Le-Chang Sun

Purification and characterization of parvalbumins from silver carp (Hypophthalmichthy molitrix)

BACKGROUND As the largest producer and consumer of freshwater fish in the world, many people suffer from allergy for consuming freshwater fish in China. However, the allergen profiles of freshwater fish are rarely known. RESULTS Parvalbumins (PVs) from the white muscle of silver carp (Hypophthalmichthy molitrix) were purified by ammonium sulfate fractionation and column chromatography including DEAE-Sepharose and Superdex 75. Three PV isoforms-PV-I, PV-II, and PV-III-were obtained and their molecular masses as estimated by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 12, 11, and 14 kDa, respectively. All the PVs could be detected by anti-frog PV monoclonal antibody. PV-I and PV-II were quite possibly glycoproteins, while PV-III was not glycosylated, as analyzed by periodic acid-Schiff (PAS) staining. Thermal stability revealed that PV-I and PV-II easily formed polymers, while these proteins were stable in a pH range of 4.0-10.0. A PV gene encoding 110 amino acid residues was cloned and it revealed high identity with PVs from other species of fish. CONCLUSION Three isotypes of PV were purified to homogeneity and one distinct PV gene was cloned in silver carp white muscle.

Biochemical characterization of chymotrypsins from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus).

Two chymotrypsins (chymotrypsins A and B) have been purified to homogeneity from the hepatopancreas of Japanese sea bass ( Lateolabrax japonicus ) by ammonium sulfate fractionation and chromatographies on DEAE-Sepharose and Phenyl-Sepharose. Two-dimensional electrophoresis (2-DE) analysis revealed that the molecular masses of chymotrypsins A and B were approximately 27.0 and 27.5 kDa, respectively. Their respective isoelectric points were 8.0 and 7.0. Purified chymotrypsins also revealed a single band on native-PAGE, whereas their mobilities were quite different. Optimum temperature and pH of chymotrypsins A and B were 45 degrees C and 8.0, respectively. Both enzymes were strongly inhibited by chymostatin, phenylmethanesulfonyl fluoride (PMSF), and Pefabloc SC, but slightly inhibited by metalloproteinase inhibitor of 1,10-phenanthroline and EDTA. Using Suc-Leu-Leu-Val-Tyr-MCA as substrate, apparent K(m) values of chymotrypsins A and B were 0.8 and 1.1 microM and k(cat) values were 2.7 and 2.0 s(-1), respectively. The N-terminal amino acid sequences of chymotrypsins A and B were determined to the 21st and 18th residues, respectively, and were identical. These sequences exhibited high identities to chymotrypsins from other animals. The digestive effect of the two chymotrypsins on myofibrillar proteins indicated their effectiveness in the degradation of food proteins.

Study on pepsinogens and pepsins from snakehead (Channa argus).

Three pepsinogens (PG1, PG2, and PG3) were highly purified from the stomach of freshwater fish snakehead (Channa argus) by ammonium sulfate fractionation, anion exchange, and gel filtration. Two-dimensional gel electrophoresis and native-PAGE analysis revealed that their molecular masses were 37, 38, and 36 kDa and their isoelectric points 4.8, 4.4, 4.0, respectively. All of the pepsinogens converted into their active form pepsins under pH 2.0 by one-step pathway or stepwise pathway. The three pepsins showed maximal activity at pH 3.0, 3.5, and 3.0 with optimum temperature at 45, 40, and 40 degrees C, respectively, using hemoglobin as substrate. All of the pepsins were completely inhibited by pepstatin A, a typical aspartic proteinase inhibitor. The N-terminal amino acid sequences of the three pepsinogens were determined to the 34th, 25th, and 28th amino acid residues, respectively. Western blot analysis of the three PGs exhibited different immunological reactions.

