Zedong Jiang

Characterization of an alkaline β-agarase from Stenotrophomonas sp. NTa and the enzymatic hydrolysates

An extracellular agarase from marine bacterium Stenotrophomonas sp. NTa was purified to homogeneity. By size exclusion chromatography and SDS-PAGE analysis, the enzyme was determined to be a homodimer with monomeric molecular mass of 89.0 kDa. The optimal temperature and pH of strain NTa agarase were 40 °C and 10.0, respectively. It exhibited striking stability across a wide pH range of 5.0-11.0. Agarase from Stenotrophomonas sp. NTa had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. The Km and Vmax for agar were 11.3mg/ml and 25.4 U/mg, respectively. Thin layer chromatography analysis, mass spectrometry, and enzyme assay using p-nitrophenyl-α/β-D-galactopyranoside revealed that strain NTa agarase was a β-agarase that degraded agarose into neoagarobiose, neoagarotetraose and neoagarohexaose as the predominant products, as well as a small amount of 3,6-anhydro-α-L-galactose. This is the first to present evidence of agarolytic activity in strain from genus Stenotrophomonas.

Expression and biochemical characterization of recombinant α-l-rhamnosidase r-Rha1 from Aspergillus niger JMU-TS528

A putative cDNA of α-l-rhamnosidase was PCR-cloned from Aspergillus niger JMU-TS528 and further extracellular over-expressed in Pichia pastoris GS115. The activity of the recombinant α-l-rhamnosidase r-Rha1 was 711.9U/mL, eightfold higher than the native α-l-rhamnosidase from A. niger JMU-TS528. r-Rha1 is a N-glycosylated protein of 90kDa and possesses broad substrate specificities by hydrolyzing α-1,2, α-1,3 α-1,4, and α-1,6 linkages to β-d-glucosides. This is the first report presenting that α-l-rhamnosidase showed activity on four kinds of glucosidic linkages. Compared with other previously characterized α-l-rhamnosidases, r-Rha1 showed a good thermostability and wide range of pH-stability with the optimum pH of 5.0 and temperature of 60°C. r-Rha1 activity was not greatly affected by representative metal ions and other detected effectors and showed excellent tolerance abilities against glucose and ethanol. These beneficial characteristics of r-Rha1 suggest that r-Rha1 should be considered a potential new biocatalyst for food and drug industrial applications.

Separation and purification of astaxanthin from Phaffia rhodozyma by preparative high-speed counter-current chromatography.

An effective high-speed counter-current chromatography (HSCCC) method was established for the preparative isolation and purification of astaxanthin from Phaffia rhodozyma. With a two-phase solvent system composed of n-hexane-acetone-ethanol-water (1:1:1:1, v/v/v/v), 100mg crude extract of P. rhodozyma was separated to yield 20.6mg of astaxanthin at 92.0% purity. By further one step silica gel column chromatography, the purity reached 99.0%. The chemical structure of astaxanthin was confirmed by thin layer chromatography (TLC), UV spectroscopy scanning, high performance liquid chromatography with a ZORBAX SB-C18 column and a Waters Nova-pak C18 column, and ESI/MS/MS.

Characterization of an arylsulfatase from a mutant library of Pseudoalteromonas carrageenovora arylsulfatase

A library of Pseudoalteromonas carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using error-prone PCR. After screening, one mutant strain was obtained whose arylsulfatase had improved thermal stability. Protein sequence analysis revealed one amino acid substitution of H260L. The mutant arylsulfatase (named H260L) retained higher residual activity than wild-type enzyme (named WT) after incubation at 45, 50, 55 and 60°C for 60min. Thermal inactivation analysis showed that the half-life (t1/2) value at 55°C for H260L was 40.6min, while that of WT was 9.1min. When p-nitrophenyl sulfate was used as a substrate, the optimal reaction temperature and pH for the mutant enzyme were 55°C and pH 8.0, respectively. H260L was stable over the pH range of 6.0-9.0. Inhibition assay with EDTA indicated that metal ions play an important role during the catalytic process of the mutant enzyme. The desulfation ratio against agar of Gracilaria lemaneiformis was 82%.

Improvement thermostability of Pseudoalteromonas carrageenovora arylsulfatase by rational design

This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A total of 10 single-site mutants were chosen using the PoPMuSiC program, and two mutants of K253N and P314T showed enhanced thermal stability. By saturation mutagenesis and thermostability analysis, K253H and P314T were the best mutants at the two sites. Combinational mutations of K253H, P314T and H260L were subsequently introduced, and the best mutant of K253H/H260L was selected. Thermal inactivation analysis showed the half-life (t1/2) value at 55°C for K253H/H260L was 7.7-fold that of the wild-type enzyme (WT), meanwhile this mutant maintained the specific enzyme activity. Structure modeling demonstrated that the additional hydrogen bonds, optimization of surface charge-charge interactions, and increasing of hydrophobic interaction could account for the improved thermostability imparted by K253H/H260L.

