Accurate, automatic annotation of peptidases with Hotpep-protease

Peptidases are essential for intracellular protein processing, signaling and homeostasis, physiological processes and for digestion of food. Moreover, peptidases are important biotechnological enzymes used in processes such as industrial food processing, leather manufacturing and the washing industry. Identification of peptidases is a crucial step in their characterization but peptidase annotation is not a trivial task due to their large sequence diversity.In the present study short, conserved sequence profiles were generated for all peptidase families with more than four members in the comprehensive Merops peptidase database. The sequence profiles were combined with the Homology to Peptide Pattern (Hotpep) method for automatic annotation of peptidases. This method is a stand-alone software that annotates protease sequences to Merops family and subgroup and is suitable for large-scale sequence analysis. Compared to the Mammalian Degradome Database Hotpep-protease had an accuracy of 92% and a sensitivity of 96% for annotation of the human degradome. Annotation by commonly used methods (Blast and conserved domains) had an accuracy of 69% and a sensitivity of 78%. For fungal genomes, there were large differences between annotation with Hotpep-protease, Blast- and Hidden Markov Model-based annotation and the Merops annotation, which confirms the difficulty of large-scale peptidase annotation. Manual annotation indicated that Hotpep-protease had a positive prediction rate of 0.90 compared to a positive prediction rate of 0.67 for Blast search. Hence, Hotpep-protease is highly accurate method for fast and accurate annotation of peptidases

Microbial decomposition of keratin in nature—a new hypothesis of industrial relevance

Discovery of keratin-degrading enzymes from fungi and bacteria has primarily focused on finding one protease with efficient keratinase activity. Recently, an investigation was conducted of all keratinases secreted from a fungus known to grow on keratinaceous materials, such as feather, horn, and hooves. The study demonstrated that a minimum of three keratinases is needed to break down keratin, an endo-acting, an exo-acting, and an oligopeptide-acting keratinase. Further, several studies have documented that disruption of sulfur bridges of the keratin structure acts synergistically with the keratinases to loosen the molecular structure, thus giving the enzymes access to their substrate, the protein structure. With such complexity, it is relevant to compare microbial keratin decomposition with the microbial decomposition of well-studied polymers such as cellulose and chitin. Interestingly, it was recently shown that the specialized enzymes, lytic polysaccharide monoxygenases (LPMOs), shown to be important for breaking the recalcitrance of cellulose and chitin, are also found in keratin-degrading fungi. A holistic view of the complex molecular self-assembling structure of keratin and knowledge about enzymatic and boosting factors needed for keratin breakdown have been used to formulate a hypothesis for mode of action of the LPMOs in keratin decomposition and for a model for degradation of keratin in nature. Testing such hypotheses and models still needs to be done. Even now, the hypothesis can serve as an inspiration for designing industrial processes for keratin decomposition for conversion of unexploited waste streams, chicken feather, and pig bristles into bioaccessible animal feed

New insights into the molecular mechanism of methanol-induced inactivation of Thermomyces lanuginosus lipase: A molecular dynamics simulation study

Methanol intolerance of lipase is a major limitation in lipase-catalyzed methanolysis reactions. In this study, to understand the molecular mechanism of methanol-induced inactivation of lipases, we performed molecular dynamics (MD) simulations of Thermomyces lanuginosus lipase (TLL) in water and methanol and compared the observed structural and dynamic properties. The solvent accessibility analysis showed that in methanol, polar residues tended to be buried away from the solvent while non-polar residues tended to be more solvent-exposed in comparison to those in water. Moreover, we observed that in methanol, the van der Waals packing of the core residues in two hydrophobic regions of TLL became weak. Additionally, the catalytically relevant hydrogen bond between Asp201 OD2 and His258 ND1 in the active site was broken when the enzyme was solvated in methanol. This may affect the stability of the tetrahedral intermediates in the catalytic cycle of TLL. Furthermore, compared to those in water, some enzyme surface residues displayed enhanced movement in methanol with higher Cα root-mean-square atomic positional fluctuation values. One of such methanol-affecting surface residues (Ile241) was chosen for mutation, and MD simulation of the I241E mutant in methanol was conducted. The structural analysis of the mutant showed that replacing a non-polar surface residue with an acidic one at position 241 contributed to the stabilization of enzyme structure in methanol. Ultimately, these results, while providing molecular-level insights into the destabilizing effect of methanol on TLL, highlight the importance of surface residue redesign to improve the stability of lipases in methanol environments

