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The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants.
This article was published in the following journal.
Name: New biotechnology
Fusarium oxysporum has recently been identified as the cause of a wilt disease affecting blackberry in California and Mexico. Thirty-six isolates of F. oxysporum obtained from symptomatic blackberry p...
To discover novel feruloyl esterases (FAEs) by the function-driven screening procedure from soil metagenome.
Isolates of the Fusarium oxysporum species complex have been characterized as plant pathogens that commonly cause vascular wilt, stunting, and yellowing of the leaves in a variety of hosts. F. oxyspor...
Standardized protocols for determining pathogenicity of Fusarium oxysporum ff. spp. canariensis and palmarum, the cause of Fusarium wilt of ornamental palms, were developed using small palm plants wit...
The fungus Fusarium oxysporum f.sp. cubense (Focub) causes Fusarium wilt of banana. Focub strains are divided into races according to their host specificity, but which virulence factors underlie these...
The aim of this study was to describe the clinical-epidemiological characteristics of a series of cases of fungal keratitis associated with Fusarium spp., In Spain during the years 2012 to...
This study evaluates the impact of a high phenolic acid intake from wholegrain wheat bread on human vascular function and plasma phenolic acid concentrations in healthy adults. All partici...
Fusarium are microscopic filamentous fungi that live as saprobiontics in outside environments. They are found in the soil, the air, in water and on numerous plants. The frequency of fusari...
The purpose of this research study is to evaluate the safety, effect, and pharmacology of C1 Esterase Inhibitor (human) in kidney transplant patients with Antibody-Mediated Rejection (AMR)...
Hereditary angioedema (HAE) is a rare disorder characterized by congenital lack of functional C1 esterase inhibitor. If not treated adequately, the acute attacks of HAE can be life-threate...
The techniques used to produce molecules exhibiting properties that conform to the demands of the experimenter. These techniques combine methods of generating structural changes with methods of selection. They are also used to examine proposed mechanisms of evolution under in vitro selection conditions.
The interdisciplinary science that studies evolutionary biology, including the origin and evolution of the major elements required for life, their processing in the interstellar medium and in protostellar systems. This field also includes the study of chemical evolution and the subsequent interactions between evolving biota and planetary evolution as well as the field of biology that deals with the study of extraterrestrial life.
Enzymes that catalyze DNA template-directed extension of the 3'-end of an RNA strand one nucleotide at a time. They can initiate a chain de novo. In eukaryotes, three forms of the enzyme have been distinguished on the basis of sensitivity to alpha-amanitin, and the type of RNA synthesized. (From Enzyme Nomenclature, 1992).
Evolution at the molecular level of DNA sequences and proteins. (Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed)
Procedures by which protein structure and function are changed or created in vitro by altering existing or synthesizing new structural genes that direct the synthesis of proteins with sought-after properties. Such procedures may include the design of MOLECULAR MODELS of proteins using COMPUTER GRAPHICS or other molecular modeling techniques; site-specific mutagenesis (MUTAGENESIS, SITE-SPECIFIC) of existing genes; and DIRECTED MOLECULAR EVOLUTION techniques to create new genes.
Polymerase Chain Reaction (PCR)
PCR (Polymerase Chain Reaction) uses the ability of DNA polymerase (enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two ident...
Enzymes are proteins that catalyze (i.e., increase the rates of) chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical re...