Search Results (10)

Proteolytic enzymes, which play a crucial role in peptide bond cleavage, are widely applied in various industries. In this study, protease-producing bacteria were isolated and characterized from marine sediments collected from the Yellow Sea, China. Comprehensive screening and 16S rDNA sequencing identified a promising G4 strain as Bacillus atrophaeus. Following meticulous optimization of fermentation conditions and medium composition via response surface methodology, protease production using strain G4 was significantly enhanced by 64%, achieving a yield of 3258 U/mL. The G4 protease exhibited optimal activity at 50 °C and pH 7.5, demonstrating moderate thermal stability with 52% residual activity after 30-min incubation at 50 °C—characteristics typical of an alkaline protease. Notably, the enzyme retained over 79% activity across a broad pH range (6–11) and exhibited excellent salt tolerance, maintaining over 50% activity in a saturated NaCl solution. Inhibition by phenylmethylsulfonyl fluoride, a serine protease inhibitor, confirmed its classification as a serine protease. The enzyme’s potential in generating bioactive peptides was further demonstrated through hydrolysis of sheep (Ovis aries) placenta, resulting in a hydrolysate with notable antioxidant properties. The hydrolysate exhibited a 64% superoxide anion scavenging activity, surpassing that of reduced glutathione. These findings expand the current understanding of Bacillus atrophaeus G4 proteases and provide a foundation for innovative sheep placenta utilization with potential industrial applications. Full article

Cyanobacteria are adaptable and dominant organisms that exist in many harsh and extreme environments due to their great ecological tolerance. They produce various secondary metabolites, including cyanotoxins. While cyanobacteria are well studied in surface waters and some aerial habitats, numerous other habitats and niches remain underexplored. We collected 61 samples of: (i) biofilms from springs, (ii) aerial microbial mats from buildings and subaerial mats from caves, and (iii) water from borehole wells, caves, alkaline, saline, sulphidic, thermal, and iron springs, rivers, seas, and melted cave ice from five countries (Croatia, Georgia, Italy, Serbia, and Slovenia). We used (q)PCR to detect cyanobacteria (phycocyanin intergenic spacer—PC-IGS and cyanobacteria-specific 16S rRNA gene) and cyanotoxin genes (microcystins—mcyE, saxitoxins—sxtA, cylindrospermopsins—cyrJ), as well as amplicon sequencing and morphological observations for taxonomic identification. Cyanobacteria were detected in samples from caves, a saline spring, and an alkaline spring. While mcyE or sxtA genes were not observed in any sample, cyrJ results showed the presence of a potential cylindrospermopsin producer in a biofilm from a sulphidic spring in Slovenia. This study contributes to our understanding of cyanobacteria occurrence in diverse habitats, including rare and extreme ones, and provides relevant methodological considerations for future research in such environments. Full article

Fuel cells are considered as efficient and environmentally ecofriendly alternatives for energy production. The oxygen-reduction reaction is important in energy-conversion systems for fuel cells. In this work, rhodium (Rh) and cobalt (Co) alloy nanoparticles were deposited on nitrogen (N)-doped carbon (C) supports (RhCo/NC) using ball milling and thermal decomposition. The RhCo/NC composites were transformed into small nanoparticles with an average diameter of approximately 4 nm. The properties of the as-synthesized RhCo/NC nanocatalyst were characterized through transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and Raman spectroscopy. The catalytic activity of the nanocatalyst for the ORR was investigated. The RhCo/NC nanocatalyst showed good activity for the ORR, long-term durability in chronoamperometry tests, and resistance to methanol crossover in an alkaline solution. This was because of the synergistic effects of the metal alloy. Chronoamperometric analysis demonstrated the remarkable durability of the RhCo/NC nanocatalyst compared to a commercial platinum (Pt)/C catalyst. Moreover, the RhCo/NC nanocatalyst exhibited good methanol tolerance. The RhCo/NC nanocatalyst can replace Pt-based catalysts in energy-conversion systems. Full article

Poly(vinyl alcohol) (PVA) displays ice recrystallization inhibition (IRI) properties as many antifreeze proteins found in cold tolerant organisms. The molecular architecture and composition (molecular weight and distribution of pendant OH and acetate groups) have been studied to improve the antifreezing properties of PVA, suggesting that the molecular architecture of PVA plays an important role in IRI activity. The present work deals with the preparation of PVA microparticles using an alkaline treatment. The effect of PVA molecular weight on the morphology and antifreezeing properties of PVA microparticles was investigated. The antifreezeing property of PVA microparticles on the susceptibility of flower bud tissues to freeze damage was also evaluated. The alkaline treatment of an aqueous PVA solution produced stable polymer chain aggregates with spherical shapes. The average size of the PVA microparticles increased significantly with the increasing molecular weight of the PVA macromolecule precursor. The PVA microparticles inhibited the growth of ice crystals and blocked ice growth at concentrations as low as 0.01 % w/v. The effect of impeding ice crystal growth by preventing the joining of adjacent ice crystals is attributed to the larger size of the PVA particles adsorbed on the ice surface compared to the aggregated PVA macromolecules in saline solution. The thermal hysteresis activity of PVA macromolecules and microparticles was not detected by differential scanning calorimetry analysis. The PVA microparticles reduced the incidence of freeze injuries in flower bud tissues by 55% and their application, considering the low toxicity of PVA, has a high potential for freeze protection in fruit crops. Full article

