Prethodna obrada biljnih stanica enzimima radi ekstrakcije ulja

Oil from oilseeds can be extracted by mechanical extraction (pressing), aqueous extraction, or by extraction with organic solvents. Although solvent extraction is the most efficient method, organic solvents are a potential hazard to the life and health for workers as well as to the environment, when solvent vapours are released and act as air pollutant with a high ozone-forming potential. Pressing is safer, environmentally friendly, and it preserves valuable natural components in the resulting oils. The problems associated with pressing are the high energy consumption and the lower yield of oil extraction, because the applied mechanical force does not completely destroy the structural cell components storing the oil. In seed cells, the oil is contained in the form of lipid bodies (oleosomes) that are surrounded by a phospholipid monolayer with a protein layer on the surface. These lipid bodies are further protected by the seed cell walls consisting mainly of polysaccharides such as pectins, hemicelluloses and cellulose, but also of glycoproteins. The use of hydrolases to degrade these barriers is a promising pretreatment strategy to support mechanical extraction and improve the oil yield. It is advisable to use a combination of enzymes with different activities when considering the multicompartment and multicomponent structure of oilseed cells. This article gives an overview of the microstructure and composition of oilseed cells, reviews enzymes capable of destroying oil containing cell compartments, and summarizes the main parameters of enzymatic treatment procedures, such as the composition of the enzyme cocktail, the amount of enzyme and water used, temperature, pH, and the duration of the treatment. Finally, it analyzes the efficiency of proteolytic, cellulolytic and pectolytic enzyme pretreatment to increase the yield of mechanically extracted oil from various types of vegetable raw materials with the main focus on oilseeds.Ulje se iz uljarica može dobiti mehaničkom ekstrakcijom (prešanjem), ekstrakcijom u vodenom mediju ili ekstrakcijom pomoću organskih otapala. Iako je ekstrakcija pomoću otapala najučinkovitija metoda, organska otapala mogu štetno utjecati na zdravlje radnika, kao i na okoliš, zbog isparavanja koja zagađuju okoliš i potiču stvaranje ozona. Prešanje je sigurnija i ekološki prihvatljiva metoda, kojom se vrijedni prirodni sastojci uljarica zadržavaju u dobivenom ulju. Nedostaci ove metode su velika potrošnja energije i manji prinos ulja, jer primijenjena mehanička sila ne uništava u potpunosti strukturne komponente stanica u kojima je pohranjeno ulje. U sjemenim stanicama ulje je prisutno u obliku lipidnih kapljica okruženih fosfolipidnim slojem te površinskim proteinskim slojem. Ta su lipidna tijela dodatno zaštićena staničnom stijenkom, koju uglavnom čine polisaharidi poput pektina, hemiceloloza i celuloza, ali i glikoproteini. Primjena hidrolaza za razgradnju takvih zaštitnih slojeva učinkovita je metoda prethodne obrade sjemena uljarica radi poboljšanja mehaničke ekstrakcije i povećanja prinosa ulja. Imajući na umu složenost strukture stanica uljarica preporučljivo je koristiti više enzima različitih načina djelovanja. U ovom je revijalnom prikazu dan opis mikrostrukture i sastava stanica uljarica, zatim pregled enzima koji mogu razgraditi stanične komponente koje sadržavaju ulje, te su sažete glavne značajke enzimskih obrada, kao što su sastav mješavine enzima, količina enzima i vode, temperatura, pH-vrijednost i trajanje postupka. Naposljetku je ispitana učinkovitost prethodne obrade sjemenki uljane repice pomoću proteolitičkih i pektolitičkih enzima, te enzima što razgrađuju celulozu, na povećanje prinosa mehanički ekstrahiranog ulja iz različitih sirovina, s primarnim fokusom na sjeme uljarica

Electron Transfer of Cellobiose Dehydrogenase in Polyethyleneimine Films

Abstract Cellobiose dehydrogenase (CDH) is applied as a bioelectrocatalyst in biosensors because its mobile cytochrome domain is capable of direct electron transfer. This study investigates the electron transfer mechanism of CDH molecules embedded in the polycation polyethyleneimine (PEI), which has been reported as a current‐boosting component of CDH‐based biosensors. By immobilizing different concentrations of CDH and its isolated cytochrome domain in PEI films, we found that increasing concentrations of cytochrome enhanced the film conductivity (up to 251±8 mS cm−1) through improved electron transfer between the protein redox centers. The increased electrical conductivity of the film contacts CDH molecules at a greater distance from the electrode. The cross‐linker poly(ethylene glycol) diglycidyl ether improves the packing and contacting of the cytochrome domains, whereas glutaraldehyde reduces the current obtained. Deglycosylation of CDH enhances the conductivity of enzyme‐polymer films by up to 34 %, implying a higher number of productive electron‐hopping events between cytochrome domains due to enhanced mobility or reduced shielding. By balancing negative charges on the CDH surface at neutral and alkaline pH, PEI increases the interdomain electron transfer and the electrical film conductivity. The resulting increased current output is relevant for in vivo bioanalytical applications

Amino Acid Residues Controlling Domain Interaction and Interdomain Electron Transfer in Cellobiose Dehydrogenase

The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD-containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure-based engineering. The electron transfer kinetics of wild-type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped-flow spectrophotometry and structural effects were studied by small-angle X-ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway - its mutation reducing the interdomain electron transfer 10-fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer