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Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae

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Abstract

We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C4-C6 and C3-C8 chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.

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References

  • Ando Y, Yoshikawa K, Yoshikawa T, Nishioka M, Ishioka R, Yakabe Y (1998) Biodegradability of poly (tetramethylene succinate-co-tetramethylene adipate). I. Enzymatic hydrolysis. Polym Degrad Stabil 61:129–137

    Article  Google Scholar 

  • Barbesgaard P, Heldt-Hansen HP, Diderichsen B (1992) On the safety of Aspergillus oryzae: a review. Appl Microbiol Biotechnol 36:569–572

    Google Scholar 

  • Christensen T, Woeldike H, Boel E, Mortensen SB, Hjortshoejk K, Thim L, Hansen MT (1988) High-level expression of recombinant genes in Aspergillus oryzae. Biotechnology 6:1419–1422

    Article  Google Scholar 

  • Cleland WW (1979) Substrate inhibition. Methods Enzymol 63:500–513

    Google Scholar 

  • Coligan JE, Krusbeek AM, Margulies DH, Shevach EM, Strober W (1991) Current protocols in immunology. Greene/Wiley, New York

    Google Scholar 

  • Egmond MR, de Vlieg J (2000) Fusarium solani pisi cutinase. Biochimie 82:1015–1021

    Article  Google Scholar 

  • Ferreira BS, Calado CR, van Keulen F, Fonseca LP, Cabral JM, da Fonseca MM (2003) Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects. Appl Microbiol Biotechnol 61:69–76

    Google Scholar 

  • Fromm HJ (1979) Use of competitive inhibitors to study substrate binding order. Methods Enzymol 63:467–486

    Google Scholar 

  • Fujimaki T (1998) Processability and properties of aliphatic polyesters, ‘BIONOLLE’, synthesized by polycondensation reaction. Polym Degrad Stabil 59:209–214

    Article  Google Scholar 

  • Gouka RJ, Punt PJ, van den Hondel CA (1997) Efficient production of secreted proteins by Aspergillus: progress, limitations and prospects. Appl Microbiol Biotechnol 47:1-11

    Google Scholar 

  • Hatamoto O, Sekine H, Nakano E, Abe K (1999) Cloning and expression of a cDNA encoding the laccase from Schizophyllum commune. Biosci Biotechnol Biochem 63:58–64

    CAS  PubMed  Google Scholar 

  • Hawthorne BT, Rees-George J, Crowhurst RN (2001) Induction of cutinolytic esterase activity during saprophytic growth of cucurbit pathogens, Fusarium solani fsp. cucurbitae races one and two (Nectria haematococca MPI and MPV, respectively). FEMS Microbiol Lett 194:135–141

    Article  Google Scholar 

  • Hirano H, Watanabe T (1990) Microsequencing of proteins electrotransferred onto immobilizing matrices from polyacrylamide gel electrophoresis: application to an insoluble protein. Electrophoresis 11:573–580

    Google Scholar 

  • Huang SJ (1995) Polymer waste management-biodegradation, incineration, and recycling. J Macromol Sci Pure A 32:593–597

    Google Scholar 

  • Ishigaki T, Sugano W, Ike M, Kawagoshi Y, Fukunaga I, Fujita M (2000) Abundance of polymers degrading microorganisms in a sea based solid waste disposal site. J Basic Microbiol 40:177–186

    Article  Google Scholar 

  • Kitakuni E, Yoshikawa K, Nakano K, Sasuga J, Nobiki M, Naoi H, Yokota Y, Ishioka R, Yakabe Y (2001) Biodegradation of poly (tetramethylene succinate-co-tetramethylene adipate) and poly (tetramethylene succinate) through water-soluble products. Environ Toxicol Chem 20:941–946

    Google Scholar 

  • Kleeberg I, Hetz C, Kroppenstedt RM, Muller RJ, Deckwer WD (1998) Biodegradation of aliphatic-aromatic copolyesters by Thermomonospora fusca and other thermophilic compost isolates. Appl Environ Microbiol 64:1731–1735

    Google Scholar 

  • Kolattukudy PE (1984) Detection of an N-terminal glucuronamide linkage in proteins. Methods Enzymol 106:210–217

    Google Scholar 

  • Kolattukudy PE, Rogers LM, Li D, Hwang CS, Flaishman MA (1995) Surface signaling in pathogenesis. Proc Natl Acad Sci USA 92:4080–4087

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Google Scholar 

  • Lin TS, Kolattukudy PE (1977) Glucuronyl glycine, a novel N-terminus in a glycoprotein. Biochem Biophys Res Commun 75:87–93

    Article  Google Scholar 

  • Lin TS, Kolattukudy PE (1980) Structural studies on cutinase, a glycoprotein containing novel amino acids and glucuronic acid amide at the N terminus. Eur J Biochem 106:341–351