An α-galactosidase-producing strain named Anoxybacillus vitaminiphilus WMF1, which catalyzed the reverse hydrolysis of d-galactose and glycerol to produce isofloridoside, was isolated from soil. The α-galactosidase (galV) gene was cloned and expressed in Escherichia coli. The galV was classified into the GH36 family with a molecular mass of 80 kDa. The optimum pH and temperature of galV was pH 7.5 and 60 °C, respectively, and it was highly stable at alkaline pH (6.0–9.0) and temperature below 65 °C. The specificity for p-nitrophenyl α-d-galactopyranoside was 70 U/mg, much higher than that for raffinose and stachyose. Among the metals and reagents tested, galV showed tolerance in the presence of various organic solvents. The kinetic parameters of the enzyme towards p-nitrophenyl α-d-galactopyranoside were obtained as K_m (0.12 mM), V_max (1.10 × 10⁻³ mM s⁻¹), and K_cat/K_m (763.92 mM⁻¹ s⁻¹). During the reaction of reverse hydrolysis, the enzyme exhibited high specificity towards the glycosyl donor galactose and acceptors glycerol, ethanol and ethylene glycol. Finally, the isofloridoside was synthesized using galactose as the donor and glycerol as the acceptor with a 26.6% conversion rate of galactose. This study indicated that galV might provide a potential enzyme source in producing isofloridoside because of its high thermal stability and activity. Full article

Wood-cement panels are becoming increasingly widely used as prefabricated building materials. In order to increase the use of renewable resources as materials for industrial applications, the use of alternative plant fibres has been gaining interest. Additionally, it is assumed that new or better board properties can be achieved due to the different chemical and mechanical properties of such alternative sources of fibres. In south-eastern Spain, the Canary Islands palm (Phoenix canariensis) is widely used in urban landscaping. Plantations attacked by red palm weevils generate abundant plant waste that must be shredded and taken to authorised landfills. This paper discusses the use of particles of Canary Islands palm for manufacturing fibre panels containing 20% cement in relation to the weight of the particles, using different proportions of starch as a plasticiser. A pressure of 2.6 MPa and a temperature of 100 °C were used in their production. Density, thickness swelling, water absorption, internal bonding strength, modulus of rupture (MOR), modulus of elasticity (MOE), and thermal conductivity were studied. The mechanical tests showed that the MOR and MOE values increased with longer setting times, meaning that the palm particles were able to tolerate the alkalinity of the cement. The board with 5% starch had a MOR of 15.76 N·mm⁻² and a MOE of 1.872 N·mm⁻² after 28 days. The boards with thicknesses of 6.7 mm had a mean thermal conductivity of 0.054 W·m⁻¹·K⁻¹. These boards achieved good mechanical properties and could be used for general use and as a thermal insulation material in building construction. Full article

Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg⁻¹) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg⁻¹), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase. Full article

Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. The Nb28-AP was produced by auto-induction expression and retained an intact antigen-binding capacity and enzymatic activity. It exhibited high thermal stability and organic solvent tolerance. Under the optimal conditions, the developed assay for OTA could be finished in 20 min with a half maximal inhibitory concentration of 0.57 ng mL⁻¹ and a limit of detection of 0.059 ng mL⁻¹, which was 1.1 times and 2.7 times lower than that of the unfused Nb28-based ELISA. The Nb28-AP exhibited a low cross-reactivity (CR) with ochratoxin B (0.92%) and ochratoxin C (6.2%), and an ignorable CR (<0.10%) with other mycotoxins. The developed Nb-AP-based one-step ELISA was validated and compared with a liquid chromatography-tandem mass spectrometry method. The results show the reliability of Nb-AP-based one-step ELISA for the detection of OTA in rice. Full article

A P, N dual doped reduced graphene oxide (PN-rGO) catalyst with high surface area (376.20 m²·g⁻¹), relatively high P-doping level (1.02 at. %) and a trace amount of N (0.35 at. %) was successfully prepared using a one-step method by directly pyrolyzing a homogenous mixture of graphite oxide (GO) and diammonium hydrogen phosphate ((NH₄)₂HPO₄) in an argon atmosphere, during which the thermal expansion, deoxidization of GO and P, N co-doping were realized simultaneously. The catalyst exhibited enhanced catalytic performances for oxygen reduction reaction (ORR) via a dominated four-electron reduction pathway, as well as superior long-term stability, better tolerance to methanol crossover than that of commercial Pt/C catalyst in an alkaline solution. Full article

Endoglucanase B (EGLB) derived from Aspergillus niger BCRC31494 has been used in the food fermentation industry because of its thermal and alkaline tolerance. It was cloned and expressed in Pichia pastoris. According to sequence analysis, the gene open reading frame comprises 1,217 bp with five introns (GenBank GQ292753). According to sequence and protein domain analyses, EGLB was assigned to glycosyl hydrolase family 5 of the cellulase superfamily. Several binding sites were found in the promoter region. The purified recombinant enzyme was induced by 0.5% methanol, and it exhibited optimal activity at 70 °C and pH 4. EGLB was stable for 3 h at temperatures below 60 °C, with more than 90% of its activity remaining. The enzyme was specific for substrates with β-1,3 and β-1,4 linkages. In Lineweaver-Burk plot analysis, the K_m and V_max values of EGLB for β-D-glucan were 134 mg/mL and 4.68 U/min/mg, respectively. The enzyme activity was increased by 1.86-fold by Co²⁺ and by 2-fold by Triton X-100 and Tween 80. These favorable properties make EGLB a potential candidate for use in laundry and textile industrial applications. Full article