SummaryA foundational study assessed effects of biochemical pathway introduction into poplar to produce eugenol, chavicol, p‐anol, isoeugenol and their sequestered storage products, from potentially available substrates, coniferyl and p‐coumaryl alcohols. At the onset, it was unknown whether significant carbon flux to monolignols vs. other phenylpropanoid (acetate) pathway metabolites would be kinetically favoured. Various transgenic poplar lines generated eugenol and chavicol glucosides in ca. 0.45% (~0.35 and ~0.1%, respectively) of dry weight foliage tissue in field trials, as well as their corresponding aglycones in trace amounts. There were only traces of any of these metabolites in branch tissues, even after ~4‐year field trials. Levels of bioproduct accumulation in foliage plateaued, even at the lowest introduced gene expression levels, suggesting limited monolignol substrate availability. Nevertheless, this level still allows foliage collection for platform chemical production, with the remaining (stem) biomass available for wood, pulp/paper and bioenergy product purposes. Several transformed lines displayed unexpected precocious flowering after 4‐year field trial growth. This necessitated terminating (felling) these particular plants, as USDA APHIS prohibits the possibility of their interacting (cross‐pollination, etc.) with wild‐type (native plant) lines. In future, additional biotechnological approaches can be employed (e.g. gene editing) to produce sterile plant lines, to avoid such complications. While increased gene expression did not increase target bioproduct accumulation, the exciting possibility now exists of significantly increasing their amounts (e.g. 10‐ to 40‐fold plus) in foliage and stems via systematic deployment of numerous ‘omics’, systems biology, synthetic biology and metabolic flux modelling approaches.

Three laccase isoenzymes (Lac1, Lac2 and Lac3) have been purified to homogeneity from Pleurotus nebrodensis in our previous study. Lac2 was shown to be the dominant isoform, capable of oxidizing the majority of laccase substrates and manifesting good thermostability and pH stability. Hence, Lac2 was selected to decolourize structurally different dyes and the colour removal efficiencies of Lac2 and the crude extract of P. nebrodensis were compared. By monitoring the λmax of the reaction system during the course of biotransformation, clear hypsochromic shifts were observed for most of the dyes examined, illustrating that at least one peak disappeared as a result of laccase treatment. In general, Lac2 was more efficient within a short time (1 h) and the crude extract, in general, could achieve similar or even higher efficiency when the duration of treatment was extended to 24 h. Malachite green (MG) was chosen to study the detoxifying potential of Lac2, because of the relatively simple structure and high toxicity of the dye towards microorganisms. The toxicity of MG towards both bacteria (Bacillus subtilis, Bacillus licheniformis, Pseudomonas fluorescens and Escherichia coli) and fungi (Fusarium graminearum and Trichoderma harzianum) was dramatically decreased and the potential mechanism was estimated by GC–MS as to remove four methyl groups firstly and the two newly formed amine groups would be degraded or polymerized further. The present study facilitates an understanding of the application of P. nebrodensis laccases and furnishes evidence for the safety of their utilization in the treatment of wastewater emanating from textile industries.

The characterization of three laccase isoforms from Pleurotus nebrodensis is described. Isoenzymes Lac1, Lac2 and Lac3 were purified to homogeneity using ion exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose and a gel filtration step on Superdex 75. The molecular weights of the purified laccases were estimated to be 68, 64 and 51 kDa, respectively. The isoenzymes demonstrated the same optimum pH at 3.0 but slightly different temperature optima: 50-60 °C for Lac1 and Lac3 and 60 °C for Lac2. Lac2 was always more stable than the other two isoforms and exposure to 50 °C for 120 min caused 30% loss in activity. Lac2 was relatively less stable than the other two isoforms when exposed to the pH range of 3.0-8.0 for 24 h, but inactivation only occurred initially, with around 70% residual activity being maintained during the whole process. Oxidative ability towards aromatic compounds varied substantially among the isoforms and each of them displayed preference toward some substrates. Kinetic constants (Km, Kcat) were determined by using a 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay, with Lac3 showing the best affinity and Lac2 displaying the highest catalytic efficiency. Amino acid sequences from peptides derived from digestion of isoenzymes showed great consistency with laccases in the databases.

The characterization of a novel protease from Amanita virgineoides is described. The A. virgineoides protease was purified to homogeneity using Q-Sepharose, carboxymethyl-cellulose, diethylaminoethyl-cellulose, and a gel filtration step on Superdex 75. The molecular mass of the purified protease was estimated to be 16.6 kDa. The protease was purified 32.1-fold, and its specific activity was 301.4 U/mg. The optimum pH was 4.0, and the optimum temperature was 50 °C. Kinetic constants (K , V ) were determined under the optimum reaction conditions, with K and V , being 3.74 mg/mL and 9.98 μg mL Min , respectively. The activity of the protease was curtailed by Cu , Pb , Fe , Cd , and Hg ions but enhanced by Mg , Ca , and K ions at low concentrations. The protease activity was adversely affected by ethylene diamine tetraacetic acid, suggesting that it is a metalloprotease. Four peptide sequences were obtained from liquid chromatography-tandem mass spectrometry, including KQALSGIR, TIAMDGTEGLVR, VALTGLTVAEYFR, and AGAGSATLSMAYAGAR, which showed 86%, 64%, 60%, and 75% identity with peptides of Hypsizygus marmoreus, Dacryopinax sp. DJM-731 SS1, Trametes versicolor FP-101664 SS1, and Paxillus involutus ATCC 200175, respectively. The newly isolated protease showed good hydrolytic activity and biochemical characteristics, which expanded the knowledge of biologically active proteins and provided further insight on this poisonous fungus.