    Google Scholar 

  • Longhi S, Czjzek M, Lamzin V, Nicolas A, Cambillau C (1997a) Atomic resolution (1.0 Å) crystal structure of Fusarium solani cutinase: stereochemical analysis. J Mol Biol 268:779–799

    Article  Google Scholar 

  • Longhi S, Mannesse M, Verheij HM, De Haas GH, Egmond M, Knoops-Mouthuy E, Cambillau C (1997b) Crystal structure of cutinase covalently inhibited by a triglyceride analogue. Protein Sci 6:275–286

    Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  • Machida M (2002) Progress of Aspergillus oryzae genomics. Adv Appl Microbiol 51:81–106

    CAS  PubMed  Google Scholar 

  • Maeda H, Sano M, Maruyama Y, Tanno T, Akao T, Totsuka Y, Endo M, Sakurada R, Yamagata Y, Machida M, Akita O, Hasegawa F, Abe K, Gomi K, Nakajima T, Iguchi Y (2004) Transcriptional analysis of genes for energy catabolism and hydrolytic enzymes in the filamentous fungus Aspergillus oryzae using cDNA microarrays and expressed sequence tags. Appl Microbiol Biotechnol 65:74–83

    Google Scholar 

  • Mannesse ML, Cox RC, Koops BC, Verheij HM, de Haas GH, Egmond MR, van der Hijden HT, de Vlieg J (1995) Cutinase from Fusarium solani pisi hydrolyzing triglyceride analogues. Effect of acyl chain length and position in the substrate molecule on activity and enantioselectivity. Biochemistry 34:6400–6407

    Google Scholar 

  • Martinez C, De Geus P, Lauwereys M, Matthyssens G, Cambillau C (1992) Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent. Nature 356:615–618

    Article  Google Scholar 

  • Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038

    Google Scholar 

  • Mayer JM, Kaplin DL (1994) Biodegradable materials: balancing degradability and performance. Trends Polym Sci 2:227–235

    Google Scholar 

  • Murphy CA, Cameron JA, Huang SJ, Vinopal RT (1996) Fusarium polycaprolactone depolymerase is cutinase. Appl Environ Microbiol 62:456–460

    Google Scholar 

  • Nakamura K, Tomita T, Abe N, Kamio Y (2001) Purification and characterization of an extracellular poly(l-lactic acid) depolymerase from a soil isolate, Amycolatopsis sp. strain K104-1. Appl Environ Microbiol 67:345–353

    Google Scholar 

  • Ohnishi K, Yoshida Y, Sekiguchi J (1994) Lipase production of Aspergillus oryzae. J Ferment Bioeng 77:490–495

    Article  Google Scholar 

  • Ohnishi K, Yoshida Y, Toida J, Sekiguchi J (1994) Purification and characterization of a novel lipolytic enzyme from Aspergillus oryzae. J Ferment Bioeng 78:413–419

    Article  Google Scholar 

  • Ohnishi K, Toida J, Nakazawa H, Sekiguchi J (1995) Genome structure and nucleotide sequence of a lipolytic enzyme gene of Aspergillus oryzae. FEMS Microbiol Lett 126:145–150

    Article  Google Scholar 

  • Potts J, Clendinning R, Ackart WB, Niegisch WD (1973) The biodegradability of synthetic polymers. In: Guillet J (eds) Polymers and ecological problems. Plenum, New York, pp 61–79

    Google Scholar 

  • Prompers JJ, van Noorloos B, Mannesse ML, Groenewegen A, Egmond MR, Verheij HM, Hilbers CW, Pepermans HA (1999) NMR studies of Fusarium solani pisi cutinase in complex with phosphonate inhibitors. Biochemistry 38:5982–5994

    Article  Google Scholar 

  • Purdy RE, Kolattukudy PE (1975a) Hydrolysis of plant cuticle by plant pathogens. Properties of cutinase I, cutinase II, and a nonspecific esterase isolated from Fusarium solani pisi. Biochemistry 14:2832–2840

    Google Scholar 

  • Purdy RE, Kolattukudy PE (1975b) Hydrolysis of plant cuticle by plant pathogens. Purification, amino acid composition, and molecular weight of two isozymes of cutinase and a nonspecific esterase from Fusarium solani f. pisi. Biochemistry 14:2824–2831

    Google Scholar 

  • Rudolph FB (1979) Product inhibition and abortive complex formation. Methods Enzymol 63:411–436

    Google Scholar 

  • Rudolph FB, Fromm HJ (1979) Plotting methods for analyzing enzyme rate data. Methods Enzymol 63:138–159

    Google Scholar 

  • Saito K, Miura N, Yamazaki M, Hirano H, Murakoshi I (1992) Molecular cloning and bacterial expression of cDNA encoding a plant cysteine synthase. Proc Natl Acad Sci USA 89:8078–8082

    Google Scholar 

  • Schafer W (1993) The role of cutinase in fungal pathogenicity. Trends Microbiol 1:69–71

