Characterization of a thermostable κ-carrageenase from a hot spring bacterium and plant protection activity of the oligosaccharide enzymolysis product.

BACKGROUND: Seaweed oligosaccharides are environmentally-friendly natural products and their use for disease control in sustainable agriculture is extremely promising. Enzymatic digestion to prepare seaweed oligosaccharides has drawn considerable interest. However, the study of enzymatically degraded products of carrageenan is still in its infancy compared with that of other hydrocolloids such as agar and alginate. To prepare degraded carrageenan on a commercial scale, it is necessary to select superior producer bacterial strains to improve the yield and thermostability of carrageenases. RESULTS: The carrageenan-degrading bacterium Bacillus sp. HT19 was isolated from sediment of a hot spring in Indonesia, and a κ-carrageenase with high activity was purified from the culture supernatant. The purified enzyme, named Car19, had maximum activity (538 U mg-1 ) at 60 °C and pH 7.0. Notably, the enzyme retained >90% of its initial activity after incubation at 60 °C for 24 h. The Ca2+ obviously improved the thermostability of Car19 at 70 °C. The Km and Vmax values of purified Car19 were 0.061 mg mL-1 and 115.13 U mg-1 , respectively, with κ-carrageenan as substrate. Thin-layer chromatography and electrospray ionization mass-spectrometry analysis of hydrolysates indicated that the enzyme exolytically depolymerized κ-carrageenan to neo-carrabiose. The hydrolysate enhanced the resistance of cucumber to cucumber mosaic virus and increased the activity of antioxidant enzymes in infected plants. CONCLUSION: To our knowledge, Car19 is the most thermostable κ-carrageenase reported so far. Its high optimal reaction temperature and thermostability, and unitary hydrolysate constituent, makes Car19 a promising candidate for the preparation of carrageenan oligosaccharides with plant protection activity. © 2018 Society of Chemical Industry.

Green Synthesis of Gold Nanoparticles Using Carrageenan Oligosaccharide and Their In Vitro Antitumor Activity.

Gold nanoparticles (AuNPs) have been widely used in catalysis, photothermal therapy, and targeted drug delivery. Carrageenan oligosaccharide (CAO) derived from marine red algae was used as a reducing and capping agent to obtain AuNPs by an eco-friendly, efficient, and simple synthetic route for the first time. The synthetic conditions of AuNPs were optimized by response surface methodology (RSM), and the CAO-AuNPs obtained were demonstrated to be ellipsoidal, stable and crystalline by means of transmission electron microscopy (TEM), scanning electron microscopy (SEM) and X-ray diffraction (XRD). The CAO-AuNPs showed localized surface plasmon resonance (LSPR) oscillation at about 530 nm with a mean diameter of 35 ± 8 nm. The zeta potential of CAO-AuNPs was around -20 mV, which was related to the negatively charged CAO around AuNPs. The CAO-AuNPs exhibited significant cytotoxic activities to HCT-116 and MDA-MB-231 cells, which could be a promising nanomaterial for drug delivery.

Influence of the Mixture of Carrageenan Oligosaccharides and Egg White Protein on the Gelation Properties of Culter alburnus Myofibrillar Protein under Repeated Freezing-Thawing Cycles.

This study aims to investigate the influence of the mixture (CGO/EWP) of carrageenan oligosaccharide (CGO) and egg white protein (EWP) (CGO/EWP, CGO: EWP = 1:1, m/m) on the functional, structural, and gelling properties of Culter alburnus myofibrillar protein (MP) during repeated freezing-thawing cycles by treating MP samples separately with EWP, CGO, or CGO/EWP based on the wet weight (1%, m/m), using samples without any cryoprotectant as the blank group. After the second repeated freezing-thawing cycle, the sulfhydryl group content was found to be significantly (p < 0.05) higher in the CGO/EWP (30.57 nmol/mg) and CGO (36.14 nmol/mg) groups than in the EWP group (23.80 nmol/mg), indicating that CGO/EWP and CGO can more effectively delay the oxidative deterioration of functional groups. Additionally, the surface hydrophobicity was shown to be significantly lower in the CGO (25.74) and CGO/EWP (27.46) groups than in the EWP (34.66) and blank (39.32) groups. Moreover, the α-helix content was higher in the CGO (35.2%) and CGO/EWP (32.3%) groups than in the EWP (29.2%) and blank (25.0%) groups. These data indicated that CGO and CGO/EWP could more effectively increase the structural stability, thereby inhibiting the exposure of hydrophobic groups and curbing the decline of α-helix content. During the heat-induced gel-forming process, EWP and CGO/EWP could enhance the gel viscoelasticity and strength. After the second freezing-thawing cycle, when compared with the blank group, the CGO/EWP group showed significantly (p < 0.05) higher water-holding capacity (66.30% versus 53.93%) and shorter T22 relaxation time (413.56 versus 474.99 ms). The integrated results indicated that CGO/EWP could more effectively delay the decrease of protein-water molecular interaction forces in the MP gel. This study shed light on the mechanism of CGO/EWP as a cryoprotective mixture in improving the deterioration of MP gelation properties during repeated freezing-thawing cycles.

