Purified dextransucrase from Pediococcus pentosaceus CRAG3 as food additive.

The extracellular crude dextransucrase (0.67 U/mg) from P. pentosaceus CRAG3 (GenBank accession number JX679020) after PEG-1500 fractionation gave specific activity, 20.0 U/mg which by gel filtration resulted in 46.0 U/mg. The purified dextransucrase displayed molecular size of approximately, 224 kDa. The optimum assay conditions for dextransucrase activity were 5% sucrose in 20 mM sodium acetate buffer (pH 5.4) and 30 oC. The dextransucrase was stable up to 40 oC and at pH range of 5.4-7.0. The metal ions such as Co2+, Ca2+, Mg2+ and Zn2+ stimulated the dextransucrase activity by 56, 44, 14 and 12%, respectively. It was most stable at -20 oC with half-life of 307 days. Amongst various additives used, glycerol and Tween 80 provided significant stability to the enzyme with half-life 15.5 and 85.5 h, respectively as compared to control (6.9 h). The solidification of sucrose supplemented milk by purified dextransucrase due to dextran synthesis displayed its application as additive for improving the texture of dairy products.

Immobilization of cyclodextrin glucanotransferase on aminopropyl-functionalized silica-coated superparamagnetic nanoparticles

Background: Cyclodextrin glycosyltransferase (CGTase) from Amphibacillus sp. NPST-10 was successfully covalently immobilized on aminopropyl-functionalized silica coated superparamagnetic nanoparticles; and the properties of immobilized enzyme were investigated. The synthesis process included preparing of core magnetic magnetite (Fe3O4) nanoparticles using solvothermal synthesis; followed by coating of Fe3O4 nanoparticles with dense amino-functionalized silica (NH2-SiO2) layer using in situ functionalization method. The structure of synthesized Fe3O4@NH2-SiO2 nanoparticles was characterized using TEM, XRD, and FT-IR analysis. Fe3O4@NH2-SiO2 nanoparticles were further activated by gluteraaldehyde as bifunctional cross linker, and the activated nanoparticles were used for CGTase immobilization by covalent attachment. Results: Magnetite nanoparticles was successfully synthesized and coated with and amino functionalized silica layer (Fe3O4/NH2-SiO2), with particle size of 50-70 nm. The silica coated magnetite nanoparticles showed with saturation magnetization of 65 emug-1, and can be quickly recovered from the bulk solution using an external magnet within 10 sec. The activated support was effective for CGTase immobilization, which was confirmed by comparison of FT-IR spectra of free and immobilized enzyme. The applied approach for support preparation, activation, and optimization of immobilization conditions, led to high yields of CGTase immobilization (92.3%), activity recovery (73%), and loading efficiency (95.2%); which is one of the highest so far reported for CGTase. Immobilized enzyme showed shift in the optimal temperature from 50 to 55ºC, and significant enhancement in the thermal stability compared with free enzyme. The optimum pH for enzyme activity was pH 8 and pH 7.5 for free and immobilized CGTase, respectively, with slight improvement of pH stability of immobilized enzyme. Furthermore, kinetic studies revealed that immobilized CGTase had higher affinity toward substrate; with k m values of 1.18 ± 0.05 mg/ml and 1.75 ± 0.07 mg/ml for immobilized and free CGTase, respectively. Immobilized CGTase retained 87% and 67 of its initial activity after 5 and 10 repeated batches reaction, indicating that immobilized CGTase on Fe3O4/NH2-SiO2 had good durability and magnetic recovery. Conclusion: The improvement in kinetic and stability parameters of immobilized CGTase makes the proposed method a suitable candidate for industrial applications of CGTase. To best of our knowledge, this is the first report about CGTase immobilization on silica coated magnetite nanoparticles.

Inhibition of dextransucrase activity in Streptococcus mutans by plant phenolics.

Streptococcus mutans is responsible for causing dental caries in humans and utilizes sucrose for its growth. The dextransucrase (EC 2.4.1.5) is responsible for sucrose metabolism, which exhibits both hydrolytic and glucosyltransferase activities. In this study, we examined the effects of the plant phenols, namely gallic, tannic and syringic acids and aqueous extracts of certain traditionally used chewing sticks (Acacia arabica, Azadirachta indica, Pongamia pinnata and Salvadora persica) for prevention of dental caries on hydrolytic activity of dextransucrsae in S. mutans. Gallic acid (4-5 mM) produced 80-90% inhibition of the enzyme, while tannic acid (0.2 mM) and syringic acid (5 mM) inhibited the enzyme activity 80% and 48%, respectively in vitro. The aqueous extracts of chewing sticks produced 35-40% inhibition of dextransucrase activity at 5 mg phenol concentration. Kinetic analysis revealed mixed-type of enzyme inhibition by polyphenols, where both Km and Vmax were altered. The value of Ki for tannic, gallic and syringic acids were 0.35, 1.6 and 1.94 mM, respectively. The enzyme inhibition by polyphenols was optimum at pH 7-7.5, while by plant extract was maximum at pH 5-6. These results suggest that plant polyphenols may find potential applications in the prevention and control of dental caries by inhibiting dextransucrase activity in S. mutans.

Production, purification and biochemical characterization of cyclodextrin glucanotrans-ferase from bacillus cereus N1

Cyclodextrin glucanotransferase [CGTase], [EC .2.4.1.19] producing bacteria were isolated from different sources of soils and identified as Bacillus cereus N1 and the best source was the soil of the National Research Centre. The maximum production of the crude CGTase enzyme was observed after 48hr of incubation at 37°C producing CGTase activity of 3.5 U/ml. The effect of nutritional requirements on the CGTasc production were carried out. Soluble starch and yeast extracts were found to be the best carbon and nitrogen sources, respectively. The enzyme was successively purified by ammonium sulphate precipitation, DEAE-cellulose and sephadex G-100 column chromatography and the final specific activity of CGTasc enzyme was increased by 24 fold. The SDS-PAGE showed that the purified CGTase enzyme was homogenous and the molecular weight of the purified enzyme was about 75 kDa. The characterization of the enzyme exhibited optimum pH and temperature at 6.0 and 40°C, respectively. The enzyme was stable at pH 6.5 to 8.0 and retained its high activity up to 45°C

Purification and characterization of trehalose-6-phosphate synthase from Saccharomycopsis fibuligera A11.

Mutant A11, a mutant of Saccharomycopsis fibuligera Sdu with low acid and neutral trehalase was found to accumulate over 18% (w/w) trehalose from starch in its cells. In this study, trehalose-6-phosphate synthase (Tps1) was purified to homogeneity from this mutant, with a 30-fold increase in the specific enzyme activity, as compared to the concentrated cell-free extract, from initial cells. The molecular mass of the purified enzyme as determined by SDS-PAGE was 66 kDa. The optimum pH and temperature of the purified enzyme were 6.6 and 37 degrees C, respectively. The enzyme was activated by Ca2+, K+ and Mg2+, with K+ showing the highest activation at 35 mM. On the other hand, Mn2+, Cu2+, Fe3+, Hg2+ and Co2+ inhibited the enzyme. The enzyme was also strongly inhibited by protease inhibitors such as iodoacetic acid, EDTA and PMSF.