CRISPR/Cas9-mediated genomic insertion of functional genes into WCFS1.

Unlabelled: a natural inhabitant of the human body, is a promising candidate vehicle for vaccine delivery. An obstacle in developing bacterial delivery vehicles is generating a production strain that lacks antibiotic resistance genes and contains minimal foreign DNA. To deal with this obstacle, we have constructed a finetuned, inducible two-plasmid CRISPR/Cas9-system for chromosomal gene insertion in .

Identification and Characterization of a New Thermophilic κ-Carrageenan Sulfatase.

Carrageenans are sulfated polysaccharides found in the cell wall of certain red seaweeds. They are widely used in the food industry for their gelling and stabilizing properties. In nature, carrageenans undergo enzymatic modification and degradation by marine organisms.

pH-mediated manipulation of the histidine brace in LPMOs and generation of a tri-anionic variant, investigated by EPR, ENDOR, ESEEM and HYSCORE spectroscopy.

Lytic Polysaccharide Monooxygenases (LPMOs) catalyze the oxidative depolymerization of polysaccharides at a monocopper active site, that is coordinated by the so-called histidine brace. In the past, this motif has sparked considerable interest, mostly due to its ability to generate and stabilize highly oxidizing intermediates during catalysis. We used a variety of advanced EPR techniques, including Electron Nuclear Double Resonance (ENDOR), Electron Spin Echo Envelope Modulation (ESEEM) and Hyperfine Sublevel Correlation (HYSCORE) spectroscopy in combination with isotopic labelling (N, H) to characterize the active site of the bacterial LPMO AA10A over a wide pH range (pH 4.

Fracture morphology and ossification process of the keel bone in modern laying hens based on radiographic imaging.

Keel bone fractures (KBF) are one of the most important welfare problems in commercial laying hens. Despite extensive research on the matter, its etiology remains unclear. Studying fracture characteristics in radiographic images can aid in the understanding of the disorder.

Revisiting the activity of two poly(vinyl chloride)- and polyethylene-degrading enzymes.

Biocatalytic degradation of non-hydrolyzable plastics is a rapidly growing field of research, driven by the global accumulation of waste. Enzymes capable of cleaving the carbon-carbon bonds in synthetic polymers are highly sought-after as they may provide tools for environmentally friendly plastic recycling. Despite some reports of oxidative enzymes acting on non-hydrolyzable plastics, including polyethylene or poly(vinyl chloride), the notion that these materials are susceptible to efficient enzymatic degradation remains controversial, partly driven by a general lack of studies independently reproducing previous observations.

Hygiene performance rating at farm level - an auditing scheme for evaluation of biosecurity measures' effect on prevalence of Campylobacter from selected broiler producers.

Background: Preventing pathogens from entering the broiler premises is the main biosecurity measure at farm level. In conventional broiler production, chickens are kept indoors during the entire production period. Pathogens can enter the broiler-producing unit from sources such as water, equipment, personnel, insects, and rodents.

Oxidized reducing ends in celluloses: Quantitative profiling relative to molar mass distribution by fluorescence labeling.

Fluorescence labeling with N-(1-naphthyl)ethylenediamine is highly effective for quantifying oxidized reducing end groups (REGs) in cellulosic materials. When combined with size exclusion chromatography in DMAc/LiCl, along with fluorescence / multiple-angle laser light scattering / refractive index detection, a detailed profile of C1-oxidized REGs relative to the molecular weight distribution of the cellulosic material can be obtained. In this work, the derivatization process was extensively optimized, to be carried out heterogeneously in the solvent N-methyl-2-pyrrolidone.

Beyond the Surface: A Methodological Exploration of Enzyme Impact along the Cellulose Fiber Cross-Section.

Despite the wide range of analytical tools available for the characterization of cellulose, the in-depth characterization of inhomogeneous, layered cellulose fiber structures remains a challenge. When treating fibers or spinning man-made fibers, the question always arises as to whether the changes in the fiber structure affect only the surface or the entire fiber. Here, we developed an analysis tool based on the sequential limited dissolution of cellulose fiber layers.

