Microbial Keratinases: Enzymes with Promising Biotechnological Applications

Keratin je netopljivi strukturni protein, koji izgrađuje čvrsta tkiva ljudi i životinja, poput perja, vune, kose, rogova i noktiju. Perje i vuna predstavljaju glavne izvore otpada bogatog proteinima koji se može preraditi u proizvode s dodanom vrijednošću, kao što su krmiva, gnojiva i biogoriva. Glavno ograničenje razgradnje keratinskih supstrata je njihova stabilna struktura, koja otežava hidrolizu s uobičajenim proteazama. Međutim, u prirodi se nalaze mikroorganizmi koji proizvode keratinolitičke enzime za razgradnju takvih supstrata. Keratinaze se upotrebljavaju u farmaceutskoj i tekstilnoj industriji te industriji kože. No, njihova je šira primjena npr. u preradi otpada iz industrije peradi ograničena, stoga se trebaju dodatno optimirati svojstva te uvjeti proizvodnje enzima. U ovom preglednom članku opisujemo molekularne značajke i svojstva keratinaza, njihovu klasifikaciju, tradicionalne i nove metode pronalaska novih enzima, proizvodnju, karakterizaciju, načine poboljšanja njihovih svojstava i biotehnološku primjenu.Keratin is a complex and structurally stable protein found in human and animal hard tissues, such as feathers, wool, hair, hoof and nails. Some of these, like feathers and wool, represent one of the main sources of protein-rich waste with significant potential to be transformed into value-added products such as feed, fertilizers or bioenergy. A major limitation impeding valorization of keratinous substrates is their recalcitrant structure and resistance to hydrolysis by common proteases. However, specialized keratinolytic enzymes produced by some microorganisms can efficiently degrade these substrates. Keratinases have already found a purpose in pharmaceutical, textile and leather industries. However, their wider implementation in other processes, such as cost-effective (pre)treatment of poultry waste, still requires optimization of production and performance of the available enzymes. Here we present a comprehensive review covering molecular properties and characteristics of keratinases, their classification, traditional and novel approaches in discovery of novel enzymes, production, characterization, improvement and biotechnological applications

Potential of Selected Rumen Bacteria for Cellulose and Hemicellulose Degradation

U probavnom se sustavu biljojeda nalaze vrlo učinkoviti mikroorganizmi što razgrađuju celulolozu i hemicelulozu iz sažvakanog biljnog materijala, te ih opskrbljuju hranjivim tvarima. Osim protozoa i gljivica, razgradnji otporne (hemi)celulozne biomase u buragu znatno pridonose i bakterije. U fokusu je ovog preglednog rada opis enzimskog sustava triju predstavnika bakterija iz buraga što proizvode celulazu, i to: Ruminococcus flavefaciens, Prevotella bryantii i Pseudobutyrivibrio xylanivorans. R. flavefaciens je poznat po proizvodnji jedne od najkompleksnijih enzimskih struktura, pa bi se mogao upotrijebiti za dizajn enzimskog kompleksa za razgradnju stanične stijenke biljaka. S druge strane, bakterije Prevotella bryantii i Pseudobutyrivibrio xylanivorans proizvode jednostavne, slobodne i vrlo aktivne ksilanaze. Bakterija P. xylanivorans ima i probiotička svojstva, pa se može upotrijebiti za proizvodnju bioplina i kao dodatak krmivu. Ispitivanje je genoma i proteoma bakterija što razgrađuju celulozu i hemicelulozu usmjereno na identifikaciju novih enzima, koji se nakon kloniranja i ekspresije u odgovarajućim domaćinima mogu upotrijebiti za izradu vrlo aktivnih rekombinantnih hidrolitičkih mikroorganizama, te primijeniti u različitim biotehnološkim procesima.Herbivorous animals harbour potent cellulolytic and hemicellulolytic microorganisms that supply the host with nutrients acquired from degradation of ingested plant material. In addition to protozoa and fungi, rumen bacteria contribute a considerable part in the breakdown of recalcitrant (hemi)cellulosic biomass. The present review is focused on the enzymatic systems of three representative fibrolytic rumen bacteria, namely Ruminococcus flavefaciens, Prevotella bryantii and Pseudobutyrivibrio xylanivorans. R. flavefaciens is known for one of the most elaborated cellulosome architectures and might represent a promising candidate for the construction of designer cellulosomes. On the other hand, Prevotella bryantii and Pseudobutyrivibrio xylanivorans produce multiple free, but highly efficient xylanases. In addition, P. xylanivorans was also shown to have some probiotic traits, which makes it a promising candidate not only for biogas production, but also as an animal feed supplement. Genomic and proteomic analyses of cellulolytic and hemicellulolytic bacterial species aim to identify novel enzymes, which can then be cloned and expressed in adequate hosts to construct highly active recombinant hydrolytic microorganisms applicable for different biotechnological tasks

