Journal: Scientific Reports

Article Title: Characterization of a GH5 endoxylanase from Penicillium funiculosum and its synergism with GH16 endo-1,3(4)-glucanase in saccharification of sugarcane bagasse

doi: 10.1038/s41598-022-21529-1

Figure Lengend Snippet: List of plasmids and strains used in the study.

Article Snippet: p PICZαA , A zeocin-resistant vector for protein expression in P. pastoris under the control of AOX1 promoter , Genscript.

Techniques: Over Expression, Plasmid Preparation, Expressing, Clone Assay

Journal: Chem & Bio Engineering

Article Title: Molecular Insights into the Activation of a Fungal Copper Radical Oxidase by Peroxidases

doi: 10.1021/cbe.5c00060

Figure Lengend Snippet: Phylogenetic analysis of the peroxidase-catalase superfamily. Position of the tandem peroxidase within the peroxidase–catalase superfamily, which includes class I (intracellular peroxidases), class II (fungal secreted peroxidases, including WT- Cor PerOx), and class III (plant secreted peroxidases, including WT-HRP). Peroxidases and related proteins from Ascomycetes and Basidiomycetes are colored in orange and blue, respectively. Hybrid B peroxidases represent a distinct group of fungal peroxidases, evolutionarily related to, but distinct from class II peroxidases. The average sequence identity is indicated within the tandem peroxidase clade (circled in green) and between this clade and the others (circled in black). The figure was adapted from Bissaro et al., (2022).

Article Snippet: The intron-free sequences of the genes from C. orbiculare MAFF 240422 coding for the alcohol oxidase ( Cor AlcOx, GenBank ID TDZ17043.1 , UniProt ID N4UTF2), the fungal tandem peroxidase (WT- Cor PerOx, GenBank ID TDZ17044.1 ; UniProt ID N4UUY4 ) and HRP C2 (WT-HRP, GenBank ID BAA14143.1 , D90115.1 ; UniProt ID P17179.1 ) were synthesized after codon optimization for heterologous expression in P. pastoris and inserted into a modified p PICZαC vector using Xho I and Not I restriction sites in frame with the α-factor secretion signal peptide sequence at the N-terminus (i.e., without native signal peptide) and with a (His) 6-tag at the C-terminus (i.e., without c-myc epitope) (Genewiz, Leipzig, Germany).

Techniques: Sequencing

Journal: Chem & Bio Engineering

Article Title: Molecular Insights into the Activation of a Fungal Copper Radical Oxidase by Peroxidases

doi: 10.1021/cbe.5c00060

Figure Lengend Snippet: Capacity of wild-type and variant peroxidases to activate Cor AlcOx . The specific activity of Cor AlcOx at 10 nM is reported using different concentrations of WT- Cor PerOx, M3- Cor PerOx, WT-HRP, and Δ HLH HRP. The activation assays were performed as follows: the assay contained 50 mM sodium phosphate buffer pH 7.0, peroxidase (1–1000 nM) and 1.5 mM benzyl alcohol. The appearance of the product was monitored at 254 nm over 10 min at 23 °C. Units (U) correspond to μM benzaldehyde/min.

Article Snippet: The intron-free sequences of the genes from C. orbiculare MAFF 240422 coding for the alcohol oxidase ( Cor AlcOx, GenBank ID TDZ17043.1 , UniProt ID N4UTF2), the fungal tandem peroxidase (WT- Cor PerOx, GenBank ID TDZ17044.1 ; UniProt ID N4UUY4 ) and HRP C2 (WT-HRP, GenBank ID BAA14143.1 , D90115.1 ; UniProt ID P17179.1 ) were synthesized after codon optimization for heterologous expression in P. pastoris and inserted into a modified p PICZαC vector using Xho I and Not I restriction sites in frame with the α-factor secretion signal peptide sequence at the N-terminus (i.e., without native signal peptide) and with a (His) 6-tag at the C-terminus (i.e., without c-myc epitope) (Genewiz, Leipzig, Germany).

Techniques: Variant Assay, Activity Assay, Activation Assay

Journal: Chem & Bio Engineering

Article Title: Molecular Insights into the Activation of a Fungal Copper Radical Oxidase by Peroxidases

doi: 10.1021/cbe.5c00060

Figure Lengend Snippet: Biochemical characterization of produced peroxidases. (A) The peroxidase activities of WT- Cor PerOx, M3- Cor PerOx, WT-HRP, and Δ HLH HRP were measured at 414 nm and performed using 100 μM H 2 O 2 and 500 μM ABTS at 23 °C for 10 min. (B) The pH dependence of peroxidase activity was determined over a pH range from 2 to 8 using citrate-phosphate buffer (pH 4–8) and tartrate buffer (pH 2–5). (C) The thermal stability of WT- Cor PerOx, M3- Cor PerOx, WT-HRP, and Δ HLH HRP was evaluated measuring the residual activity after 30 min of incubation in a temperature range of 4–80 °C.

Article Snippet: The intron-free sequences of the genes from C. orbiculare MAFF 240422 coding for the alcohol oxidase ( Cor AlcOx, GenBank ID TDZ17043.1 , UniProt ID N4UTF2), the fungal tandem peroxidase (WT- Cor PerOx, GenBank ID TDZ17044.1 ; UniProt ID N4UUY4 ) and HRP C2 (WT-HRP, GenBank ID BAA14143.1 , D90115.1 ; UniProt ID P17179.1 ) were synthesized after codon optimization for heterologous expression in P. pastoris and inserted into a modified p PICZαC vector using Xho I and Not I restriction sites in frame with the α-factor secretion signal peptide sequence at the N-terminus (i.e., without native signal peptide) and with a (His) 6-tag at the C-terminus (i.e., without c-myc epitope) (Genewiz, Leipzig, Germany).

Techniques: Produced, Activity Assay, Incubation