Quantitative at-line monitoring of enzymatic hydrolysis using benchtop diffusion nuclear magnetic resonance spectroscopy.

Benchtop diffusion nuclear magnetic resonance (NMR) spectroscopy was used to perform quantitative monitoring of enzymatic hydrolysis. The study aimed to test the feasibility of the technology to characterize enzymatic hydrolysis processes in real time. Diffusion ordered spectroscopy (DOSY) was used to measure the signal intensity and apparent self-diffusion constant of solubilized protein in hydrolysate.

A novel approach to analyze the impact of lytic polysaccharide monooxygenases (LPMOs) on cellulosic fibres.

Enzymatic treatment of cellulosic fibres is a green alternative to classical chemical modification. For many applications, mild procedures for cellulose alteration are sufficient, in which the fibre structure and, therefore, the mechanical performance of cellulosic fibres are preserved. Lytic polysaccharide monooxygenases (LPMOs) bear a great potential to become a green reagent for such targeted cellulose modifications.

Antigen surface display in two novel whole genome sequenced food grade strains, Lactiplantibacillus pentosus KW1 and KW2.

Background: Utilization of commensal bacteria for delivery of medicinal proteins, such as vaccine antigens, is an emerging strategy. Here, we describe two novel food-grade strains of lactic acid bacteria, Lactiplantibacillus pentosus KW1 and KW2, as well as newly developed tools for using this relatively unexplored but promising bacterial species for production and surface-display of heterologous proteins.

Results: Whole genome sequencing was performed to investigate genomic features of both strains and to identify native proteins enabling surface display of heterologous proteins.

The "life-span" of lytic polysaccharide monooxygenases (LPMOs) correlates to the number of turnovers in the reductant peroxidase reaction.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that degrade the insoluble crystalline polysaccharides cellulose and chitin. Besides the HO cosubstrate, the cleavage of glycosidic bonds by LPMOs depends on the presence of a reductant needed to bring the enzyme into its reduced, catalytically active Cu(I) state. Reduced LPMOs that are not bound to substrate catalyze reductant peroxidase reactions, which may lead to oxidative damage and irreversible inactivation of the enzyme.

Climate, host and geography shape insect and fungal communities of trees.

Non-native pests, climate change, and their interactions are likely to alter relationships between trees and tree-associated organisms with consequences for forest health. To understand and predict such changes, factors structuring tree-associated communities need to be determined. Here, we analysed the data consisting of records of insects and fungi collected from dormant twigs from 155 tree species at 51 botanical gardens or arboreta in 32 countries.

FTIR-based prediction of collagen content in hydrolyzed protein samples.

Fourier transform infrared spectroscopy (FTIR) is a powerful analytical tool that has been used for protein and peptide characterization for decades. In the present study, the objective was to investigate if FTIR can be used to predict collagen content in hydrolyzed protein samples. All samples were obtained from enzymatic protein hydrolysis (EPH) of poultry by-products providing a span in collagen content from 0.

Causes of carcass condemnation in Norwegian aviary housed layers.

Background: Meat inspection data is commonly used to monitor health and welfare in commercial broiler production; however, less used in layers. Slaughterhouse records can provide insight into animal and herd health and identify important health and welfare challenges. To gain knowledge of health issues in commercial aviary housed laying hens, the aim of this repeated cross-sectional study was to describe the occurrence and causes of carcass condemnation, including dead-on-arrivals (DOA), in commercial aviary housed layers in Norway, and to explore seasonal patterns and correlation between DOA and number of carcass condemnations.

Investigation of the functions of -3 very-long-chain PUFAs in skin using Atlantic salmon and human and fish skin models.