Biochemical characterization of trypsins from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus)

A cationic trypsin (trypsin A) and an anionic trypsin (trypsin B) were highly purified from the hepatopancreas of the Japanese sea bass (Lateolabrax japonicus) by ammonium sulfate precipitation, column chromatographies of DEAE-Sepharose and Sephacryl S-200 HR. Purified trypsins revealed single band on SDS-PAGE and their molecular masses were 21 kDa and 21.5 kDa, respectively. Trypsins A and B exhibited maximal activity at 40°C, and shared the same optimal pH at 9.0 using Boc-Phe-Ser-Arg-MCA as the substrate. The two trypsins were stable up to 45°C and in the pH range from 7.0 to 11.0. Trypsin inhibitors such as Pefabloc SC, PMSF and benzamidine are effective to these two enzymes and their susceptibilities were similar. Apparent K(m)s of trypsins A and B were 1.12 and 0.7 μM and k(cat)s of them were 72.08 and 67.79 S(-1) for Boc-Phe-Ser-Arg-MCA, respectively. The N-terminal amino acid sequences of the two trypsins were determined to the 24th residues, which were highly identical to trypsins from other species of fish while trypsins A and B only shared 45.8% identity. The digestive effect of the two trypsins on native shrimp muscular proteins indicated their effectiveness in the degradation of food proteins.

Antibacterial activity and mechanisms of depolymerized fucoidans isolated from Laminaria japonica

Fucoidans, sulfated polysaccharides in brown algae, were depolymerized though high-pressure hydrolysis, and their antibacterial activity, structural properties, and antibacterial mechanisms were investigated in this work. The fucoidans from Laminaria japonica show no antibacterial activity before depolymerization; however, their depolymerized products can effectively (p<0.05) inhibit the proliferation of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The structure-activity study demonstrated that lower molecular weight and stronger polyanionic property can promote the antibacterial activity. And the depolymerized fucoidans exhibited better antibacterial activity against E. coli than against S. aureus. The results also indicated that the bactericidal pathway of depolymerized fucoidans should be through destruction of the cytomembranes and the target molecules are the membrane proteins, which can result in changed membrane fluidity and/or activated autophagocytosis. Therefore, the depolymerized fucoidans possess potential appliance values in partly or totally replacing antibiotics in our daily life.

Visible Light-Induced Lipid Peroxidation of Unsaturated Fatty Acids in the Retina and the Inhibitory Effects of Blueberry Polyphenols

The lipid peroxidation of unsaturated fatty acids (UFAs) in the retina not only threatens visual cells but also affects the physiological health of the retina. In this work, the potential damages caused by daily visible light exposure on retinal UFAs were evaluated via a simulated in vitro model. At the same time, the benefits of dietary supplementation of blueberries to the eyes were also assessed. After prolonged light exposure, lipid peroxidation occurred for both docosahexaenoic and arachidonic acids (DHA and AA, respectively). The oxidized UFAs presented obvious cytotoxicity and significantly inhibited cell growth in retinal pigment epithelium cells. Among the different blueberry polyphenol fractions, the flavonoid-rich fraction, in which quercetin was discovered as the main component, was considerably better in preventing visible light-induced DHA lipid peroxidation than the anthocyanin- and phenolic acid-rich fractions. Then the retinal protective activity of blueberry polyphenols against light-induced retinal injury was confirmed in vivo. On the basis of the above results, inhibiting lipid peroxidation of UFAs in the retina is proposed to be another important function mechanism for antioxidants to nourish eyes.

Mung bean trypsin inhibitor is effective in suppressing the degradation of myofibrillar proteins in the skeletal muscle of blue scad (Decapterus maruadsi).

Mung bean trypsin inhibitor (MBTI) of the Bowman-Birk family was purified to homogeneity with a molecular mass of approximately 9 kDa on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 8887.25 Da as determined by matrix-assisted laser desorption/ionization-quadrupole ion trap-time-of-flight mass spectrometry (MALDI-QIT-TOF MS). Using blue scad myofibrillar proteins as targets, it was found that, in the absence of MBTI, proteolysis of myofibrillar proteins, especially myosin heavy chain (MHC), could be identified after incubation at 55 °C for 2 h, while in the presence of MBTI, with a final concentration of 25 ng/mL, proteolysis of these proteins was greatly suppressed even after incubation for 3 h. Although cysteine proteinase inhibitor E-64 was also effective in preventing protein degradation, inhibitors for metallo- and asparatic proteinases did not reveal obvious inhibitory effects. Our present results strongly suggested that the naturally occurring legume bean seed protein MBTI can be used as an effective additive in preventing marine fish blue scad surimi gel softening, which is quite possibly caused by myofibril-bound serine proteinase (MBSP).