Structural characteristics of carrageenans of red alga Mastocarpus pacificus from sea of Japan.

The sulfated polysaccharide from sterile alga Mastocarpus pacificus was investigated. Partial reductive hydrolysis and NMR spectroscopy showed that the extracted polysaccharides were only carrageenans. According to FT-IR- and NMR spectroscopy this polysaccharide was a hybrid kappa/iota-carrageenan with a predominance of kappa-type units. According to MALDI-TOFMS, oligosaccharide fragments obtained by mild acid hydrolysis had a polymerization degree of 1-9, while chains built up of galactose residues were up to 3. Tandem ESI mass spectrometry together with innovative 18O-labelling method showed that the polymer chain of the carrageenan included kappa-carrabiose, kappa-carratetraose, iota-carrabiose, hybrid kappa/iota oligosaccharide units and contained minor insertions of mu-carrageenan (the precursor of kappa-carrageenan). Parallel artificial membrane permeability assay shown that the studied carrageenan inhibited bile salts permeation through an artificial membrane imitating the gastrointestinal barrier by 50 % on average compared to negative control independent of incubation time. However, its action was less pronounced than the hindering ability of cholestyramine.

Influence of temperature fluctuations on growth and recrystallization of ice crystals in frozen peeled shrimp (Litopenaeus vannamei) pre-soaked with carrageenan oligosaccharide and xylooligosaccharide.

Temperature fluctuation is a common problem in the frozen storage of shrimp products. This study investigated the influence of carrageenan oligosaccharide (CO) and xylooligosaccharide (XO) on the growth and recrystallization of ice crystals in frozen peeled shrimp exposed to temperature fluctuations. Shrimp soaked with water and 3.0% (w/v) Na4P2O7 solution were designated as the negative and positive controls, respectively. Our data revealed that both CO- and XO-soaked shrimp had significant improvements in thawing and cooking loss, myofibrillar protein content, Ca2+-ATPase activity, and textural variables when exposed to temperature fluctuations compared to control samples. Microstructural imaging indicated that soaking the shrimp in CO and XO slowed the progression of damage caused to tissue myofibrils by large ice crystals, as well as inhibited the growth and recrystallization of ice crystals in muscle tissues. SDS-PAGE analysis confirmed that treatment with the oligosaccharides exhibited marked effects on the stability of muscle proteins and inhibited the degradation of muscle proteins affected by the temperature fluctuations. Based on these data, we hypothesize that the incorporated CO and XO may bind to muscle proteins and capture water molecules in the myofibrillar network through hydrogen bonding, thereby suppressing the myofibrillar denaturation and tissue structure destruction induced by the growth and recrystallization of ice crystals.

Enhanced Production of κ-Carrageenase and κ-Carrageenan Oligosaccharides through Immobilization of Thalassospira sp. Fjfst-332 with Magnetic Fe3O4-Chitosan Microspheres.

In this study, immobilized bacteria (IMB) microsphere was prepared by embedding κ-carrageenase-producing Thalassospira sp. Fjfst-332 (TF332) onto a magnetic Fe3O4-chitosan carrier. The performance of Fe3O4-chitosan carrier was optimized by comparing its bacteria immobilization capacity at different Fe3O4:chitosan ratios and temperatures, while the functions of IMB microspheres were characterized by examining their κ-carrageenase production at different temperatures, pH's, and reuse cycles. At the 1:1 (w:w) Fe3O4:chitosan ratio, the Fe3O4-chitosan carriers possessed sufficient anchoring capacity for bacterial immobilization without significant compromise of their magnetism for magnetic separation of IMB from culture media. The spectroscopic analysis of IMB microspheres indicated that the immobilization of TF332 might affect the amide groups in chitosan. Compared to free bacteria, IMB can produce κ-carrageenase at higher temperature, wider pH range, and faster rate. More importantly, the κ-carrageenase-producing activity was sustained for at least seven reuse cycles. The major κ-carrageenan degradation products of IMB-derived κ-carrageenase were the oligosaccharides containing two to six monosaccharide units. Overall, this Fe3O4-chitosan-TF-332 microsphere has the potential to become a stable and reusable platform for large-scale production of κ-carrageenan oligosaccharides.