Alginate enhances Toll-like receptor 4-mediated phagocytosis by murine RAW264.7 macrophages

Alginate is a naturally acidic polysaccharide consisting alternately of β-d-mannuronic acid and α-l-guluronic acid with 1, 4-glycosidic linkages and is derived from brown seaweeds. Herein, the effect of alginate on the promotion of macrophage phagocytosis and the corresponding molecular mechanisms were investigated in murine RAW264.7 cells. Alginate could enhance the intracellular phagocytosis of gold nanoparticles (AuNPs), fluorescent microspheres and immunoglobulin G (IgG)-opsonized Staphylococcus aureus (S. aureus). Moreover, alginate increased Toll-like receptor 4 (TLR4) expression and activated the Akt/nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signalling pathways. Alginate-promoted phagocytosis was suppressed by the addition of inhibitors of TLR4, NF-κB and p38 MAPK and by TLR4 gene knockdown, indicating the involvement of these key components. This work is the first to propose that alginate promotes phagocytosis via upregulating TLR4 expression and stimulating the Akt/NF-κB and p38 MAPK signalling pathways, which may contribute to the capacity of alginate to activate macrophages.

An improved high performance liquid chromatography method for the separation of carotenoids extracted from Phaffia rhodozyma

Separation of analytes is an important factor affecting the effectiveness of carotenoids analysis from a complex extraction. In this study, a high performance liquid chromatography procedure with improved separation was developed to determine carotenoids extracted from P. rhodozyma. Mixture design experiment was employed to optimize the organic solvent constitution, which showed that the optimal organic phase was composed (v/v) of 61% of methanol, 32% of tetrahydrofuran, and 7% of acetonitrile. Furthermore, the gradient elution procedure optimization demonstrated that the gradient elution started at the optimal organic phase concentration of 60% lead to the best separation effect. Meanwhile, 18 compounds in total 25 detected peaks were separated. Based on their absorption spectra, astaxanthin, ß-carotene, zeaxanthin, β-cryptoxanthin and lycopene were identified by matching their retention times with standards. Interestingly, our improved HPLC method was effective to classify P. rhodozyma strains and monitor the degradation of astaxanthin in accordance with their carotenoids composition.

β-Agarase immobilized on tannic acid-modified Fe3O4 nanoparticles for efficient preparation of bioactive neoagaro-oligosaccharide

β-Agarase was immobilized by using tannic acid modified-Fe3O4 magnetic nanoparticles (TA-MNPs) as a supportmaterial. The MNPs were synthesized by improved chemical coprecipitation method and modified with TA for agarase immobilization. TA-MNPs and immobilized β-agarase were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and thermal gravimetric analysis (TGA), all of which indicated the successful surface modification of MNPs with TA and the immobilization of β-agarase. The optimal immobilization conditions for 25 mg TA-MNPs included 100 r/min oscillation speed, immobilization time of 2 h, immobilization temperature of 15 °C, and initial β-agarase concentration of 3 mL (480 U). Immobilized β-agarase showed better pH and thermal stability and excellent reusability than the free enzyme. Results revealed the promising application of β-agarase-TA-MNPs for the preparation of neoagaro-oligosaccharides with different averagepolymerizationdegrees and varying activities in the antioxidant.

Effect of oxygen and heating on aromas of pummelo (Citrus maxima) essential oil

Abstract Objective: To investigate effects of oxygen and heating on aromas and volatile components of pummelo essential oil (PEO). Method: The PEOs were analyzed by sensory evaluation, gas chromatography coupled with mass spectrometry (GC-MS), GC-olfactometry (GC-O) and sensory reconstruction analysis. Results: The PEO co-treated by oxygen and heating exhibited significant differences in both aromas and volatile compositions from those of the fresh, the nitrogen-protected, the heated and the oxygen-exposed PEOs. The strong sweet and floral notes of the oxygen and heating co-treated PEO were attributed to high concentration of limonene oxides; the strong minty note was resulted from the increase of L-carvone. Conclusion: The results indicate that limonene oxides and L-carvone could significantly change the aroma of PEO co-treatment by oxygen and heating, providing valuable information for the production and storage of aromatic products of PEO.

Improving the thermostability by introduction of arginines on the surface of α-L-rhamnosidase (r-Rha1) from Aspergillus niger

To improve the thermostability of α-L-rhamnosidase (r-Rha1), an enzyme previously identified from Aspergillus niger JMU-TS528, multiple arginine (Arg) residues were introduced into the r-Rha1 sequence to replace several lysine (Lys) residues that located on the surface of the folded r-Rha1. Hinted by in silico analysis, five surface Lys residues (K134, K228, K406, K440, K573) were targeted to produce a list of 5 single-residue mutants and 4 multiple-residue mutants using site-directed mutagenesis. Among these mutants, a double Lys to Arg mutant, i.e. K406R/K573R, showed the best thermostability improvement. The half-life of this mutants enzyme activity increased 3 h at 60 °C, 23 min at 65 °C, and 3.5 min at 70 °C, when compared with the wild type. The simulated protein structure based interaction analysis and molecular dynamics calculation indicate that the thermostability improvement of the mutant K406R-K573R was possibly due to the extra hydrogen bonds, the additional cation-π interactions, and the relatively compact conformation. With the enhanced thermostability, the α-L-rhamnosidase mutant, K406R-K573R, has potentially broadened the r-Rha1 applications in food processing industry.