Minimizing anti-nutritional factors in wet protein extraction from Swedish faba beans through the application of response surface methodology.

Faba beans, rich in protein and ideal for Swedish cultivation, are limited in food industry use due to anti-nutritional factors (ANFs) that hinder nutrient absorption. An extraction method was developed in our study to mitigate ANFs in faba beans, using aqueous alkaline methods and isoelectric precipitation with differential salt concentration. This method yielded 15.

Protein Nanofibrils from Fava Bean and Its Major Storage Proteins: Formation and Ability to Generate and Stabilise Foams.

Protein nanofibrils (PNFs) have potential for use in food applications as texture inducers. This study investigated the formation of PNFs from protein extracted from whole fava bean and from its two major storage proteins, globulin fractions 11S and 7S. PNFs were formed by heating (85 °C) the proteins under acidic conditions (pH 2) for 24 h.

Comparison of glycoside hydrolase family 3 β-xylosidases from basidiomycetes and ascomycetes reveals evolutionarily distinct xylan degradation systems.

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) β-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite.

Season does not have a deleterious effect on proportions of stallion seminal plasma proteins.

The mechanism by which the content of the major groups of seminal plasma proteins in stallion semen changes between the breeding and non-breeding seasons remains unknown. Here, we investigated the proportions of non-heparin-binding, phosphorylcholine-binding, and heparin-binding proteins in seminal plasma with the aim of relating them to sperm quality and testosterone levels in good and bad freezer stallions. Only minor variations in the major protein groups were found between the breeding and non-breeding seasons.

Structural studies of a glycoside hydrolase family 3 β-glucosidase from the model fungus Neurospora crassa.

The glycoside hydrolase family 3 (GH3) β-glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β-Glucosidases hydrolyse the β-1,4 bond at the nonreducing end of cellodextrins.

Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars.

Natural carbohydrate polymers such as starch, cellulose, and chitin provide renewable alternatives to fossil fuels as a source for fuels and materials. As such, there is considerable interest in their conversion for industrial purposes, which is evidenced by the established and emerging markets for products derived from these natural polymers. In many cases, this is achieved via industrial processes that use enzymes to break down carbohydrates to monomer sugars.

High-resolution structure of a lytic polysaccharide monooxygenase from reveals a predicted linker as an integral part of the catalytic domain.

For decades, the enzymes of the fungus have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all cellulose-degrading enzymes are available, except for LPMO9A, belonging to the AA9 family of lytic polysaccharide monooxygenases (LPMOs). These enzymes enhance the hydrolytic activity of cellulases and are essential for cost-efficient conversion of lignocellulosic biomass.

Improving the thermal stability of cellobiohydrolase Cel7A from by directed evolution.

Secreted mixtures of cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C.

Structural and functional studies of the glycoside hydrolase family 3 β-glucosidase Cel3A from the moderately thermophilic fungus Rasamsonia emersonii.

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. β-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the β-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the β-glucosidase from H.

Crystal structure of linoleate 13R-manganese lipoxygenase in complex with an adhesion protein.

The crystal structure of 13R-manganese lipoxygenase (MnLOX) of Gaeumannomyces graminis (Gg) in complex with zonadhesin of Pichia pastoris was solved by molecular replacement. Zonadhesin contains β-strands in two subdomains. A comparison of Gg-MnLOX with the 9S-MnLOX of Magnaporthe oryzae (Mo) shows that the protein fold and the geometry of the metal ligands are conserved.

Crystal Structure of Manganese Lipoxygenase of the Rice Blast Fungus Magnaporthe oryzae.

Lipoxygenases (LOX) are non-heme metal enzymes, which oxidize polyunsaturated fatty acids to hydroperoxides. All LOX belong to the same gene family, and they are widely distributed. LOX of animals, plants, and prokaryotes contain iron as the catalytic metal, whereas fungi express LOX with iron or with manganese.

Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus Humicola grisea var. thermoidea.

Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling in nature. They are typically the most abundant enzymes expressed by potent cellulolytic fungi, and are also responsible for the majority of hydrolytic potential in enzyme cocktails for industrial processing of plant biomass. The thermostability of the enzyme is an important parameter for industrial utilization.

Biochemical characterization and crystal structures of a fungal family 3 β-glucosidase, Cel3A from Hypocrea jecorina.

Cellulase mixtures from Hypocrea jecorina are commonly used for the saccharification of cellulose in biotechnical applications. The most abundant β-glucosidase in the mesophilic fungus Hypocrea jecorina is HjCel3A, which hydrolyzes the β-linkage between two adjacent molecules in dimers and short oligomers of glucose. It has been shown that enhanced levels of HjCel3A in H.

Kinetic investigation of the rate-limiting step of manganese- and iron-lipoxygenases.

Lipoxygenases (LOX) oxidize polyunsaturated fatty acids to hydroperoxides, which are generated by proton coupled electron transfer to the metal center with FeIIIOH- or MnIIIOH-. Hydrogen abstraction by FeIIIOH- of soybean LOX-1 (sLOX-1) is associated with a large deuterium kinetic isotope effect (D-KIE). Our goal was to compare the D-KIE and other kinetic parameters at different temperatures of sLOX-1 with 13R-LOX with catalytic manganese (13R-MnLOX).

