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

A purified lipase from the yeast Cryptococcus sp. strain S-2exhibited remote homology to proteins belonging to the cutinasefamily rather than to lipases. This enzyme could effectivelydegrade the high-molecular-weight compound polylactic acid,as well as other biodegradable plastics, including polybutylenesuccinate, poly ( ${varepsilon}$ -caprolactone), and poly(3-hydroxybutyrate).

INTRODUCTION

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Introduction

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

The yeast Cryptococcus sp. strain S-2 isolated in our laboratorycould be used for various wastewater treatment processes (6),and it produced a lipase (8) which could be used effectivelyin the production of methyl esters, which were excellent substitutesfor diesel fuel (9). In the present study, the purified lipasefrom Cryptococcus sp. strain S-2 was analyzed to determine itsamino acid sequence, and it was compared with lipases and otherrelated enzymes in the database. An attempt was made to testthe potency of the purified enzyme for the degradation of high-molecular-weightPLA and other biodegradable plastics. The enzyme was producedand purified as described previously (8). The purified enzymeproduced a single band on sodium dodecyl sulfate-polyacrylamidegel electrophoresis gels.

Nucleotide sequencing.

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Nucleotide sequencing.

The open reading frame encoding the enzyme contains 720 nucleotides,including a start codon (ATG) and a stop codon (TAA). N-terminalsequencing of the purified mature protein revealed that thefirst 34 amino acids are a secretion signal sequence. The deducedamino acid sequence of the mature protein contains 205 aminoacids with an estimated molecular mass of 20.9 kDa, which wassimilar to the molecular mass estimated from sodium dodecylsulfate-polyacrylamide gel electrophoresis of the purified protein(8).

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

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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.

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Hydrolysis of plastics.

The plastics and enzymes used in this study are listed in Table1. The CLE, proteinase K from Tritirachium album, and commerciallipases were employed for hydrolysis of emulsified biodegradableplastics (Table 2). Enzymatic degradation was carried out at30°C with continuous shaking at 50 rpm for 10 days. Thereaction 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 plasticswere prepared as described by Nisida and Tokiwa (11). The degradationratio was calculated by measuring the decrease in turbidityat 660 nm of solutions before and after addition of the enzyme.Two of the seven commercial lipases (lipases from Burkholderiasp. and Rhizopus oryzae) could degrade only poly( ${varepsilon}$ -caprolactone)(PCL) completely, and none of the lipases could degrade PLAand polybutylene succinate (PBS). Proteinase K could degradeonly PLA, while CLE exhibited strong activities with all biodegradableplastics. Lipases are known to decompose various polyesters(16). Hoshino and Isono (5) compared the degradation of PLAusing 18 commercially available lipases; however, these enzymeswere unable to degrade high-molecular-weight PLA. Our resultsalso indicated that the commercial lipases from various microorganismswere unable to degrade high-molecular-weight PLA.

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TABLE 1. Biodegradable plastics and enzymes

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

Degradation of PLA and other plastics.

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Degradation of pla and...

The hydrolytic activity of CLE with PLA was compared with thatof proteinase K, which at present is the most effective enzymefor PLA degradation (12, 19) (Fig. 2A). Degradation of PLA wasalso carried out with 0.8 and 400 µg/ml of proteinaseK, while 400µg/ml of proteinase K was used for degradationof PBS and PCL. The concentration of CLE in the reaction mixtureswas 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 PCLand PBS.

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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 ( ${triangleup}$ ) and 0.8 µg/ml ( ${blacktriangleup}$ ) for proteinase K from T. album or 0.8 µg/ml for CLE from Cryptococcus sp. strain S-2 ( ${blacklozenge}$ ). A reaction mixture incubated without any enzyme served as the control (x). 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 degradedhigh-molecular-weight PLA in 60 h, while proteinase K degradedPLA completely at a concentration of 400 µg/ml in 88 h.Thus, the CLE was more than 500 times more effective than proteinaseK for degradation of high-molecular-weight PLA. The ratio ofdegradation of PLA was less than 20% with proteinase K at aconcentration of 0.8 µg/ml at 100 h. The CLEdegraded PLAcompletely even at a concentration of 0.08µg/ml aftera 10-day reaction. The degradation was confirmed by the increasein the concentration of lactate monomer in the reaction mixtureas 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 moreeffective than proteinase K for degradation of high-molecular-weightPLA. One strain of Fusarium moniliforme was able to grow ona polylactic acid-glycolic acid copolymer after 2 months ofincubation at 28°C on synthetic agar medium, but the enzymeinvolved in the degradation was not identified (17). Althoughthere have been reports of the degradation of PLA by bacteria(10, 13, 15), only a few enzymes from the bacteria were foundto degrade PLA (2, 10). Until now, there have been no reportsof degradation of biodegradable plastics using enzymes fromyeasts. Akutsu-Shigeno et al. (2) cloned a gene encoding poly(DL-lacticacid) depolymerase from Paenibacillus amylolyticus strain TB-13in Escherichia coli and found that the enzyme (PlaA) was a typeof lipase. Although some enzymatic characteristics of PlaA weresimilar 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 biodegradableplastics are shown in Fig. 2B and C. The CLE effectively degradedthe 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 concentrationof 400 µg/ml; similar results were reported for PLA depolymerasefrom Amycolatopsis sp., which could degrade the high-molecular-weightPLA but did not hydrolyze PHB and PCL (10). Although CLE coulddegrade PHB, the degradation of PHB was not complete with CLEunder these conditions (data not shown).

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

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

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

FOOTNOTES

* Corresponding author. Mailing address: Environmental Research Division, National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima 739-0046, Japan. Phone: 81-82-420-0818. Fax: 81-82-420-0806. E-mail: masaki{at}nrib.go.jp.

Present address: Department of Biotechnology, Central LeatherResearch Institute, Adyar, Chennai 600 025, India.

REFERENCES

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