    Article  Google Scholar 

  • Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379

    PubMed  Google Scholar 

  • Shibuya I, Tsuchiya K, Tamura G, Ishikawa T, Hara S (1992) Overproduction of an alpha-amylase/glucoamylase fusion protein in Aspergillus oryzae using a high expression vector. Biosci Biotechnol Biochem 56:1674–1675

    Google Scholar 

  • Suyama T, Hosoya H, Tokiwa Y (1998a) Bacterial isolates degrading aliphatic polycarbonates. FEMS Microbiol Lett 161:255–261

    Article  Google Scholar 

  • Suyama T, Tokiwa Y, Ouichanpagdee P, Kanagawa T, Kamagata Y (1998b) Phylogenetic affiliation of soil bacteria that degrade aliphatic polyesters available commercially as biodegradable plastics. Appl Environ Microbiol 64:5008–5011

    Google Scholar 

  • Sweigard JA, Chumley FG, Valent B (1992a) Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea. Mol Gen Genet 232:174–182

    Google Scholar 

  • Sweigard JA, Chumley FG, Valent B (1992b) Disruption of a Magnaporthe grisea cutinase gene. Mol Gen Genet 232:183–190

    Google Scholar 

  • Takahashi T, Chang PK, Matsushima K, Yu J, Abe K, Bhatnagar D, Cleveland TE, Koyama Y (2002) Nonfunctionality of Aspergillus sojae aflR in a strain of Aspergillus parasiticus with a disrupted aflR gene. Appl Environ Microbiol 68:3737–3743

    Google Scholar 

  • Teeraphatpornchai T, Nakajima-Kambe T, Shigeno-Akutsu Y, Nakayama M, Nomura N, Nakahara T, Uchiyama H (2003) Isolation and characterization of a bacterium that degrades various polyester-based biodegradable plastics. Biotechnol Lett 25:23–28

    Article  Google Scholar 

  • Tsuchiya K, Nagashima T, Yamamoto Y, Gomi K, Kitamoto K, Kumagai C, Tamura G (1994) High level secretion of calf chymosin using a glucoamylase-prochymosin fusion gene in Aspergillus oryzae. Biosci Biotechnol Biochem 58:895–899

    Google Scholar 

  • Tsuchiya K, Tada S, Gomi K, Kitamoto K, Kumagai C, Jigami Y, Tamura G (1992) High level expression of the synthetic human lysozyme gene in Aspergillus oryzae. Appl Microbiol Biotechnol 38:109–114

    Google Scholar 

  • Uchida H, Nakajima-Kambe T, Shigeno-Akutsu Y, Nomura N, Tokiwa Y, Nakahara T (2000) Properties of a bacterium which degrades solid poly (tetramethylene succinate)-co-adipate, a biodegradable plastic. FEMS Microbiol Lett 189:25–29

    Article  Google Scholar 

  • Yu J, Proctor RH, Brown DW, Abe K, Gomi K, Machida M, Hasegawa F, Nierman WC, Bhatnagar D, Cleveland TE (2004). Genomics of economically significant Aspergillus and Fusarium species. In: Arora KD, Khachatourians GG (eds) Applied mycology and biotechnology, vol 4. Fungal genomics. Elsevier, Amsterdam

    Google Scholar 

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Acknowledgements

This work has been supported by Innovation Plaza Miyagi of JST (Japan Science and Technology Agency), and was supported in part by a grant-in-aid (Bio Design Program) from the Ministry of Agriculture, Forestry and Fisheries of Japan (BDP-03-VI-1-6)

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Authors and Affiliations

  1. Tohoku Technoarch, 2–1-1 Katahira, Aoba-ku, Sendai, 980-8577, Japan

    Hiroshi Maeda

  2. Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan

    Hiroshi Maeda, Youhei Yamagata, Keietsu Abe & Tasuku Nakajima

  3. New Industry Creation Hatchery Center, Tohoku University, 468 Aramaki-aza-aoba, Aoba-ku, Sendai, 981-0845, Japan

    Hiroshi Maeda, Youhei Yamagata, Keietsu Abe, Fumihiko Hasegawa & Katsuya Gomi

  4. Gene Regulation Group, Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1 Higashi, Tsukuba, 305-8566, Japan

    Masayuki Machida

  5. Showa Highpolymer, Kanda Chuo Bldg. 20, Kanda Nishiki-cho 3-chome, Chiyoda-ku, Tokyo, 101-0054, Japan

    Ryoji Ishioka

  6. Laboratory of Bioindustrial Genomics, Division of Bioscience and Biotechnology for Future Bioindustries, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan

    Katsuya Gomi

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  1. Hiroshi Maeda
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Correspondence to Youhei Yamagata.

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Maeda, H., Yamagata, Y., Abe, K. et al. Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae . Appl Microbiol Biotechnol 67, 778–788 (2005). https://doi.org/10.1007/s00253-004-1853-6

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  • DOI: https://doi.org/10.1007/s00253-004-1853-6

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