Protective Effect of Fucoxanthin Isolated from Laminaria japonica against Visible Light-Induced Retinal Damage Both in Vitro and in Vivo.

With increasingly serious eye exposure to light stresses, such as light-emitting diodes, computers, and widescreen mobile phones, efficient natural compounds for preventing visible light-induced retinal damages are becoming compelling needs in the modern society. Fucoxanthin, as the main light absorption system in marine algae, may possess an outstanding bioactivity in vision protection because of its filtration of blue light and excellent antioxidative activity. In this work, both in vitro and in vivo simulated visible light-induced retinal damage models were employed. The in vitro results revealed that fucoxanthin exhibited better bioactivities than lutein, zeaxanthin, and blueberry anthocyanins in inhibiting overexpression of vascular endothelial growth factor, resisting senescence, improving phagocytic function, and clearing intracellular reactive oxygen species in retinal pigment epithelium cells. The in vivo experiment also confirmed the superiority of fucoxanthin than lutein in protecting retina against photoinduced damage. This excellent bioactivity may be attributed to its unique structural features, including allenic, epoxide, and acetyl groups. Fucoxanthin is expected to be an important ocular nutrient in the future.

Mechanism study of high browning degree of mantle muscle meat from Japanese common squid Todarodes pacificus during air-drying

Mantle meat from the Japanese common squid (Todarodes pacificus) browns more than other squid meats during air-drying. The factors contributing to the browning of Japanese common squid, long-finned squid (Photololigo edulis) and bigfin reef squid (Sepioteuthis lessoniana) were studied in boiled and raw meat both before and after air-drying. Dried raw meat from the Japanese common squid browned more than dried boiled meat (b(∗) value, from 4.7 to 28.5). The results from SDS-PAGE showed significant degradation of myosin heavy chain (MHC) suggesting that protease activity in raw Japanese common squid meat was higher than in the other two species. The concentration of arginine (1932.0mg/100g) and ribose (28.8μmol/g) in Japanese common squid meat was higher than in the other two species. These results suggest that high protease activity and high concentrations of arginine and ribose increase the browning discoloration of Japanese common squid during air-drying.

Cloning, Expression, and the Effects of Processing on Sarcoplasmic-Calcium-Binding Protein: An Important Allergen in Mud Crab

Shellfish allergy is a prevalent, long-lasting disorder usually persisting throughout life. However, the allergen information is incomprehensive in crab. This study aimed to identify a novel allergen in crab, show its potential in diagnosis and reduce the allergenicity by food processing. A 21-kDa protein was purified from Scylla paramamosain and confirmed as sarcoplasmic calcium binding protein (SCP) by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Total RNA was isolated from crab muscle, and a rapid amplification of cDNA was performed to obtain an ORF of 579 bp that coded for 193 amino acid residues. According to the results of circular dichroism analysis and ELISA assay, the recombinant SCP (rSCP) expressed in Escherichia coli showed similar physicochemical and immunoreactive properties to native SCP (nSCP). Additionally, the extensive cross reactivity of SCP among different species and the bidirectional IgE cross-reactivity between nSCP and rSCP were detected by iELISA. The allergenicity of rSCP was reduced via Maillard reaction or enzymatic cross-linking reaction, which was confirmed by the results of scanning electron microscopy, dot blot, and digestion assay. A straightforward and reproducible way was developed to obtain high yields of rSCP that maintains structural integrity and full IgE reactivity, which could compensate the low specific IgE-titers of most patient sera for future diagnosis. Furthermore, the Maillard reaction and enzymatic cross-linking reaction were effective approaches for the production of hypoallergenic seafood.