sp. S-2 for use in wastewater treatment and found that the strain excreated the strong lipase activity (25). They cloned the gene for a lipase. The gene had higher homology with cutinase (EC 3.1.1.74) than with lipase, which showed stronger degradation ability toward PLA than proteinase K. Therefore we could say that the enzyme is the strongest PLA depolymerase so far known. This is astonishing, since the strain was not intended to show PLA degradation. Another cutinase-type enzyme from Aspergillus oryzae RIB40 also had weak activity toward PLA, compared with those toward PBS and PBSA (30). 409 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 1. Chemical structure of L-lactic acid (A) and L-alanine (B)

Figure 2. Reaction of lipase-type (A) and protease-type (B) PLA depolymerases with PLLA and PDLA. (A) CLE from Cryyptococcus sp. S-1 (20 µg/ml, pH 7.0); (B) purified recombinant PLA depolymerase from Amycolatopsis sp. K104-1 (20 µg/ml, pH 9,0). Reactions were performed at 37 °C with shaking using PLLA or PDLA (a final concentration of approximately 0.1 wt%). Degradation rate was measured by change of absorbance at 600 nm. Open circle, PLLA; closed circle, PDLA All the PLA-degraders and their enzymes acted on PLLA or DL-PLA except Bacillus stearothermophilus that was reported to degrade PDLA at 60 °C (16), but degradation of PLLA and the enzyme activity was not mentioned. Unavailability of PDLA through regular commercial routes and extremely high price of PDLA seemed to have restricted research on degradation of PDLA.

Degradation of PLA by Hydrolytic Enzymes The biodegradation of PLA was reported first by Williams in 1981 using proteases including proteinase K (1). Degradation of PLA by lipases was unfeasible in his report. Later the feasibility of lipases was reported (2, 3), but they were active on low molecular weights (4) or poly(DL-lactide) (5). However, active proteases are widely distributed in Pseudonocardiaceae and their activities are in general higher than those of lipases (esterases) (26–28). Most of commercially available proteases can catalyze hydrolysis of PLA (8): Microbial proteases (proteinase K and subtilisin) and mammalian proteases (α-chymotrypsin, trypsin and elastase) could degrade PLA, but plant proteases could not. This result suggested that PLA as bioabsorbable material used in human body must partly be hydrolyzed by proteases in vivo. As proteinase K has the best activity among commercially available enzymes, it is often used for enzymatic degradation of PLA. As the chemical structure of PLA is a polyester or PHA, lipases (esterases) 410 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


are predicted to be most probable degrading enzymes, but actually proteases are the most potential degrading enzymes, which is in accordance with that PLA depolymerases purified or cloned from PLA-degrading Amycolatopsis species are proteases (Table 1 (11–14)). It is already well known that the nature does not discriminate natural or synthetic compounds, but the most importance is chemical structures themselves recognizable by enzymes. In general, polyesters are degraded by lipases (esterases) with increased rates in turn of aromatic ones, aliphatic-aromatic ones and aliphatic ones. Tokiwa et al. suggested degradation rates of polyesters as polyhydroxybutyrate (PHB=plycaprolactone (PCL)>polybutylene succinate (PBS) >PLA (4). Biodegradation test in field also showed low biodegradability of PLA compared with PHB/V (valerate), PCL and PBS. Biodegadation of polyesters by lipases is dependent on either chemical structure or melting point (mp) (4). Accordingly, low biodegradability of PLA is possibly due to its short carbon length of monomer and high mp (PLLA and PDLA: approximately 175 °C).

