Sea Cucumber Intestinal Peptide Induces the Apoptosis of MCF-7 Cells by Inhibiting PI3K/AKT Pathway.

Sea cucumbers are one of many marine echinoderm animals that contain valuable nutrients and medicinal compounds. The bioactive substances in sea cucumbers make them have promising biological and pharmacological properties, including antioxidant, anti-bacterial, and anti-tumor effects. In this study, sea cucumber intestinal peptide (SCIP) is a small molecular oligopeptide (<1,000 Da) extracted from sea cucumber intestines hydrolyzed by alkaline protease. The analysis of amino acid composition showed that hydrophobic amino acids and branched-chain amino acids were rich in SCIP. Nowadays, although increasing studies have revealed the biological functions of the sea cucumber active substances, there are few studies on the function of SCIP. Furthermore, due to the anti-cancer activity being an essential characteristic of sea cucumber active substances, we also investigated the anti-cancer potential and the underlying mechanism of SCIP in vivo and in vitro. The results indicate that SCIP inhibits the growth of MCF-7 tumor cells in zebrafish and increases the apoptosis of human breast cancer MCF-7 cells. Further mechanism studies confirm that SCIP promotes the expression of apoptosis-related proteins and thus promotes the breast cancer cells (MCF-7) apoptosis via inhibition of PI3K/AKT signal transduction pathway.

A novel chondroitin AC lyase from Pedobacter xixiisoli: Cloning, expression, characterization and the application in the preparation of oligosaccharides.

Chondroitin AC lyase can efficiently hydrolyze chondroitin sulfate (CS) to low molecule weight chondroitin sulfate, which has been widely used in clinical therapy, including anti-tumor, anti-oxidation, hypolipidemic, and anti-inflammatory. In this work, a novel chondroitin AC lyase from Pedobacter xixiisoli (PxchonAC) was cloned and overexpressed in Escherichia coli BL21 (DE3). The characterization of PxchonAC showed that it has specific activities on chondroitin sulfate A, Chondroitin sulfate C and hyaluronic acid with 428.77, 270.57, and 136.06 U mg-1, respectively. The Km and Vmax of PxchonAC were 0.61 mg mL-1 and 670.18 U mg-1 using chondroitin sulfate A as the substrate. The enzyme had a half-life of roughly 660 min at 37 °C in the presence of Ca2+ and remained a residual activity of 54 % after incubated at 4 °C for 25 days. Molecular docking revealed that Asn123, His223, Tyr232, Arg286, Arg290, Asn372, and Glu374 were mainly involved in the substrate binding. The enzymatic hydrolysis product was analyzed by gel permeation chromatography, demonstrating PxchonAC could hydrolyze CS efficiently.

Role of introduced surface cysteine of NADH oxidase from Lactobacillus rhamnosus.

Cysteine, a critical residue for catalytic process but also vulnerable to oxidative damage, was conventionally expressed as a buried catalytic site in most redox enzymes. In the present work, specific surface-exposed sites of a NADH oxidase from Lactobacillus rhamnosus (LrNox) were selected and mutated to cysteine to investigate its effects on catalytic function because LrNox has a buried catalytic cysteine but no surface-exposed one. The results showed that exception of the sites on dimer interface, the activities of LrNox mutants were improved to vary degrees when the polar uncharged and alanine residues were mutated to cysteine. But the cysteine mutations of polar charged and nonpolar residues except alanine showed obvious decline in catalytic activity. Substituting of Ala85 and Thr96 with other residues suggested that the cysteine mutation showed the highest activity. Structural analysis suggested that even a single cysteine mutation on the specific non-conserved surface area of LrNox could induce changes on the conformation of catalytic cysteine and lower the activation free energy to improve the catalytic activity.

Cloning, expression, characterization and homology modeling of a novel water-forming NADH oxidase from Streptococcus mutans ATCC 25175.

A novel nicotinamide adenine dinucleotide (NADH) oxidase from Streptococcus mutans ATCC 25175 (SmNox) was cloned and overexpressed in Escherichia coli BL21 (DE3). Sequence analysis revealed an open reading frame of 1374bp, capable of encoding a polypeptide of 457 amino acid residues. The molecular mass of the purified SmNox was estimated to be ∼49.9kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified SmNox had the highest specific activity of 281.2U·mg-1 at optimal pH and temperature of 7.0 and 35°C, with a Km of 57.7µM and a Vmax of 154.3U·mg-1. The good stability at room temperature was observed. Homology modeling and substrate docking were performed to evaluate the catalytic characteristics. The results indicated that Nicotinamide ring of NADH extends vertically toward to re-face of coenzyme (FAD), and the specific conformation of NADH suggested that the charges transfer in SmNox complex could be easier than in its homologous enzyme (LbNox) under alkaline environment. The characterization of the SmNox indicated it has potential in industrial regeneration of coenzyme NAD+ for coupling with dehydrogenases.

Cloning and characterization of a thermostable H2O-forming NADH oxidase from Lactobacillus rhamnosus.

NADH oxidase (Nox) catalyzes the conversion of NADH to NAD(+). A previously uncharacterized Nox gene (LrNox) was cloned from Lactobacillus rhamnosus and overexpressed in Escherichia coli BL21(DE3). Sequence analysis revealed an open reading frame of 1359 bp, capable of encoding a polypeptide of 453 amino acid residues. The molecular mass of the purified LrNox enzyme was estimated to be ~50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 100 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had optimal activity at pH 5.6 and temperature 65 °C, and k(cat)/K(m) of 3.77×10(7) s(-1) M(-1), the highest ever reported. Heat inactivation studies revealed that LrNox had high thermostability, with a half-life of 120 min at 80 °C. Molecular dynamics simulation studies shed light on the factors contributing to the high activity of LrNox. Although the properties of Nox from several microorganisms have been reported, this is the first report on the characterization of a recombinant H(2)O-forming Nox with high activity and thermostability. The characteristics of the LrNox enzyme could prove to be of interest in industrial applications such as NAD(+) regeneration.