Production of a thermostable metal-tolerant laccase from Trametes versicolor and its application in dye decolorization

Production of laccase using a submerged culture of Trametes versicolor sdu-4 was optimized using a central composite design of the Response Surface Methodology. Optimized conditions gave a laccase yield of 4,213 U/L which was approximately three times of that in basal medium. The laccase was purified to homogeneity using a three-step process. The overall yield of the purification was 58%, with a purification fold of 11.4 and a specific activity of 1320.7 U/mg protein. The molecular mass of the laccase was 60 kDa. The optimum pH values of the enzyme were 2.2, 3.7, and 7 for the oxidations of ABTS, DMP, and syringaldazine, respectively. The enzyme had adaptability to a broad pH range and high temperature and wsa stable at pH 3.0 ∼ 10.0. The half-life of this laccase at 70°C was 2.2 h. Methyl red, 2-bromophenol, and 4-bromophenol were oxidized by the purified laccase in the absence of mediators. Purified laccase was effective in the decolorization of several dyes and was not inhibited by Cu²⁺, Mn²⁺, Zn²⁺, Na⁺, K⁺, Mg²⁺, Ba²⁺, and Ca²⁺ at 5 mM. These excellent characteristics made it a highly attractive candidate for industrial use.     

Improved eicosapentaenoic acid production in Pythium splendens RBB-5 based on metabolic regulation analysis

Applied Microbiology and Biotechnology - Eicosapentaenoic acid (EPA) is an essential polyunsaturated fatty acid for human beings. At present, the production of commercially available long-chain...     

Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli

Chemerin is a novel chemokine that binds to the G protein-coupled receptor (GPCR) ChemR23, also known as chemokine-like receptor 1 (CMKLR1). It is secreted as a precursor and executes pro-inflammatory functions when the last six amino acids are removed from its C-terminus by serine proteases. After maturation, Chemerin attracts dendritic cells and macrophages through binding to ChemR23. We report a new method for expression and purification of mature recombinant human Chemerin (rhChemerin) using a prokaryotic system. After being expressed in bacteria, rhChemerin in inclusion bodies was denatured using 6 M guanidine chloride. Soluble rhChemerin was prepared by the protein-specific renaturation solution under defined conditions. It was subsequently purified using ion-exchange columns to more than 95% purity with endotoxin level <1.0 EU/μg. We further demonstrated its biological activities for attracting migration of human dendritic cells and murine macrophages in vitro using established chemotaxis assays.     

Towards precise classification of cancers based on robust gene functional expression profiles

Background

Despite the continuous production of genome sequence for a number of organisms, reliable, comprehensive, and cost effective gene prediction remains problematic. This is particularly true for genomes for which there is not a large collection of known gene sequences, such as the recently published chicken genome. We used the chicken sequence to test comparative and homology-based gene-finding methods followed by experimental validation as an effective genome annotation method.

Results

We performed experimental evaluation by RT-PCR of three different computational gene finders, Ensembl, SGP2 and TWINSCAN, applied to the chicken genome. A Venn diagram was computed and each component of it was evaluated. The results showed that de novo comparative methods can identify up to about 700 chicken genes with no previous evidence of expression, and can correctly extend about 40% of homology-based predictions at the 5' end.

Conclusions

De novo comparative gene prediction followed by experimental verification is effective at enhancing the annotation of the newly sequenced genomes provided by standard homology-based methods.     

Coadministration of interleukin 2 plasmid DNA with combined DNA vaccines significantly enhances the protective efficacy against Mycobacterium tuberculosis

Coadministration of interleukin 2(IL-2) plasmid DNA with combined DNA vaccines enhanced Th1-type cellular responses by producing higher amounts of IFN-gamma with a higher ratio of antigen-specific IgG2a/IgG1. The IFN-gamma specific for Ag85B, MPT64, and MPT83 in this group was 415, 267, and 255 U/ml, respectively, and was 1.6-, 1.8-, and 2.5-fold higher than that of the same vaccine without adding IL-2. The IgG2a/IgG1 ratio for Ag85B, MPT64, and MPT83 was 4, 8, and 4, respectively, upon addition of the genetic adjuvant in the DNA vaccine, which was four times higher for every antigen when IL-2 was not included. Fluorescence activated cell sorter (FACS) analysis showed that, in the presence of IL-2, CD8+ and CD4+ T cells increased significantly, whereas in the absence of the genetic adjuvant, only a mild increase was observed for CD8+ T cells compared to the vector DNA-treated group. Bacterial CFU was reduced to less than 1/100 in the lung and to about 1/10 in the spleen relative to the same combined DNA vaccine without IL-2. The lungs of this group of mice showed much less damage due to an influx of epithelioid macrophages and less lymphocytes. RT-PCR showed that antigen genes could be detected in more organs and for a longer period of time when treated with combined DNA vaccine formulated in IL-2. We suggest that IL-2 enhanced the immunigencity and protective efficacy in immunized mice by improving the Th1-type response and also by prolonging the antigen gene expression in different organs.     

Methyl lucidenate F isolated from the ethanol-soluble-acidic components of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> is a novel tyrosinase inhibitor

Tyrosinase is a key enzyme in the biosynthesis of melanin, and the use of inhibitors against tyrosinase can prevent hyperpigmentation by inhibiting enzymatic oxidation. However, the current use of tyrosine inhibitors is limited by their low activities and high toxicities. The aim of the present research was to develop novel whitening agents, or tyrosinase-targeted medicine, from a submerged culture of the fungus Ganoderma lucidum. Methyl lucidenate F was isolated from the ethanol-soluble-acidic components (ESACs) of G. lucidum, with the structure of ESACs elucidated via UV, LC-MS, and ¹³C-NMR spectral analysis. The tyrosinase inhibitory activity was measured using catechol as a substrate. Methyl lucidenate F displayed uncompetitive inhibition of the potato tyrosinase activity, for which Lineweaver-Burk plots revealed a maximum reaction rate (V _max) of 0.4367/min, Michaelis constant (K _m) of 6.765 mM and uncompetitive inhibition constant (K _i) of 19.22 μM. Meanwhile, methyl lucidenate F (tetra cyclic triterpenoid) exhibited high tyrosinase inhibitory activity, with an IC₅₀ of 32.23 μM. These results suggest that methyl lucidenate F may serve as a potential candidate for skin-whitening agents.     

A transversely isotropic viscoelastic constitutive equation for brainstem undergoing finite deformation

The objective of this study was to define the constitutive response of brainstem undergoing finite shear deformation. Brainstem was characterized as a transversely isotropic viscoelastic material and the material model was formulated for numerical implementation. Model parameters were fit to shear data obtained in porcine brainstem specimens undergoing finite shear deformation in three directions: parallel, perpendicular, and cross sectional to axonal fiber orientation and determined using a combined approach of finite element analysis (FEA) and a genetic algorithm (GA) optimizing method. The average initial shear modulus of brainstem matrix of 4-week old pigs was 12.7 Pa, and therefore the brainstem offers little resistance to large shear deformations in the parallel or perpendicular directions, due to the dominant contribution of the matrix in these directions. The fiber reinforcement stiffness was 121.2 Pa, indicating that brainstem is anisotropic and that axonal fibers have an important role in the cross-sectional direction. The first two leading relative shear relaxation moduli were 0.8973 and 0.0741, respectively, with corresponding characteristic times of 0.0047 and 1.4538 s, respectively, implying rapid relaxation of shear stresses. The developed material model and parameter estimation technique are likely to find broad applications in neural and orthopaedic tissues.