Catalytic Aspect of PLA Depolymerase Both of proteases and lipases belong to a serine hydrolase family, but differ in the substrate specificities. All the PLA depolymerases from Pseudonocardiaceae are proteases active on PLLA, as shown in Table 1. Proteinase K and a protease-type enzyme from Tritirachium album ATCC22563 were PLLA-spccific (15, 23). PLA depolymerases from thermotolerant Bacillus sinithii strain PL21 and Paenibacillus amylolyticus strain TB-13 were an esterase active on PLLA and a lipase active on DL-PLA, respectively (18, 21), although their exact biodegradation ability are ambiguous. Commercially available lipases (esterases) act on DL-PLA, but not on optically active PLLA or D-PHB (4). Protease-type PLA depolymerases can hydrolyze PLLA, but not D-PHB. PHB depolymerases do not act on PLLA. Cutinase from Cryptococcus sp. S-2 showed higher activity toward PLLA than that of proteinase K, suggesting that this acts exceptionally as an esterase on PLLA (25). These results suggested that PLA-degrading enzymes are distinctly different from PHB depolymerases. Specificities of hydrolytic enzymes are in general wide, but recognition of the polyester (PLA) by proteases is biochemically interesting to understand what specificity means. Tokiwa et al. explained that the origin of a protease-type PLA depolymerase from Actinomycetes is a protease working on silk fibroin, based on homology of L-lactic acid with L-alanine as the major component of silk fibroin (Fig. 1) and induction of the enzyme by proteinous compounds such as gelatin (4). Reeve et al disclosed that proteinase K was PLLA-specific and did not act on PDLA at all (15). Matsuda et al. confirmed that a recombinant PLA depolymerase from Amycolatopsis did not work on PCL and PHB (13). PHB depolymerase does not act on PLLA (a kind of hydroxyalkanoate), due to differences in optical activities of both substrates and in carbon chain length of 2-hydroxyalkanoate and 3-hydroxyalkanoate. Thus PLA depolymerases have to be categorized differently from PHB depolymerases and PLA is considered as the third type-polyester following synthetic polyesters and polyhydroxyalkanoate (PHA) including PHB. 411 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Using the recombinant purified PLA-degrading enzyme from Amycolatopsis sp. K104-1 (13) and the recombinant purified cutinase-like enzyme (CLE) from Cryptococcus sp. S-2 (25), we examined their enantioselectivity toward PLLA and PDLA, as shown in Fig. 2. PLA-degrading enzyme was absolutely PLLAspecific. Together with the report on the enatioselectivity of proteinase K (15) and the fact that proteases originally recognize polymer of L-amino acids, we could conclude that protease-type PLA depolymerases are PLLA-specific. On the other hand, CLE acted on both PLLA and PDLA, but the activity was higher on PDLA than on PLLA (PDLA-preferential). The enantioselectivity of two types of PLA depolymerases will be useful for the biological recycling of PLA and the recovery of lactic acid (especially expensive D-lactic acid). We found that some lipases are also PDLA-preferential (unpublished data). The enantioselectivity of crude enzymes could be a good indicator for prediction of the type of enzymes, either protease or cutinase, which would lead to the successful cloning of enzyme genes, based on the conserved regions of each group. Commercially available true lipases did not act on PLLA and PDLA. True lipases possess a lid covering an active site to lead to the interfacial activation (31), but some lipases, esterases and cutinases have neither lid nor interfacial activation. To cover an active site by a lid leading to an interfacial activation, the size of an active site inlet cannot be too big. On the other hand, the inlet of PLA depolymerase has to be big enough to accommodate a macromolecular PLA. Accordingly, lipase-type PLA depolymerases are probably not a typical true lipase, but an esterase (cutinase) without a lid useful for interfacial activation and with an active cavity big enough to accommodate a polymer substrate (cutin is a rather big molecule of a complex structure).

Conclusion From the aforementioned information, the following conclusions can be obtained for the enzymatic degradation of PLLA and PDLA by PLA depolymerases. 1) Polyester-degrading enzymes are categorized into three groups; synthetic (aliphatic) polyester-degrading group, PHA-degrading groups and PLAdegrading groups. 2) PLA depolymerases are categorized into two enzyme groups; proteasetype (type I) and lipase (cutinase)-type (type II). 3) Type I-PLA depolymerases are PLLA-specific, but lipase (cutinase)-type PLA depolymerases are PDLA-preferencial. 4) The presence of a lid in true lipases (causing the interfacial activation) interferes the access of PLA to the catalytic amino acid in the active cavity. 5) Absence of a lid and the geometry of the active site are the two important factors in the catalysis of the lipase family. Therefore, cutinase-type enzymes in the lipase superfamily are most probably type II-PLA depolymerase. 412 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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Polylactic Acid (PLA)-Degrading Microorganisms and PLA

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