The purpose of this study was to investigate the effect of dietary -3 very-long-chain PUFA (-3 VLC-PUFA) on the maturation and development of skin tissue in juvenile Atlantic salmon r) , as well as their effects on skin keratocyte and human skin fibroblast cell migration Atlantic salmon were fed different dietary levels of -3 VLC-PUFA from an initial weight of 6 g to a final weight of 11 g. Changes in skin morphology were analysed at two time points during the experiment, and the effects on skin tissue fatty acid composition were determined. Additionally, experiments using human dermal fibroblasts and primary Atlantic salmon keratocytes were conducted to investigate the effect of VLC-PUFA on the migration capacity of the cells.

Reductants fuel lytic polysaccharide monooxygenase activity in a pH-dependent manner.

Polysaccharide-degrading mono-copper lytic polysaccharide monooxygenases (LPMOs) are efficient peroxygenases that require electron donors (reductants) to remain in the active Cu(I) form and to generate the H O co-substrate from molecular oxygen. Here, we show how commonly used reductants affect LPMO catalysis in a pH-dependent manner. Between pH 6.

Engineering cellulases for conversion of lignocellulosic biomass.

Lignocellulosic biomass is a renewable source of energy, chemicals and materials. Many applications of this resource require the depolymerization of one or more of its polymeric constituents. Efficient enzymatic depolymerization of cellulose to glucose by cellulases and accessory enzymes such as lytic polysaccharide monooxygenases is a prerequisite for economically viable exploitation of this biomass.

Looking at LPMO reactions through the lens of the HRP/Amplex Red assay.

Lytic polysaccharide monooxygenases (LPMOs) are unique redox enzymes capable of disrupting the crystalline surfaces of industry-relevant recalcitrant polysaccharides, such as chitin and cellulose. Historically, LPMOs were thought to be slow enzymes relying on O as the co-substrate, but it is now clear that these enzymes prefer HO, allowing for fast depolymerization of polysaccharides through a peroxygenase reaction. Thus, quantifying HO in LPMO reaction set-ups is of a great interest.

Enzymatic debranching is a key determinant of the xylan-degrading activity of family AA9 lytic polysaccharide monooxygenases.

Background: Previous studies have revealed that some Auxiliary Activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) oxidize and degrade certain types of xylans when incubated with mixtures of xylan and cellulose. Here, we demonstrate that the xylanolytic activities of two xylan-active LPMOs, TtLPMO9E and TtLPMO9G from Thermothielavioides terrestris, strongly depend on the presence of xylan substitutions.

Results: Using mixtures of phosphoric acid-swollen cellulose (PASC) and wheat arabinoxylan (WAX), we show that removal of arabinosyl substitutions with a GH62 arabinofuranosidase resulted in better adsorption of xylan to cellulose, and enabled LPMO-catalyzed cleavage of this xylan.

Flock Factors Correlated with Elevated Mortality in Non-Beak Trimmed Aviary-Housed Layers.

The use of non-cage housing systems for layers is increasing in Europe and elsewhere. Knowledge of factors that may affect mortality in these systems is important to be able to improve animal welfare, reduce mortality and enhance sustainability. The aim of this study was to investigate factors that may contribute to increased mortality in non-beak trimmed aviary-housed laying hens in Norway.

On the impact of carbohydrate-binding modules (CBMs) in lytic polysaccharide monooxygenases (LPMOs).

Lytic polysaccharide monooxygenases (LPMOs) have revolutionized our understanding of how enzymes degrade insoluble polysaccharides. Compared with the substantial knowledge developed on the structure and mode of action of the catalytic LPMO domains, the (multi)modularity of LPMOs has received less attention. The presence of other domains, in particular carbohydrate-binding modules (CBMs), tethered to LPMOs has profound implications for the catalytic performance of the full-length enzymes.

End of lay postmortem findings in aviary housed laying hens.

Good health and low mortality are constitutive elements of good animal welfare. In laying hens, mortality and pathological findings are usually reported as cumulative proportions from onset of lay to culling. However, knowledge of pathological lesions and causes of death specifically toward the end of the production period are scarce.