Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae.
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.
Maeda
Hiroshi
H
Tohoku Technoarch, 2-1-1 Katahira, Aoba-ku, Sendai, 980-8577, Japan.
Yamagata
Youhei
Y
Abe
Keietsu
K
Hasegawa
Fumihiko
F
Machida
Masayuki
M
Ishioka
Ryoji
R
Gomi
Katsuya
K
Nakajima
Tasuku
T
D000272:Adipates; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000254:growth & development; D001673:Biodegradation, Environmental; D002072:Butylene Glycols / Q000378:metabolism; D020148:Butyric Acid / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003470:Culture Media; D004655:Emulsions; D000441:Hexanols / Q000378:metabolism; D019344:Lactic Acid; D008970:Molecular Weight; D010969:Plastics / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D013379:Substrate Specificity; D013386:Succinates / Q000378:metabolism
D000272:Adipates; D002072:Butylene Glycols; D003470:Culture Media; D004655:Emulsions; D000441:Hexanols; D010969:Plastics; D011108:Polymers; D013386:Succinates; C089797:bionole; C475864:poly(butylene succinate-co-butylene adipate); D020148:Butyric Acid; D019344:Lactic Acid; C036260:1-hexanol; D002265:Carboxylic Ester Hydrolases; C019952:cutinase