Assessment of the National Test Strategy on the Development of the COVID-19 Pandemic in Denmark

During the COVID-19 pandemic, Denmark has pursued a mass testing strategy culminating in the testing of 12.167 individuals per 100,000 inhabitants per day during the spring of 2021. The strategy included free access to COVID-19 testing, and since 2021, compulsory documentation for negative tests or vaccination has been required for access to workplace, educational institutions, restaurants, and many other places. Testing and subsequent isolation if testing was positive were voluntary. The present study provides an analysis of whether testing frequency in Denmark showed any correlation to hospitalizations throughout the relevant stages of the pandemic. Mass testing was found not to correlate significantly with the number of hospitalizations during the pandemic. Interestingly, during the highest level of testing in spring 2021 the fraction of positive tests increased slightly; thus, the Danish mass testing strategy, at its best, failed to reduce the prevalence of COVID-19. Furthermore, the relationship between positives in antigen testing and in rt-PCR testing indicated that many patients were not tested early in their infection when the risk of transmission was at the highest. In conclusion, the Danish mass testing strategy for COVID-19 does not appear to have a detectable correlation to the number of hospitalizations due to COVID-19

Genome and secretome analyses provide insights into keratin decomposition by novel proteases from the non-pathogenic fungus <i>Onygena corvina</i>

Poultry processing plants and slaughterhouses produce huge quantities of feathers and hair/bristle waste annually. These keratinaceous wastes are highly resistant to degradation. Onygena corvina, a non-pathogenic fungus, grows specifically on feathers, hooves, horn, and hair in nature. Hence, the proteases secreted by O. corvina are interesting in view of their potential relevance for industrial decomposition of keratinaceous wastes. We sequenced and assembled the genome of O. corvina and used a method called peptide pattern recognition to identify 73 different proteases. Comparative genome analysis of proteases in keratin-degrading and non-keratin-degrading fungi indicated that 18 putative secreted proteases from four protease families (M36, M35, M43, and S8) may be responsible for keratin decomposition. Twelve of the 18 predicted protease genes could be amplified from O. corvina grown on keratinaceous materials and were transformed into Pichia pastoris. One of the recombinant proteases belonging to the S8 family showed high keratin-degrading activity. Furthermore, 29 different proteases were identified by mass spectrometry in the culture broth of O. corvina grown on feathers and bristle. The culture broth was fractionated by ion exchange chromatography to isolate active fractions with five novel proteases belonging to three protease families (S8, M28, and M3). Enzyme blends composed of three of these five proteases, one from each family, showed high degree of degradation of keratin in vitro. A blend of novel proteases, such as those we discovered, could possibly find a use for degrading keratinaceous wastes and provide proteins, peptides, and amino acids as valuable ingredients for animal feed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-015-6805-9) contains supplementary material, which is available to authorized users

A New Functional Classification of Glucuronoyl Esterases by Peptide Pattern Recognition

Glucuronoyl esterases are a novel type of enzymes believed to catalyze the hydrolysis of ester linkages between lignin and glucuronoxylan in lignocellulosic biomass, linkages known as lignin carbohydrate complexes. These complexes contribute to the recalcitrance of lignocellulose. Glucuronoyl esterases are a part of the microbial machinery for lignocellulose degradation and coupling their role to the occurrence of lignin carbohydrate complexes in biomass is a desired research goal. Glucuronoyl esterases have been assigned to CAZymes family 15 of carbohydrate esterases, but only few examples of characterized enzymes exist and the exact activity is still uncertain. Here peptide pattern recognition is used as a bioinformatic tool to identify and group new CE15 proteins that are likely to have glucuronoyl esterase activity. 1024 CE15-like sequences were drawn from GenBank and grouped into 24 groups. Phylogenetic analysis of these groups made it possible to pinpoint groups of putative fungal and bacterial glucuronoyl esterases and their sequence variation. Moreover, a number of groups included previously undescribed CE15-like sequences that are distinct from the glucuronoyl esterases and may possibly have different esterase activity. Hence, the CE15 family is likely to comprise other enzyme functions than glucuronoyl esterase alone. Gene annotation in a variety of fungal and bacterial microorganisms showed that coprophilic fungi are rich and diverse sources of CE15 proteins. Combined with the lifestyle and habitat of coprophilic fungi, they are predicted to be excellent candidates for finding new glucuronoyl esterase genes