Cutinase-Like Enzyme from the Yeast Cryptococcus sp. Strain S-2 Hydrolyzes Polylactic Acid and Other Biodegradable Plastics

Abstract

A purified lipase from the yeast Cryptococcus sp. strain S-2 exhibited remote homology to proteins belonging to the cutinase family rather than to lipases. This enzyme could effectively degrade the high-molecular-weight compound polylactic acid, as well as other biodegradable plastics, including polybutylene succinate, poly (ɛ-caprolactone), and poly(3-hydroxybutyrate).

Polylactic acid (PLA) is a plastic obtained from renewable resources that has attracted attention in response to increasing concerns about the environmental effects of disposal of nonbiodegradable plastics (4). However, the infrastructure for the disposal of biodegradable waste plastics is still not complete (4). Enzymatic degradation is an ideal waste treatment method because enzymes accelerate hydrolysis of PLA and other biodegradable plastics and can be incorporated into a natural cycle of organic materials. Furthermore, the hydrolysate can be recycled as material for polymers. Degradation of low-molecular-weight PLA has been investigated with lipase from Rhizopus delemer (3) and polyurethane esterase from Comamonas acidovorans TB-35 (1). Attempts to degrade PLA with other enzymes have resulted in only modest success (12, 19). Studies on the degradation of high-molecular-weight PLA have been performed with strains of Amycalotopsis sp. (7, 13). However, the enzymes secreted by these microorganisms were not identified.

The yeast Cryptococcus sp. strain S-2 isolated in our laboratory could be used for various wastewater treatment processes (6), and it produced a lipase (8) which could be used effectively in the production of methyl esters, which were excellent substitutes for diesel fuel (9). In the present study, the purified lipase from Cryptococcus sp. strain S-2 was analyzed to determine its amino acid sequence, and it was compared with lipases and other related enzymes in the database. An attempt was made to test the potency of the purified enzyme for the degradation of high-molecular-weight PLA and other biodegradable plastics. The enzyme was produced and purified as described previously (8). The purified enzyme produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.

Nucleotide sequencing.

The open reading frame encoding the enzyme contains 720 nucleotides, including a start codon (ATG) and a stop codon (TAA). N-terminal sequencing of the purified mature protein revealed that the first 34 amino acids are a secretion signal sequence. The deduced amino acid sequence of the mature protein contains 205 amino acids with an estimated molecular mass of 20.9 kDa, which was similar to the molecular mass estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein (8).

A BLAST search revealed that the deduced amino acid sequence of this protein did not exhibit homology with any of the known lipases, and the enzyme was found to be weakly (12 to 20%) homologous to proteins belonging to the cutinase family. Alignment of sequences around putative catalytic residues in known cutinases and this enzyme indicated that the three residues (S119, D199, H214) which form a catalytic triad in lipases, esterases, and serine proteases are conserved in this enzyme along with the consensus sequence lipase box GXSXG motif around a catalytic serine (Fig. 1). Most of the cutinases and this enzyme contain the consensus pentapeptide sequence GYSQG in this region. In addition, the positions of four cysteine residues that form disulfide bonds in cutinases are also conserved in this enzyme, suggesting that the tertiary structure of this enzyme might be similar to that of proteins belonging to the cutinase family. Hence, this enzyme appears to be more similar to a cutinase than to a lipase. Since the level of homology of the enzyme is too low to categorize it as a cutinase, we refer to it here as cutinase-like enzyme (CLE) (formerly called a lipase). The cutinase activity of CLE was estimated as described by Sagt et al. (14) and Van Gemeren et al. (18) using p-nitrophenyl butyrate as the model substrate.

FIG. 1.

Comparison of conserved amino acid motifs in cutinases. P11373, Colletotrichum gloeosporioides; P30272, Magnaporthe grisea; P41744, Alternaria brassicicola; P29292, Ascochyta rabiei; P52956, Aspergillus oryzae; P00590, Fusarium solani; Q00298, Botrytis cinerea; Q10837, Mycobacterium tuberculosis; CLE, Cryptococcus sp. strain S-2 CLE. Three residues belonging to the catalytic triad are indicated by boldface type. A nucleophilic serine is located in a highly conserved GXSXG pentapeptide consensus motif.

Hydrolysis of plastics.

The plastics and enzymes used in this study are listed in Table 1. The CLE, proteinase K from Tritirachium album, and commercial lipases were employed for hydrolysis of emulsified biodegradable plastics (Table 2). Enzymatic degradation was carried out at 30°C with continuous shaking at 50 rpm for 10 days. The reaction mixture contained 0.04% (wt/vol) emulsified plastic, 0.0008% (wt/vol) plysurf A210G (a surfactant), 20 mM Tris-HCl (pH 8.0), and an enzyme (100 μg/ml). The emulsified plastics were prepared as described by Nisida and Tokiwa (11). The degradation ratio was calculated by measuring the decrease in turbidity at 660 nm of solutions before and after addition of the enzyme. Two of the seven commercial lipases (lipases from Burkholderia sp. and Rhizopus oryzae) could degrade only poly(ɛ-caprolactone) (PCL) completely, and none of the lipases could degrade PLA and polybutylene succinate (PBS). Proteinase K could degrade only PLA, while CLE exhibited strong activities with all biodegradable plastics. Lipases are known to decompose various polyesters (16). Hoshino and Isono (5) compared the degradation of PLA using 18 commercially available lipases; however, these enzymes were unable to degrade high-molecular-weight PLA. Our results also indicated that the commercial lipases from various microorganisms were unable to degrade high-molecular-weight PLA.