Expression of Cellulosome Components and Type IV Pili within the Extracellular Proteome of Ruminococcus flavefaciens 007

Funding: The Rowett Institute receives funding from SG-RESAS (Scottish Government Rural and Environmental Science and Analysis Service). Visit of M.V. was supported by research grants from FEMS and Slovene human resources development and scholarship funds. Parts of this work were funded by grants from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel – BSF Energy Research grant to E.A.B. and B.A.W. and Regular BSF Research grants to R.L. and B.A.W. – and by the Israel Science Foundation (grant nos 966/09 and 159/07 291/08). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

How Can We Advance Integrative Biology Research in Animal Science in 21st Century?:Experience at University of Ljubljana from 2002 to 2022

In this perspective analysis, we strive to answer the following question: how can we advance integrative biology research in the 21st century with lessons from animal science? At the University of Ljubljana, Biotechnical Faculty, Department of Animal Science, we share here our three lessons learned in the two decades from 2002 to 2022 that we believe could inform integrative biology, systems science, and animal science scholarship in other countries and geographies. Cultivating multiomics knowledge through a conceptual lens of integrative biology is crucial for life sciences research that can stand the test of diverse biological, clinical, and ecological contexts. Moreover, in an era of the current COVID-19 pandemic, animal nutrition and animal science, and the study of their interactions with human health (and vice versa) through integrative biology approaches hold enormous prospects and significance for systems medicine and ecosystem health

Ruminococcus flavefaciens 007C cellulosomes and cellulase consortium

Ruminococcus flavefaciens is among the most important cellulolytic bacterial species in rumen and gastrointestinal tract of monogastric herbivorous animals. Its efficiency in degradation of (hemi)cellulosic substrates is associated with the production of remarkably intricate extracellular multienzyme complexes, named cellulosomes. In the present work we investigated the cellulolytic system of 007C. The bioinformatic analysis of the draft genome sequence revealed identical organization of sca gene cluster as has previously been found in four other strains of R. flavefaciens. The cluster consists of five genes in the following order: scaC-scaA-scaB-cttA-scaE. The cellulases of R. flavefaciens 007C belong to four families of glycoside hydrolases, namely GH48, GH44, GH9 in GH5. Majority of these enzymes are putative endoglucanases, belonging to families GH5 and GH9, whereas only one gene encoding GH44 and GH48 was found. Apart from catalytic domains, most of these proteins also contain dockerins – signature sequences, which indicate their attachement to cellulosomes. On the other hand, carbohydrate-binding modules were only found coupled to GH9 catalytic domains. Zymogram analysis showed that larger endoglucanases were mostly constitutively expressed, wheras smaller enzymes were only detected in later phases of Avicel-grown cultures

Microbial Keratinases: Enzymes with Promising Biotechnological Applications

Keratin is a complex and structurally stable protein found in human and animal hard tissues, such as feathers, wool, hair, hoof and nails. Some of these, like feathers and wool, represent one of the main sources of protein-rich waste with significant potential to be transformed into value-added products such as feed, fertilizers or bioenergy. A major limitation impeding valorization of keratinous substrates is their recalcitrant structure and resistance to hydrolysis by common proteases. However, specialized keratinolytic enzymes produced by some microorganisms can efficiently degrade these substrates. Keratinases have already found a purpose in pharmaceutical, textile and leather industries. However, their wider implementation in other processes, such as cost-effective (pre)treatment of poultry waste, still requires optimization of production and performance of the available enzymes. Here we present a comprehensive review covering molecular properties and characteristics of keratinases, their classification, traditional and novel approaches in discovery of novel enzymes, production, characterization, improvement and biotechnological applications

Using different growth medium greatly improves distinction of Butyrivibrio fibrisolvens and Pseudobutyrivibrio xylanivorans strains by the cellular fatty acids and aldehydes profiles