Crystallization and preliminary crystallographic analysis of manganese lipoxygenase.

Lipoxygenases constitute a family of nonhaem metal enzymes with catalytic iron or, occasionally, catalytic manganese. Lipoxygenases oxidize polyunsaturated fatty acids with position specificity and stereospecificity to hydroperoxides, which contribute to inflammation and the development of cancer. Little is known about the structural differences between lipoxygenases with Fe or Mn and the metal-selection mechanism.

The crystal structure of the core domain of a cellulose induced protein (Cip1) from Hypocrea jecorina, at 1.5 Å resolution.

In an effort to characterise the whole transcriptome of the fungus Hypocrea jecorina, cDNA clones of this fungus were identified that encode for previously unknown proteins that are likely to function in biomass degradation. One of these newly identified proteins, found to be co-regulated with the major H. jecorina cellulases, is a protein that was denoted Cellulose induced protein 1 (Cip1).

The structure of a bacterial cellobiohydrolase: the catalytic core of the Thermobifida fusca family GH6 cellobiohydrolase Cel6B.

Cellulases, glycoside hydrolases that catalyze the degradation of cellulose, are classified as either endoglucanases or cellobiohydrolases (CBHs) based on their architecture and mode of action on the cellulose. CBHs bind the cellulose chain in a more or less closed tunnel and cleave off cellobiose units processively from one end of the cellulosic polymer, while endoglucanases have their active sites in a more or less open cleft and show a higher tendency to cut bonds internally in the polymer. The CBH Cel6A (also called CBH2) from the ascomycete Hypocrea jecorina has a much shorter substrate-binding tunnel and seems less processive than the CBH Cel7A (CBH1), from the same fungus.

High resolution crystal structure of the endo-N-Acetyl-β-D-glucosaminidase responsible for the deglycosylation of Hypocrea jecorina cellulases.

Endo-N-acetyl-β-D-glucosaminidases (ENGases) hydrolyze the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. The endo-N-acetyl-β-D-glucosaminidases classified into glycoside hydrolase family 18 are small, bacterial proteins with different substrate specificities. Recently two eukaryotic family 18 deglycosylating enzymes have been identified.

The first structure of a glycoside hydrolase family 61 member, Cel61B from Hypocrea jecorina, at 1.6 A resolution.

The glycoside hydrolase (GH) family 61 is a long-recognized, but still recondite, class of proteins, with little known about the activity, mechanism or function of its more than 70 members. The best-studied GH family 61 member, Cel61A of the filamentous fungus Hypocrea jecorina, is known to be an endoglucanase, but it is not clear if this represents the main activity or function of this family in vivo. We present here the first structure for this family, that of Cel61B from H.

Three-dimensional structure of an intact glycoside hydrolase family 15 glucoamylase from Hypocrea jecorina.

The three-dimensional structure of a complete Hypocrea jecorina glucoamylase has been determined at 1.8 A resolution. The presented structure model includes the catalytic and starch binding domains and traces the course of the 37-residue linker segment.

Structural and functional consequences of the replacement of proximal residues Cys(172) and Cys(192) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii.

Proximal Cys(172) and Cys(192) in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.

Structural analysis of altered large-subunit loop-6/carboxy-terminus interactions that influence catalytic efficiency and CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase.

The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-barrel of ribulose-1,5-bisphosphate carboxylase/oxygenase plays a key role in discriminating between CO2 and O2. Genetic screening in Chlamydomonas reinhardtii previously identified a loop-6 V331A substitution that decreases carboxylation and CO2/O2 specificity. Revertant selection identified T342I and G344S substitutions that restore photosynthetic growth by increasing carboxylation and specificity of the V331A enzyme.

Chimeric small subunits influence catalysis without causing global conformational changes in the crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Comparison of subunit sequences and X-ray crystal structures of ribulose-1,5-bisphosphate carboxylase/oxygenase indicates that the loop between beta-strands A and B of the small subunit is one of the most variable regions of the holoenzyme. In prokaryotes and nongreen algae, the loop contains 10 residues. In land plants and green algae, the loop is comprised of approximately 22 and 28 residues, respectively.

Altered intersubunit interactions in crystal structures of catalytically compromised ribulose-1,5-bisphosphate carboxylase/oxygenase.

Substitution of Leu290 by Phe (L290F) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from the unicellular green alga Chlamydomonas reinhardtii causes a 13% decrease in CO(2)/O(2) specificity and reduced thermal stability. Genetic selection for restored photosynthesis at the restrictive temperature identified an Ala222 to Thr (A222T) substitution that suppresses the deleterious effects of the original mutant substitution to produce a revertant enzyme with improved thermal stability and kinetic properties virtually indistinguishable from that of the wild-type enzyme. Because the mutated residues are situated approximately 19 A away from the active site, they must affect the relative rates of carboxylation and oxygenation in an indirect way.

Calcium supports loop closure but not catalysis in Rubisco.

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses CO(2) assimilation in biology. A prerequisite for catalysis is an activation process, whereby an active site lysine is selectively carbamylated. The carbamyl group is then stablised by a metal ion, which in vivo is Mg(2+).