TABLE 1.

Biodegradable plastics and enzymes

Plastic or enzyme	Characteristics and/or source
Plastics
PCL	Mol wt, 1.0 × 104; Wako Pure Chemical Industries, Ltd., Osaka, Japan
PBS	Bionolle; no. 1001; Showa Highpolymer Co., Ltd., Tokyo, Japan
PLA	LACEA; HT-100; mol wt, 1.4 × 105; Mitsui Chemicals, Inc., Tokyo, Japan
Serine protease (proteinase K from Tritirachium album)	No. 1.24568; chromatographically purified; Merck KgaA, Darmstadt, Germany
Lipases
Lipase A from Aspergillus niger	Amano Enzyme Inc., Nagoya, Japan
Lipase AY from Candida rugosa	Amano Enzyme Inc., Nagoya, Japan
Lipase F from Rhizopus oryzae	Amano Enzyme Inc., Nagoya, Japan
Lipase PS from Burkholderia sp.	Amano Enzyme Inc., Nagoya, Japan
Candida antarctica	Fluka Chemie AG, Buchs, Switzerland
Penicillium roqueforti	Fluka Chemie AG, Buchs, Switzerland
Mucor javanicus	Fluka Chemie AG, Buchs, Switzerland

TABLE 2.

Hydrolysis of biodegradable plastics by CLE from Cryptococcus sp. strain S-2, commercial lipases, and a serine protease

Enzyme	Hydrolysis of plasticsa
	PLA	PBS	PCL
CLE from Cryptococcus sp. strain S-2	+	+	+
Lipases
Rhizopus oryzae	−	−	+
Burkholderia sp.	−	−	+
Penicillium roqueforti	−	−	−
Mucor javanicus	−	−	−
Aspergillus niger	−	−	−
Candida rugosa	−	−	−
Candida antarctica	−	−	−
Serine protease from Tritirachium album	+	−	−

^a+, degraded; −, not degraded.

Degradation of PLA and other plastics.

The hydrolytic activity of CLE with PLA was compared with that of proteinase K, which at present is the most effective enzyme for PLA degradation (12, 19) (Fig. 2A). Degradation of PLA was also carried out with 0.8 and 400 μg/ml of proteinase K, while 400μg/ml of proteinase K was used for degradation of PBS and PCL. The concentration of CLE in the reaction mixtures was 0.8 μg/ml for the degradation of PLA and poly(3-hydroxybutyrate) (PHB), and 8 ng/ml of CLE was used for the degradation for PCL and PBS.

FIG. 2.

Degradation of PLA (A), PBS (B), and PCL (C) by CLE and proteinase K. PLA degradation (A) was carried out with enzyme concentrations of 400 μg/ml (▵) and 0.8 μg/ml (▴) for proteinase K from T. album or 0.8 μg/ml for CLE from Cryptococcus sp. strain S-2 (⧫). A reaction mixture incubated without any enzyme served as the control (×). Degradation of PBS (B) and degradation of PCL (C) were carried out with a CLE concentration of 8 ng/ml.

The CLE at a concentration of 0.8 μg/ml completely degraded high-molecular-weight PLA in 60 h, while proteinase K degraded PLA completely at a concentration of 400 μg/ml in 88 h. Thus, the CLE was more than 500 times more effective than proteinase K for degradation of high-molecular-weight PLA. The ratio of degradation of PLA was less than 20% with proteinase K at a concentration of 0.8 μg/ml at 100 h. The CLEdegraded PLA completely even at a concentration of 0.08μg/ml after a 10-day reaction. The degradation was confirmed by the increase in the concentration of lactate monomer in the reaction mixture as PLA was degraded (data not shown).

PLA degradation by proteinase K has been reported previously (19), and our results confirmed this; however, the CLE was more effective than proteinase K for degradation of high-molecular-weight PLA. One strain of Fusarium moniliforme was able to grow on a polylactic acid-glycolic acid copolymer after 2 months of incubation at 28°C on synthetic agar medium, but the enzyme involved in the degradation was not identified (17). Although there have been reports of the degradation of PLA by bacteria (10, 13, 15), only a few enzymes from the bacteria were found to degrade PLA (2, 10). Until now, there have been no reports of degradation of biodegradable plastics using enzymes from yeasts. Akutsu-Shigeno et al. (2) cloned a gene encoding poly(dl-lactic acid) depolymerase from Paenibacillus amylolyticus strain TB-13 in Escherichia coli and found that the enzyme (PlaA) was a type of lipase. Although some enzymatic characteristics of PlaA were similar to characteristics of CLE, the lipase box of PlaA, AHSMG, was different from that of CLE.

The effects of CLE on the degradation of other biodegradable plastics are shown in Fig. 2B and C. The CLE effectively degraded the plastics PBS and PCL at an enzyme concentration of 8 ng/ml, and complete degradation was observed at 5 h and 28 h, respectively, while proteinase K could not degrade PBS and PCL even at a concentration of 400 μg/ml; similar results were reported for PLA depolymerase from Amycolatopsis sp., which could degrade the high-molecular-weight PLA but did not hydrolyze PHB and PCL (10). Although CLE could degrade PHB, the degradation of PHB was not complete with CLE under these conditions (data not shown).

The CLE exhibits broad substrate specificity with biodegradable plastics. This characteristic of CLE makes it useful for disposal of various biodegradable plastics. Furthermore, future studies of CLE may lead to improvements in the degradation of waste plastics, as well as the development of novel biodegradable plastics.

Nucleotide sequence accession numbers.

The DDBJ accession number of the cDNA sequence of CLE is AB102945.