A total of 11 ruminal strains currently assigned to Butyrivibrio fibrisolvens and Pseudobutyrivibrio xylanivorans were cultivated in two different media, rumen fluid containing M2 and short-chain fatty acid (SCFA ) containing M330, and their cellular fatty acid methyl esters (FA ME) and dimethylacetals (DMA) were analyzed using gas chromatography. A comparison of the FA ME/DMA compositions revealed that the difference in SCFA contents in the growth medium induced a pronounced quantitative effect on the cellular branched-chainfatty acid and aldehydes proportions only in the P. xylanivorans strains. This study shows that FA ME/DMA analysis is a powerful chemotaxonomic tool in the group of phenotypically similar rumen butyrivibria especially when the influence of the growth medium is evaluated.Skupno smo gojili 11 vampnih sevov iz vrst Butyrivibrio fibrisolvens in Pseudobutyrivibrio xylanivorans v dveh različnih gojiščih: v M2 z vampnim sokom in v M330 z mešanico kratkoverižnih maščobnih kislin (SCFA ). S plinsko kromatografijo smo analizirali njihove metilne estre celičnih maščobnih kislin (FA ME) in dimetilacetale (DMA). Primerjava sestave FA ME/ DMA je razkrila, da razlika v vsebnosti SCFA v gojišču povzroči izrazit kvantitativen učinek na deleže celičnih razvejanih maščobnih kislin in aldehidov le pri sevih vrste P. xylanivorans. Naša raziskava dokazuje, da lahko FA ME/DMA analizo učinkovito uporabljamo v kemotaksonomiji fenotipsko podobnih butirivibrijev z ustreznim ovrednotenjem vpliva gojišča

Probiotics in animal nutrition

Probiotiki so živi mikroorganizmi, ki zaužiti v ustreznem številu, ugodno vplivajo na zdravje gostitelja. Njihovi učinki so praviloma povezani z vzpostavitvijo ugodnega mikrobnega ravnovesja v prebavilih gostitelja ter uravnavanjem njegovega imunskega odziva. Pri domačih živalih so ključni učinki probiotikov povezani z izboljšano učinkovitostjo prireje. Poleg ugodnega vpliva na zdravstveno stanje (predvsem mladih) živali, slednje obsega tudi izboljšano konverzijo krme, povečano hitrost rasti in nekatere druge. Probiotični krmni dodatki registrirani v EU vsebujejo predvsem Gram-pozitivne bakterije iz rodov Bacillus, Enterococcus, Lactobacillus, Pediococcus, Streptococcus ter kvasovke Saccharomyces cerevisiae in Kluyveromyces sp. Medtem, ko je večina omenjenih mikroorganiymov načeloma varnih, imajo nekateri lastnosti, ki so lahko škodljive tako za živali, kot tudi za ljudi. Tak primer so enterokoki, pri katerih pogosto opažajo zapise za prenosljive determinante odpornosti proti antibiotikom. Slednji tako predstavljajo potencialno nevarnost za širjenje odpornosti v patogenih mikrobnih populacijah ljudi in živali. Hiter napredek na področju sintetične in sistemske biologije združen s podporo bioinformatike in novimi orodji genskega inženirstva v prihodnosti obeta skoraj neskončne možnosti za pripravo probiotičnih sevov s poljubnimi lastnostmi, vendar pa bodo le-ti lahko prestopili meje laboratorijev šele ob ustrezni spremembi zakonodaje in javnega mnenja.Probiotics are defined as living micro-organisms, that upon ingestion in certain numbers, exert health benefits to the host. Their use is linked to proven efficacy on the gastrointestinal microbial equilibrium as well as immunomodulation. The positive effect in animals exerts not only in an improved health status, especially in young animals, but also in improved animal performance, including growth rate and feed conversion efficiency. Microorganisms that are used in animal feeds in the EU are mainly Gram-positive bacteria belonging to genera Bacillus, Enterococcus, Lactobacillus, Pediococcus and Streptococcus and yeasts, such as Saccharomyces cerevisiae or Kluyveromyces species. While most of the species are apparently safe, certain microorganisms may exert harmful properties for animals as well as humans. Enterococci, for example, might harbour transmissible antibiotic resistance determinants, which have the potential to spread in animal and human-associated pathogenic microbial populations. Recent developments in synthetic and systems biology, coupled with bioinformatics and novel tools for genetic engineering, will soon enable the construction of \u27artificial\u27 probiotic microorganisms with virtually any combination of properties. Whether and when these \u27designer probiotics\u27 will reach out of the labs depends on legislation as well as public opinion