A muramidase from Acremonium alcalophilum hydrolyse peptidoglycan found in the gastrointestinal tract of broiler chickens.

This study evaluates peptidoglycan hydrolysis by a microbial muramidase from the fungus Acremonium alcalophilum in vitro and in the gastrointestinal tract of broiler chickens. Peptidoglycan used for in vitro studies was derived from 5 gram-positive chicken gut isolate type strains. In vitro peptidoglycan hydrolysis was studied by three approaches: (a) helium ion microscopy to identify visual phenotypes of hydrolysis, (b) reducing end assay to quantify solubilization of peptidoglycan fragments, and (c) mass spectroscopy to estimate relative abundances of soluble substrates and reaction products. Visual effects of peptidoglycan hydrolysis could be observed by helium ion microscopy and the increase in abundance of soluble peptidoglycan due to hydrolysis was quantified by a reducing end assay. Mass spectroscopy confirmed the release of hydrolysis products and identified muropeptides from the five different peptidoglycan sources. Peptidoglycan hydrolysis in chicken crop, jejunum, and caecum samples was measured by quantifying the total and soluble muramic acid content. A significant increase in the proportion of the soluble muramic acid was observed in all three segments upon inclusion of the microbial muramidase in the diet.

Anomeric Selectivity and Product Profile of a Processive Cellulase.

Cellobiohydrolases (CBHs) make up an important group of enzymes for both natural carbon cycling and industrial deconstruction of lignocellulosic biomass. The consecutive hydrolysis of one cellulose strand relies on an intricate pattern of enzyme-substrate interactions in the long, tunnel-shaped binding site of the CBH. In this work, we have investigated the initial complexation mode with cellulose of the most thoroughly studied CBH, Cel7A from Hypocrea jecorina (HjCel7A). We found that HjCel7A predominantly produces glucose when it initiates a processive run on insoluble microcrystalline cellulose, confirming the validity of an even and odd product ratio as an estimate of processivity. Moreover, the glucose released from cellulose was predominantly α-glucose. A link between the initial binding mode of the enzyme and the reducing end configuration was investigated by inhibition studies with the two anomers of cellobiose. A clear preference for ß-cellobiose in product binding site +2 was observed for HjCel7A, but not the homologous endoglucanase, HjCe7B. Possible relationships between this anomeric preference in the product site and the prevalence of odd-numbered initial-cut products are discussed, and a correlation between processivity and anomer selectivity is proposed.

Biofuels. Altered sterol composition renders yeast thermotolerant.

Ethanol production for use as a biofuel is mainly achieved through simultaneous saccharification and fermentation by yeast. Operating at ≥40°C would be beneficial in terms of increasing efficiency of the process and reducing costs, but yeast does not grow efficiently at those temperatures. We used adaptive laboratory evolution to select yeast strains with improved growth and ethanol production at ≥40°C. Sequencing of the whole genome, genome-wide gene expression, and metabolic-flux analyses revealed a change in sterol composition, from ergosterol to fecosterol, caused by mutations in the C-5 sterol desaturase gene, and increased expression of genes involved in sterol biosynthesis. Additionally, large chromosome III rearrangements and mutations in genes associated with DNA damage and respiration were found, but contributed less to the thermotolerant phenotype.

Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus.

Animals and higher plants express endogenous peptide antibiotics called defensins. These small cysteine-rich peptides are active against bacteria, fungi and viruses. Here we describe plectasin-the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin has primary, secondary and tertiary structures that closely resemble those of defensins found in spiders, scorpions, dragonflies and mussels. Recombinant plectasin was produced at a very high, and commercially viable, yield and purity. In vitro, the recombinant peptide was especially active against Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin showed extremely low toxicity in mice, and cured them of experimental peritonitis and pneumonia caused by S. pneumoniae as efficaciously as vancomycin and penicillin. These findings identify fungi as a novel source of antimicrobial defensins, and show the therapeutic potential of plectasin. They also suggest that the defensins of insects, molluscs and fungi arose from a common ancestral gene.