Purificacion Y Caracterizacion De Un Plastico Biodegradable

Appl Microbiol Biotechnol (2005) 67: 778–788 DOI 10.1007/s00253-004-1853-6

BIOTECHNOLOG ICA LLY RELEVANT ENZYMES A ND PRO TEINS

Hiroshi Maeda . Youhei Yamagata . Keietsu Abe . Fumihiko Hasegawa . Masayuki Machida . Ryoji Ishioka . Katsuya Gomi . Tasuku Nakajima

Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae
Received: 13 August 2004/ Revised: 25 October 2004 / Accepted: 12 November 2004 / Published online: 27 January 2005 # Springer-Verlag 2005

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 super-

H. Maeda Tohoku Technoarch, 2–1-1 Katahira, Aoba-ku, Sendai, 980-8577, Japan H. Maeda . Y. Yamagata (*) . K. Abe . T. Nakajima Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of AgriculturalScience, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan e-mail: yamagata@biochem.tohoku.ac.jp Tel.: +81-022717-8776 Fax: +81-022717-8778 H. Maeda . Y. Yamagata . K. Abe . F. Hasegawa . K. Gomi New Industry Creation Hatchery Center, Tohoku University, 468 Aramaki-aza-aoba, Aoba-ku, Sendai, 981-0845, Japan M. Machida Gene Regulation Group, Molecular and Cell Biology,National Institute of Advanced Industrial Science and Technology, Central 6, 1-1 Higashi, Tsukuba, 305-8566, Japan R. Ishioka Showa Highpolymer, Kanda Chuo Bldg. 20, Kanda Nishiki-cho 3-chome, Chiyoda-ku, Tokyo, 101-0054, Japan K. Gomi Laboratory of Bioindustrial Genomics, Division of Bioscience and Biotechnology for Future Bioindustries, Graduate School of Agricultural Science, TohokuUniversity, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan

natant, 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 enzymerecognizes 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.

Introduction
In recent times, the excessive consumption of synthetic plastics derived from petroleum has had an adverse impact on the environment because the majority of these synthetic plastics do not degrade in theenvironment, and incineration of plastics generates CO2 and dioxin (Huang 1995; Potts et al. 1973). These molecules increase the warming of the Earth and environmental pollution. In view of this, some aliphatic polymer types have been developed as biodegradable plastics (Mayer and Kaplin 1994). Biodegradable plastics have several excellent properties and may provide solutions to global environmentalproblems. First, biodegradable plastics are degraded by microorganisms in the natural environment (Ishigaki et al. 2000; Suyama et al. 1998a,b). Second, they can be composted, and burn with a lower calorific value than that of synthetic plastic materials. To date, some aliphatic polyester types have been developed. Poly-(lactic acid) (PLA), poly(butylene succinate) (PBS), and poly-(butylene...