Insects are considered a nutritionally valuable source of alternative proteins, and their efficient protein extraction is a prerequisite for large-scale use. The protein content is usually calculated from total nitrogen using the nitrogen-to-protein conversion factor (Kp) of 6.25. This factor overestimates the protein content, due to the presence of nonprotein nitrogen in insects. In this paper, a specific Kp of 4.76 ± 0.09 was calculated for larvae from Tenebrio molitor, Alphitobius diaperinus, and Hermetia illucens, using amino acid analysis. After protein extraction and purification, a Kp factor of 5.60 ± 0.39 was found for the larvae of three insect species studied. We propose to adopt these Kp values for determining protein content of insects to avoid overestimation of the protein content.

Protease inhibitors from potato juice of cv. Elkana were purified and quantified. The protease inhibitors represent ca. 50% of the total soluble proteins in potato juice. The protease inhibitors were classified into seven different families: potato inhibitor I (PI-1), potato inhibitor II (PI-2), potato cysteine protease inhibitor (PCPI), potato aspartate protease inhibitor (PAPI), potato Kunitz-type protease inhibitor (PKPI), potato carboxypeptidase inhibitor (PCI), and "other serine protease inhibitors". The most abundant families were the PI-2 and PCPI families, representing 22 and 12% of all proteins in potato juice, respectively. Potato protease inhibitors show a broad spectrum of enzyme inhibition. All the families (except PCI) inhibited trypsin and/or chymotrypsin. PI-2 isoforms exhibit 82 and 50% of the total trypsin and chymotrypsin inhibiting activity, respectively. A strong variation within the latter activities was shown within one family and between protease inhibitor families.

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The reaction mechanism of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) was studied by site-directed mutagenesis and x-ray crystallography. An inactive mutant of BiSP (E232Q) was co-crystallized with sucrose. The structure revealed a substrate-binding mode comparable with that seen in other related sucrose-acting enzymes. Wild-type BiSP was also crystallized in the presence of sucrose. In the dimeric structure, a covalent glucosyl intermediate was formed in one molecule of the BiSP dimer, and after hydrolysis of the glucosyl intermediate, a ␤-D-glucose product complex was formed in the other molecule. Although the overall structure of the BiSP-glucosyl intermediate complex is similar to that of the BiSP(E232Q)-sucrose complex, the glucose complex discloses major differences in loop conformations. Two loops (residues 336 -344 and 132-137) in the proximity of the active site move up to 16 and 4 Å , respectively. On the basis of these findings, we have suggested a reaction cycle that takes into account the large movements in the active-site entrance loops.

There is an increasing interest to positively influence the human intestinal microbiota through the diet by the use of prebiotics and/or probiotics. It is anticipated that this will balance the microbial composition in the gastrointestinal tract in favor of health promoting genera such as Bifidobacterium and Lactobacillus. Carbohydrates like non-digestible oligosaccharides are potential prebiotics. To understand how these bacteria can grow on these carbon sources, knowledge of the carbohydrate-modifying enzymes is needed. Little is known about the carbohydrate-modifying enzymes of bifidobacteria. The genome sequence of Bifidobacterium adolescentis and Bifidobacterium longum biotype longum has been completed and it was observed that for B. longum biotype longum more than 8% of the annotated genes were involved in carbohydrate metabolism. In addition more sequence data of individual carbohydrases from other Bifidobacterium spp. became available. Besides the degradation of (potential) prebiotics by bifidobacterial glycoside hydrolases, we will focus in this review on the possibilities to produce new classes of non-digestible oligosaccharides by showing the presence and (transglycosylation) activity of the most important carbohydrate modifying enzymes in bifidobacteria. Approaches to use and improve carbohydrate-modifying enzymes in prebiotic design will be discussed.

An algae-based biorefinery relies on the efficient use of algae biomass through its fractionation of several valuable/bioactive compounds that can be used in industry. If this biorefinery includes green platforms as downstream processing technologies able to fulfill the requirements of green chemistry, it will end-up with sustainable processes. In the present study, a downstream processing platform has been developed to extract bioactive compounds from the microalga Isochrysis galbana using various pressurized green solvents. Extractions were performed in four sequential steps using (1) supercritical CO2 (ScCO2), (2) ScCO2/ethanol (Gas Expanded Liquid, GXL), (3) pure ethanol, and (4) pure water as solvents, respectively. The residue of the extraction step was used as the raw material for the next extraction. Optimization of the ScCO2 extraction was performed by factorial design in order to maximize carotenoid extraction. During the second step, different percentages of ethanol were evaluated (15%, 45% and 75%) in order to maximize the extraction yield of fucoxanthin, the main carotenoid present in this alga; the extraction of polar lipids was also an aim. The third and fourth steps were performed with the objective of recovering fractions with high antioxidant activity, eventually rich in carbohydrates and proteins. The green downstream platform developed in this study produced different extracts with potential for application in the food, pharmaceutical and cosmetic industries. Therefore, a good approach for complete revalorization of the microalgae biomass is proposed, by using processes complying with the green chemistry principlesThe authors acknowledge funding from the EU MIRACLES project (7th Framework Program - Grant Agreement No. 613588). B.G.L. thanks MINECO (Ministerio de Economía y Competitividad) for her Juan de la Cierva postdoctoral research contract. M.H. thanks MINECO for his Ramón y Cajal postdoctoral research contract

Hyaluronan is a polysaccharide with multiple functions in the human body being involved in creating flexible and protective layers in tissues and in many signalling pathways during embryonic development, wound healing, inflammation, and cancer. Hyaluronan is an important component of active pharmaceutical ingredients for treatment of, for example, arthritis and osteoarthritis, and its commercial value far exceeds that of other microbial extracellular polysaccharides. Traditionally hyaluronan is extracted from animal waste which is a well-established process now. However, biotechnological synthesis of biopolymers provides a wealth of new possibilities. Therefore, genetic/metabolic engineering has been applied in the area of tailor-made hyaluronan synthesis. Another approach is the controlled artificial (in vitro) synthesis of hyaluronan by enzymes. Advantage of using microbial and enzymatic synthesis for hyaluronan production is the simpler downstream processing and a reduced risk of viral contamination. In this paper an overview of the different methods used to produce hyaluronan is presented. Emphasis is on the advancements made in the field of the synthesis of bioengineered hyaluronan.

Around 80 enzymes are implicated in the generic starch and sucrose pathways. One of these enzymes is sucrose phosphorylase, which reversibly catalyzes the conversion of sucrose and orthophosphate to d-Fructose and alpha-d-glucose 1-phosphate. Here, we present the crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) refined at 1.77 A resolution. It represents the first 3D structure of a sucrose phosphorylase and is the first structure of a phosphate-dependent enzyme from the glycoside hydrolase family 13. The structure of BiSP is composed of the four domains A, B, B', and C. Domain A comprises the (beta/alpha)(8)-barrel common to family 13. The catalytic active-site residues (Asp192 and Glu232) are located at the tips of beta-sheets 4 and 5 in the (beta/alpha)(8)-barrel, as required for family 13 members. The topology of the B' domain disfavors oligosaccharide binding and reduces the size of the substrate access channel compared to other family 13 members, underlining the role of this domain in modulating the function of these enzymes. It is remarkable that the fold of the C domain is not observed in any other known hydrolases of family 13. BiSP was found as a homodimer in the crystal, and a dimer contact surface area of 960 A(2) per monomer was calculated. The majority of the interactions are confined to the two B domains, but interactions between the loop 8 regions of the two barrels are also observed. This results in a large cavity in the dimer, including the entrance to the two active sites.