Simple production of resilin-like protein hydrogels using the Brevibacillus secretory expression system and column-free purification.

Resilin, an insect structural protein, has excellent flexibility, photocrosslinking properties, and temperature responsiveness. Recombinant resilin-like proteins (RLPs) can be fabricated into three-dimensional (3D) structures for use as cell culture substrates and highly elastic materials. A simplified, high-yielding production process for RLPs is required for their widespread application. This study proposes a simple production process combining extracellular expression using Brevibacillus choshinensis (B. choshinensis) and rapid column-free purification. Extracellular production was tested using four representative signal peptides; B. choshinensis was found to efficiently secrete Rec1, an RLP derived from Drosophila melanogaster, regardless of the type of signal peptide. However, it was suggested that Rec1 is altered by an increase in the pH of the culture medium associated with prolonged incubation. Production in a jar fermentor with controllable pH yielded 530 mg Rec1 per liter of culture medium, which is superior to productivity using other hosts. The secreted Rec1 was purified from the culture supernatant via (NH4 )2 SO4 and ethanol precipitations, and the purified Rec1 was applied to ring-shaped 3D hydrogels. These results indicate that the combination of secretory production using B. choshinensis and column-free purification can accelerate the further application of RLPs.

Analysis of Four Chitin-Active Lytic Polysaccharide Monooxygenases from Streptomyces griseus Reveals Functional Variation.

Lytic polysaccharide monooxygenases (LPMOs) are redox-active enzymes that cleave insoluble polysaccharides by an oxidative reaction. In the present study, we have characterized four recombinant putative chitin-active LPMOs from Streptomyces griseus (SgLPMO10B, -C, -D, and -F) and evaluated their potential in enhancing hydrolysis of α- and ß-chitin by three families of 18 chitinases of Serratia marcescens, SmChiA, -B, and -C. All four recombinant SgLPMO10s showed oxidative activity toward both α- and ß-chitin but exhibited different abilities to promote the release of chitobiose from chitin by chitinases depending on both the chitinase and the chitin type. These effects were observed under conditions where the amount of LPMO in the reaction was not rate-limiting, showing that the observed functional differences relate to different abilities of the LPMOs to interact with and act on the substrate. These results show that four seemingly similar LPMOs carrying out the same reaction, cleavage of chitin by C1 oxidation, may have different roles in natural chitin conversion, which provides a rationale for the multiplicity of these enzymes within the same organism. The ability of the LPMOs to act on more natural substrates was demonstrated by showing that SgLPMO10B improved chitin solubilization in dried powdered shrimp shells.

Bioprospecting medicinal plants for antioxidant components.

OBJECTIVE: To evaluate antioxidant activities of seven medicinal plant species and their fractions, and to identify their phenolic compounds. METHODS: Two extractions were processed and further fractionated by column chromatography to evaluate the concentration that inhibit 50% of 2,2'-azinobis (3-ethylbenzothiazoline-6-suslfonic acid, 1,1-diphenyl-2-picryl-hydrazyl radicals, and their ferric reducing antioxidant power. The identification of the fractions of phenolic compounds was done by ultra performance liquid chromatography. RESULTS: The aqueous-acetone extracts of Feretia apodanthera and Ozoroa insignis exhibited the highest antioxidant potentials comparable to those of the standard quercetin. Their subsequently silica gel column fractionation showed three most active fractions from which the major constituents quercetin, myricetin, kampferol, rutin and isoquercetin were identified. CONCLUSIONS: These plant species have potent antioxidant profiles and polyphenol compounds that may help to manage with radical related disease and aging.