Simultaneous enhancement of thermostability and catalytic activity of κ-carrageenase from Pseudoalteromonas tetraodonis by rational design.

κ-Carrageenase provides an attractive enzymatic approach to preparation of κ-carrageenan oligosaccharides. Pseudoalteromonas tetraodonis κ-carrageenase is active at the alkaline conditions but displays low thermostability. To further improve its enzymatic performance, two mutants of Q42V and I51H exhibiting both improved thermostability and enzyme activity were screened by the PoPMuSiC algorithm. Compared with the wild-type κ-carrageenase (WT), Q42V and I51H increased the enzyme activity by 20.9% and 25.4%, respectively. After treatment at 50 â„ƒ for 40 min, Q42V and I51H enhanced the residual activity by 31.1% and 25.9%, respectively. The Tm values of Q42V, I51H, and WT determined by differential scanning calorimetry were 58.2 â„ƒ, 54.8 â„ƒ, and 51.2 â„ƒ, respectively. Compared with untreated and HCl-treated κ-carrageenans, Q42V-treated κ-carrageenan exhibited higher pancreatic lipase inhibitory activity. Molecular docking analysis indicated that the additional pi-sigma force and hydrophobic interaction in the enzyme-substrate complex could account for the increased catalytic activity of Q42V and I51H, respectively. Molecular dynamics analysis indicated that the improved thermostability of mutants Q42V and I51H could be attributed to the less structural deviation and the flexible changes of enzyme conformation at high temperature. This study provides new insight into κ-carrageenase performance improvement and identifies good candidates for their industrial applications.

A bifunctional exolytic alginate lyase from Microbulbifer sp. ALW1 with salt activation and calcium-dependent catalysis.

Alginate lyases can depolymerize alginate to oligomers with potential applications in many fields. Here a new alginate lyase, namely AlgL6, was characterized from Microbulbifer sp. ALW1, phylogenetically classified into the polysaccharide lyase family 6 (PL6). The recombinant alginate lyase AlgL6 exerted enzymatic activities towards polymannuronate, polyguluronate, and sodium alginate in an exolytic manner. AlgL6 had an optimum temperature of 35 °C and good stability at 30 °C or below. Its optimum pH was 8.0, and it had good stability over the pH range of 5.0-9.0. AlgL6 exhibited excellent halo-stability against Na+, and its activity can be increased up to about 1.8 times by 0.5 M NaCl. AlgL6 also showed strong stability in the presence of some nonionic detergents such as Tween 20 and Tween 80. The degradation products of sodium alginate by AlgL6 exhibited more effective antioxidant activities than the undigested polysaccharides. Structure analysis illustrated the catalytic mechanism defined by the coordination of the acid/base residues Arg269 and Lys248 of AlgL6. The replacement of Ca2+-interacting amino acid residues in AlgL6 and depletion of Ca2+ suggested the involvement of Ca2+ in the enzyme's catalytic activity. These properties of AlgL6 supply support to its industrial application for development of alginate bioresource.

Characterization of a Thermostable and Surfactant-Tolerant Chondroitinase B from a Marine Bacterium Microbulbifer sp. ALW1.

Chondroitinase plays an important role in structural and functional studies of chondroitin sulfate (CS). In this study, a new member of chondroitinase B of PL6 family, namely ChSase B6, was cloned from marine bacterium Microbulbifer sp. ALW1 and subjected to enzymatic and structural characterization. The recombinant ChSase B6 showed optimum activity at 40 °C and pH 8.0, with enzyme kinetic parameters of Km and Vmax against chondroitin sulfate B (CSB) to be 7.85 µg/mL and 1.21 U/mg, respectively. ChSase B6 demonstrated thermostability under 60 °C for 2 h with about 50% residual activity and good pH stability under 4.0-10.0 for 1 h with above 60% residual activity. In addition, ChSase B6 displayed excellent stability against the surfactants including Tween-20, Tween-80, Trion X-100, and CTAB. The degradation products of ChSase B6-treated CSB exhibited improved antioxidant ability as a hydroxyl radical scavenger. Structural analysis and site-directed mutagenesis suggested that the conserved residues Lys248 and Arg269 were important for the activity of ChSase B6. Characterization, structure, and molecular dynamics simulation of ChSase B6 provided a guide for further tailoring for its industrial application for chondroitin sulfate bioresource development.

Characterization of a glucose-stimulated ß-glucosidase from Microbulbifer sp. ALW1.

Glucose-tolerant and/or glucose-stimulated ß-glucosidase is of great interest for its industrial utilization in enzymatic digestion of lignocellulosic biomass for biofuel production. In this study, a new gene of ß-glucosidase MaGlu1A was cloned from an alginate-degrading marine bacterium Microbulbifer sp. ALW1. The gene of MaGlu1A encoded a 472-amino acid protein classified into the glycosyl hydrolase family 1 (GH1). The recombinant ß-glucosidase was overexpressed and purified from Escherichia coli with a molecular mass of 65.0 kDa. Structure analysis illustrated the catalytic acid/base residue Glu186 and nucleophilic residue Glu370 in the enzyme. MaGlu1A displayed optimal activity at 40 °C and pH 4.5, respectively. It had substrate preference to the aryl-ß-glycosidic bonds with glucose, fucose, and galactose moieties, in addition to cellobiose. MaGlu1A demonstrated strong stimulation to the supplemental glucose. Site-directed mutagenesis suggested an essential role of Asn242 in glucose stimulation. The enzymatic characterization of MaGlu1A provides general information about its catalytic properties facilitating its practical applications.

Structural characterization and anti-lung cancer activity of a sulfated glucurono-xylo-rhamnan from Enteromorpha prolifera.

A sulfated glucurono-xylo-rhamnan (EP-3-H) was purified from a green alga, Enteromorpha prolifera. EP-3-H and its oligomers were characterized by high performance liquid chromatography, mass spectrometry and one and two-dimensional nuclear magnetic resource spectroscopy. The structural analysis showed EP-3-H has a backbone of glucurono-xylo-rhamnan, branches with glucuronic acid and sulfated at C3 of rhamnose and/or C2 of xylose. The inhibition of EP-3-H on human lung cancer A549 cell proliferation in vitro and its therapeutic effects in BALB/c-nu mice in vivo were determined to evaluate the anti-lung cancer activity of EP-3-H. The tumor inhibition level was 59 %, suggesting that EP-3-H might be a good candidate for the treatment of lung cancer. Surface plasmon resonance (SPR) studies revealed the IC50 on the binding of fibroblast growth factors, (FGF1 and FGF2), to heparin were 0.85 and 1.47 mg/mL, respectively. These results suggest that EP-3-H inhibits cancer proliferation by interacting with these growth factors.