Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases

We cloned lipG, which encoded a lipolytic enzyme, from a Korean tidal flat metagenomic library. LipG was related to six putative lipases previously identified only in bacterial genome sequences. These enzymes comprise a new family. We partially characterized LipG, providing the first experimental data for a member of this family.     

Immobilization of Horseradish Peroxidase on Multi-Wall Carbon Nanotubes and its Electrochemical Properties

Horseradish peroxidase (HRP) was immobilized on carboxylated multi-wall carbon nanotubes in the presence of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The immobilized HRP maintained its oxidative activity for guaiacol over a broad range of pH values (4–9). An electrode of graphite rod, 6 mm diam. was fabricated using the immobilized HRP. Cyclic voltammetry of the enzyme electrode confirmed electron transfer between the immobilized HRP and the electrode in the presence of H₂O₂ but without an added mediator or a reducing substrate.     

Optimal Production of Poly-γ-glutamic Acid by Metabolically Engineered Escherichia coli

Metabolically-engineered Escherichia coli strains were developed by cloning poly-γ-glutamic acid (γ-PGA) biosynthesis genes, consisting of pgsB, pgsC and pgsA, from Bacillus subtilis The metabolic and regulatory pathways of γ-PGA biosynthesis in E. coli were analyzed by DNA microarray. The inducible trc promoter and a constitutive promoter (P_HCE) derived from the d-amino acid aminotransferase (D-AAT) gene of Geobacillus toebii were employed. The constitutive HCE promoter was more efficient than inducible trc promoter for the expression of γ-PGA biosynthesis genes. DNA microarray analysis showed that the expression levels of several NtrC family genes, glnA, glnK, glnG, yhdX, yhdY, yhdZ, amtB, nac, argT and cbl were up-regulated and sucA, B, C, D genes were down-regulated. When (NH₄)₂SO₄ was added at 40 g/l into the feeding solution, the final γ-PGA concentration reached 3.7 g/l in the fed-batch culture of recombinant E. coli/pCOpgs.     

Extracellular production of a glycolipid biosurfactant, mannosylerythritol lipid, by Candida sp. SY16 using fed-batch fermentation

Candida sp. strain SY16 produces a glycolipid-type biosurfactant, mannosylerythritol lipid (MEL-SY16), which can reduce the surface tension of a culture broth from 72 to 30 dyne cm⁻¹ and highly emulsify hydrocarbons when cultured in soybean-oil-containing media. As such, laboratory-scale fermentation for MEL-SY16 production was performed using optimized conditions. In batch fermentation, MEL-SY16 was mainly produced during the stationary phase of growth, and the concentration of MEL-SY16 reached 37 g l⁻¹ after 200 h. The effect of pH control on the production of MEL-SY16 was also examined in batch fermentation. The highest production yield of MEL-SY16 was when the pH was controlled at 4.0, and the production was significantly improved compared to batch fermentation without pH control. In fed-batch fermentation, glucose and soybean oil (1:1, w/w) were used in combination as the initial carbon sources for cell growth, and soybean oil was used as the feeding carbon source during the MEL production phase. The feeding of soybean oil resulted in the disappearance of any foam and a sharp increase in the MEL production until 200 h, at which point the concentration of MEL-SY16 was 95 g l⁻¹. Among the investigated culture systems, the highest MEL-SY16 production and volumetric production rate were achieved with fed-batch fermentation.     

A membrane-associated metalloprotease of Taenia solium metacestode structurally related to the FACE-1/Ste24p protease family

The CaaX proteases are intimately involved in the post-translational modification of prenylated proteins and play a critical role in the activation/stabilization of membrane-bound or secreted molecules constituting the CAAX protein family. In this study, we have isolated a full-length cDNA putatively encoding a type I CaaX protease of the Taenia solium metacestode (TsM), which an agent causative of human neurocysticercosis. The cDNA, designated TsSte24p, comprised 1,505 bp and coded for an open reading frame of 472 amino acids with predicted Mr 54.5 kDa. This monoexonic TsSte24p gene existed as a single copy within the TsM genome and constantly expressed in the parasite from metacestode to adult stages. The TsSte24p exhibited the typical CaaX protease topology, including seven transmembrane domains and a metalloprotease segment with a zinc-binding motif. It shared a significant degree of sequence identity with the type I CaaX proteases such as Saccharomyces cerevisiae Ste24p and Caenorhabditis elegans CeFACE-1. A comparative phylogenetic analysis demonstrated that this protein family is tightly conserved across taxa, from bacteria to mammals. The bacterially expressed recombinant TsSte24p showed proteolytic activity, with an optimal pH of 7.5. The enzyme activity was significantly inhibited by EDTA. Its activity was increased in the presence of low concentrations of the Zn2+(0.001-0.01 mM); but was reversibly down-regulated at high doses (over 0.1 mM). The native TsSte24p appeared to function as a homodimer, the subunits of which were linked to each other via covalent disulfide bond. The protein was localized in the bladder wall and scolex with differential patterns of distribution. Our results indicated that TsSte24p is a zinc-dependent metalloprotease, which belongs to the FACE-1/Ste24p protease family.     

Gelastatins and their hydroxamates as dual functional inhibitors for TNF-alpha converting enzyme and matrix metalloproteinases: synthesis, biological evaluation, and mechanism studies

The hydroxamic acid analogues (2) of the natural product gelastatins (1) were prepared by 1 step conversion reaction. The synthetic analogues (2) showed potent enzymatic inhibitory activities against MMP-2, MMP-9, and TACE IC50's of 6, 23, and 28 nM, respectively. In addition, 2 were able to inhibit TNF-alpha production effectively in mice as well as in a macrophage cell line, RAW 264.7. The protective effect of 2 also was examined on LPS-induced acute septic shock model. The mechanism of TNF-alpha inhibition was examined by RT-PCR and Western blot analyses. The relation of TACE and alpha-secretase was examined using cellular alpha-secretase assays on IMR-32 and SH-SY5Y cell lines. The docking mode of 2 with the catalytic domain of TACE was illustrated to analyze the binding mode for the further analogue design.