ABSTRACT
Trichoderma genus fungi present great potential for the production of carbohydrate-active enzymes (CAZYmes), including glycoside hydrolase (GH) family members. From a renewability perspective, CAZYmes can be biotechnologically exploited to convert plant biomass into free sugars for the production of advanced biofuels and other high-value chemicals. GH54 is an attractive enzyme family for biotechnological applications because many GH54 enzymes are bifunctional. Thus, GH54 enzymes are interesting targets in the search for new enzymes for use in industrial processes such as plant biomass conversion. Herein, a novel metal-dependent GH54 arabinofuranosidase (ThABF) from the cellulolytic fungus Trichoderma harzianum was identified and biochemically characterized. Initial in silico searches were performed to identify the GH54 sequence. Next, the gene was cloned and heterologously overexpressed in Escherichia coli. The recombinant protein was purified, and the enzyme's biochemical and biophysical properties were assessed. GH54 members show wide functional diversity and specifically remove plant cell substitutions including arabinose and galactose in the presence of a metallic cofactor. Plant cell wall substitution has a major impact on lignocellulosic substrate conversion into high-value chemicals. These results expand the known functional diversity of the GH54 family, showing the potential of a novel arabinofuranosidase for plant biomass degradation.
Subject(s)
Cations, Divalent/chemistry , Fungal Proteins/isolation & purification , Glycoside Hydrolases/isolation & purification , Hypocreales/enzymology , Multigene Family , Amino Acid Sequence , Base Sequence , Biodegradation, Environmental , Computer Simulation , Consensus Sequence , Data Mining , Fungal Proteins/classification , Fungal Proteins/genetics , Fungal Proteins/metabolism , Glycoside Hydrolases/classification , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Hydrogen-Ion Concentration , Hypocreales/genetics , Models, Molecular , Phylogeny , Polysaccharides/metabolism , Protein Conformation , Protein Folding , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Sugars/metabolism , TemperatureABSTRACT
INTRODUCTION: Plant products are sources for drug development against multidrug resistant bacteria. METHODS: The antimicrobial activity of Origanum vulgare L. essential oil (OVeo) against carbapenem-resistant strains was assessed by disk-diffusion, microdilution (REMA-Resazurin Microtiter Assay), and time kill assays. RESULTS: Carbapenemase production was confirmed for all strains. OVeo exhibited a minimum inhibitory concentration of 0.059% v/v for Klebsiella pneumoniae and Serratia marcescens, and of 0.015 % v/v for Acinetobacter baumannii. A decrease in cell count was observed after a 4 h treatment. CONCLUSIONS: OVeo antimicrobial effect was rapid and consistent, making it a candidate for developing alternative therapeutic options against carbapenem-resistant strains.
Subject(s)
Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/pharmacology , Gram-Negative Bacteria/drug effects , Klebsiella pneumoniae/drug effects , Oils, Volatile/pharmacology , Origanum/chemistry , Serratia marcescens/drug effects , Acinetobacter baumannii/growth & development , Anti-Bacterial Agents/classification , Bacterial Proteins , Carbapenems/pharmacology , Drug Resistance, Multiple, Bacterial , Gram-Negative Bacteria/growth & development , Humans , Klebsiella pneumoniae/growth & development , Microbial Sensitivity Tests , Serratia marcescens/growth & development , beta-LactamasesABSTRACT
Abstract INTRODUCTION: Plant products are sources for drug development against multidrug resistant bacteria. METHODS The antimicrobial activity of Origanum vulgare L. essential oil (OVeo) against carbapenem-resistant strains was assessed by disk-diffusion, microdilution (REMA-Resazurin Microtiter Assay), and time kill assays. RESULTS Carbapenemase production was confirmed for all strains. OVeo exhibited a minimum inhibitory concentration of 0.059% v/v for Klebsiella pneumoniae and Serratia marcescens, and of 0.015 % v/v for Acinetobacter baumannii. A decrease in cell count was observed after a 4 h treatment. CONCLUSIONS OVeo antimicrobial effect was rapid and consistent, making it a candidate for developing alternative therapeutic options against carbapenem-resistant strains.
