Biochemical and molecular characterization of Coriolopsis rigida laccases involved in transformation of the solid waste from olive oil production

Abstract

Two laccase isoenzymes were purified and characterized from the basidiomycete Coriolopsis rigida during transformation of the water-soluble fraction of "alpeorujo" (WSFA), a solid residue derived from the olive oil production containing high levels of toxic compounds. Zymogram assays of laccases secreted by the fungus growing on WSFA and WSFA supplemented with glucose showed two bands with isoelectric points of 3.3 and 3.4. The kinetic studies of the two purified isoenzymes showed similar affinity on 2,6-dimethoxyphenol and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), used as phenolic and non-phenolic model substrate, respectively. The molecular mass of both proteins was 66 kDa with 9% N-linked carbohydrate. Physico-chemical properties of the purified laccases from media containing WSFA were similar to those obtained from medium with glucose as the main carbon source. In-vitro studies performed with the purified laccases revealed a 42% phenol reduction of WSFA, as well as changes in the molecular mass distribution. These findings indicate that these laccases are involved in the process of transformation, via polymerization by the oxidation of phenolic compounds present in WSFA. A single laccase gene, containing an open reading frame of 1,488 bp, was obtained in PCR amplifications performed with cDNA extracted from mycelia grown on WSFA. The product of the gene shares 90% identity (95% similarity) with a laccase from Trametes trogii and 89% identity (95% similarity) with a laccase from Coriolopsis gallica. This is the first report on purification and molecular characterization of laccases directly involved in the transformation of olive oil residues.

Optimization of the production, purification and characterization of a laccase from the native fungus Xylaria sp.

“…However, it is important to underscore that the yield obtained for the purification process in different reports, which apply different strategies, is highly variable and is related to the specific characteristics for each crude extract and enzyme. Among this variability, excellent yields such as 77.6% have been obtained by Patel et al, (2014) working with P. ostreatus, but low yields have been reported by other researchers such as Sahay et al, (2008) with 3,46% yield using Pleorotus sajor-caju MTCC 141; or Diaz et al, (2010) with 2,2 and 2,6% yield for laccase isoforms I and II from Coriolopsis rigida; or Junghans et al, (2009) with 0,95% yield using Phoma sp. In contrast, the specific activity value obtained in this study was superior to the activity value obtained for the purified enzymes from other organisms.…”

Integrated biovalorization of wine and olive mill by-products to produce enzymes of industrial interest and soil amendments

“…DOR strongly stimulated Lac production and slightly altered MnP and LiP secretion. Olive oil residues have been reported to stimulate Lac, MnP and LiP in polyporal fungi (Díaz et al, 2010;Reina et al, 2013Reina et al, , 2014. No previous evidence of GPS induction on LME has been reported, although it is capable of stimulating lipase secretion in Aspergillus sp.…”

“…At week 4 of incubation with T. versicolor in a barley-based media, the phytotoxicity of GPS was totally eliminated. T0 T1 T2 T3 T4 T5 Control DOR 5% T0 T1 T2 T3 T4 T5 Control DOR 5% T0 T1 T2 T3 T4 T5 Control DOR 5% T0 T1 T2 T3 T4 T5 9 Integral biovalorization of agricultural by-product proteins and phenols (Díaz et al, 2010), the latter are likely to be responsible for the stimulating effect of Lac on T. versicolor, as has been demonstrated with the addition of phenolic extracts from corn steep liquor (Wang et al, 2014). However, the induction levels achieved by the addition of DOR have not so far been reported in relation to additional agro-industrial wastes.…”

“…DOR was obtained from the Sierra Sur olive oil company in Granada, Spain (2009-2010. It was sieved, autoclaved in three cycles and stored at 4 °C before use.…”

Enzymatic mechanisms and detoxification of dry olive-mill residue by Cyclocybe aegerita, Mycetinis alliaceus and Chondrostereum purpureum

“…The phytotoxicity of OMW has been attributed mostly to phenolic acids, while the decrease in phytotoxicity has been ascribed to a polymerization process that avoids passage of phenolics through the plant cell wall (Barakat et al, 2009). The bioremediation of OMW by WRF has also been observed in fungi such as Coriolopsis rigida, Phanerochaete flavido-alba, Trametes versicolor, Bjerkandera adusta, and Auricularia auricula-judae, among others (Linares et al, 2003;Ergül et al, 2009;Díaz et al, 2010;Reina et al, 2013).…”