Improving thermostability of (R)-selective amine transaminase from Aspergillus terreus through introduction of disulfide bonds.

To improve the thermostability of (R)-selective amine transaminase from Aspergillus terreus (AT-ATA), we used computer software Disulfide by Design and Modelling of Disulfide Bonds in Proteins to identify mutation sites where the disulfide bonds were most likely to form. We obtained three stabilized mutants (N25C-A28C, R131C-D134C, M150C-M280C) from seven candidates by site-directed mutagenesis. Compared to the wild type, the best two mutants N25C-A28C and M150C-M280C showed improved thermal stability with a 3.1- and 3.6-fold increase in half-life (t1/2 ) at 40 °C and a 4.6 and 5.1 °C increase in T5010 . In addition, the combination of mutant R131C-D134C and M150C-M280C displayed the largest shift in thermostability with a 4.6-fold increase in t1/2 at 40 °C and a 5.5 °C increase in T5010 . Molecular dynamics simulation indicated that mutations of N25C-A28C and M150C-M280C lowered the overall root mean square deviation for the overall residues at elevated temperature and consequently increased the protein rigidity. The stabilized mutation of R131C-D134C was in the region of high mobility and on the protein surface, and the disulfide bond constraints the flexibility of loop 121-136.

Effect of quaternization degree on physiochemical and biological activities of chitosan from squid pens.

Chitosan was prepared by alkaline N-deacetylation of ß-chitin from squid pens, and N-(2-hydroxy) propyl-3-trimethyl ammonium chitosan chloride (HTCC) derivatives, with different degrees of quaternization (DQ) ranging from 0.77 to 1.06, were synthesized. It was identified by FT-IR, 1H NMR and XRD analysis. All of the HTCC showed good water solubility in a wide pH range. The moisture absorption and retention abilities of all the HTCC were much better than that of the chitosan. The moisture absorption and retention values of all the HTCC at 43% RH for 24 h were above 49% and 92%, respectively. The scavenging ability of HTCC against hydroxyl and ABTS radicals improved with increasing concentration. The effectiveness of HTCC against hydroxyl radicals was lower than that of chitosan. These results indicated that HTCC, which has a much better moisture absorption and retention capacity, may act as a potential moisturizer in vitro.

Optimization of total polysaccharide extraction from Herba Lophatheri using RSM and antioxidant activities.

Effects of extraction temperature, extraction time and ratio of water to material as well as their interactions on the yield of total polysaccharide from Herba lophatheri were studied by response surface methodology (RSM). The optimal conditions for the extraction of polysaccharides were determined to be the ratio of liquid to solid of 39.01, extraction time of 2.11h and extraction temperature of 97.09°C. Under these optimal conditions, the yield of polysaccharides obtained was 5.73±0.10%, which was well matched with the value predicted by the model. In vitro antioxidant assays showed that the polysaccharides HLP possessed significant inhibitory effects on superoxide radical. It also exhibited strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities, hydroxyl radical scavenging activities and its reducing power, ferrous ions chelating effect was also strong. These results suggested that H. lophatheri polysaccharides could be a suitable natural antioxidant and may be the functional foods for humans.

Effects of hydroxypropyl degree on physiochemical activities of chitosan from squid pens.

Chitosan was prepared by alkaline N-deacetylation of ß-chitin and hydroxypropyl derivatives with different degrees of substitution (DS) were synthesized. It was characterized by Fourier transform infrared (FT-IR) and elemental analysis. The DS of hydroxypropyl chitosan (HPCS) calculated by an element analyzer were 0.42, 0.75, 1.20, 1.82 and 2.25. HPCS showed better foam capacity and stability than that of chitosan, and the effectiveness correlated well with the DS of HPCS. The highest bile acid-binding capacity of all five HPCS reached 56.02 mg/g, which was 4.0-fold higher than that of chitosan. The scavenging ability of HPCS against hydroxyl and ABTS radicals improved with increasing concentration. The correlation between the hydroxypropyl content (DS) of HPCS and scavenging ABTS radical ability was positive. The hydroxyl radicals scavenging activity of HPCS correlated well with its increasing concentration, and EC50 values were below 12.5 mg/mL. These results indicated that hydroxypropylation is a possible approach to obtain chitosan derivatives with desirable physiochemical properties.

In vitro antioxidant activities of polysaccharides extracted from Moso Bamboo-Leaf.

The objective of this study was to evaluate in vitro antioxidant capacities of polysaccharides extracted from Moso Bamboo-Leaf (BLP). The molecular weight of the polysaccharides were obtained by SEC-LLS and their molecular masses were 13.4×10(4)Da, 2.8×10(4)Da and 1.9×10(4)Da, respectively. Available data obtained with in vitro models suggested WB1 (precipitated by 30% ethanol) showed significant inhibitory effects on superoxide radical, hydroxyl radical, DPPH radical and ferrous metal-chelating capacities. These results clearly establish the possibility that polysaccharides extracted from Moso Bamboo-Leaf could be effectively employed as ingredient in health or functional food, to alleviate oxidative stress.