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

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.

Characterization of poly(L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175

“…One unit (U) of PCL-degrading activity and hydrolysis of olive oil was defined as a 1 U·ml-1 decrease in absorbance at 630 nm under the assay conditions described by Nakamura et al (2001). Esterase activity was assayed as described by Maeda et al (2005), using five types of p-nitrophenyl (pNP) esters: pNP-acetate (C2), pNP-butyrate (C4), pNP-decanoate (C10), pNP-palmitate (C16) and pNP-stearate (C18). Fifty mM of N-succinyl-L-alanyl-Lalanyl-L-alanine 4-nitroanilide (Suc-(Ala) 3 -pNA) was dissolved in 100 mM Tris-HCl buffer (pH 9.0).…”

Bioethanol Production from Brewers Spent Grains Using a Fungal Consolidated Bioprocessing (CBP) Approach

“…If one were to consider application of the sake CBP system for bioethanol production from BSG, it is of interest that other applications of A. oryzae might be developed. Since A. oryzae is able to decompose biodegradable plastic, such as polybutylene succinate (PBS; [35]), it could perhaps be used in the recycling of biodegradable bottles. A. oryzae has both cutinase (CutL1) and hydrophobin genes (such as rolA) within its genome which have been shown to be responsible for the deconstructive mechanism.…”

“…A. oryzae has both cutinase (CutL1) and hydrophobin genes (such as rolA) within its genome which have been shown to be responsible for the deconstructive mechanism. Cutinase facilitates the actual decomposition of the plastic, with hydrophobins acting as 'scaffolding' for the specific site recruitment of the cutinase onto the hydrophobic surfaces of biodegradable plastics [27,35].…”

Phylloplane Fungal Enzyme Accelerate Decomposition of Biodegradable Plastic Film in Agricultural Settings

“…Previous studies have examined the capacities of purified enzymes secreted by various fungi to degrade several BP materials (Murphy et al 1996;Maeda et al 2005;Baker et al 2012;). However, agricultural BP mulch films are blends of several polymers and additives, such as an ultraviolet scattering agent, and no previous studies have investigated the enzymatic degradation of these films in agricultural fields (Brodhagen et al 2015).…”