Enhancement and stabilization of desulfurization activity of Rhodococcus erythropolis KA2-5-1 by feeding ethanol and sulfur components

We developed a fed-batch culture system fed with ethanol and restricted amounts of sulfur compounds to enhance and stabilize the desulfurizing activity in bacterial cells. In this system using dibenzothiophene (DBT) as the sole sulfur source, a desulfurizing bacterium Rhodococcus erythropolis KA2-5-1 cultivated with small amounts of sulfur showed stable desulfurizing activity and a low rate of growth. However, the cells cultured with excess amounts of sulfur showed unstable activity and a high growth rate. DBT had disadvantages as a sulfur source for cultivation because it is immiscible with water and toxic to cells. We then investigated water-soluble sulfur compounds for use as the sole sulfur source for the cultivation of R. erythropolis KA2-5-1 with desulfurizing activity, and found 2-aminoethanesulfonic acid to be the most effective. When 2-aminoethanesulfonic acid was used instead of DBT as the sole sulfur source in the fed-batch fermentation system, R. erythropolis KA2-5-1 showed the highest desulfurizing activity, 111 mmol of 2-HBP/kg-cells/h, a high growth rate (mu = 0.37/h) and a final cell concentration of 20.0 g-dry cells/l during 89 h of cultivation. The production levels of the desulfurizing enzymes in the bacterial cells cultivated with DBT or 2-aminoethanesulfonic acid were evaluated by immunoblot analysis with specific antisera, indicating that the same quantity of desulfurizing enzymes was expressed in both cases.     

Production of anti-prion scFv-Fc fusion proteins by recombinant animal cells

We constructed a replication-defective retroviral vector plasmid for the expression of a single-chain antibody fragment (scFv), derived from a chicken anti-human prion protein monoclonal antibody, fused with the Fc region of human IgG1. CHO-K1 and NS-1 cells were transformed with the viral vector pseudotyped with vesicular stomatitis virus G protein (VSV-G), and scFv-Fc producer clones were established. Among the established clones, CHO-2A9 cells produced a large amount of the product with an antibody-like dimerized structure in serum-free culture that facilitated the purification of scFv-Fc. The scFv-Fc specifically recognized the epitope sequence of prion protein in solid-phase enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The injection test into quails revealed that the scFv became more stable in vivo by fusion with the Fc region. The scFv-Fc will be a useful tool for the detection of mammalian prion proteins.     

Characterization of lytic enzyme activities of Lactobacillus gasseri with special reference to autolysis

Lactobacillus gasseri JCM 1130 and JCM 1131(T) exhibited autolytic activity in agar containing autoclaved cells of each strain as substrate. By zymogram analysis of JCM 1131(T), two lytic bands with apparent molecular masses of 54.5 and 35 kDa, were detected. Similarly, JCM 1130 yielded two lytic bands with apparent molecular masses of 35 and 33.5 kDa. In simple buffers as well, JCM 1131(T) suffered a drastic decrease in cell turbidity, but JCM 1130 did not undergo the decrease. The optimal pH for autolysis of JCM 1131(T) was in the range of 6.0-7.0, and the lysis was completely inhibited at pH 4-5. The lysis of JCM 1131(T) was suppressed by NaCl, in a concentration-dependent way. When subjected to UV irradiation or mitomycin C (MMC) treatment, cultures of both strains elicited conspicuous turbidity decrease after 2-4 h of growth, suggesting the occurrence of prophage induction. The 35-kDa lytic band of JCM 1131(T) and the 33.5-kDa protein of JCM 1130 were considerably increased by UV irradiation.     

Heterologous expression and functional analysis of the gene cluster for the biosynthesis of and immunity to the lantibiotic, nukacin ISK-1

Nukacin ISK-1 is a lantibiotic produced by Staphylococcus warneri ISK-1. The gene cluster of nukacin ISK-1 consists of at least nukAMTFEG, ORF1 and ORF7. In this study, we demonstrated the heterologous production of nukacin ISK-1 in Lactococcus lactis by the artificial polycistronic expression of nukAMTFEG-ORF7 under the control of the nisin-controlled expression (NICE) system. Consequently, the recombinant L. lactis showed antimicrobial activity. Mass analysis clarified the presence of nukacin ISK-1 produced in the culture supernatant. These results suggested that the recombinant L. lactis produced nukacin ISK-1 heterologously. Inactivation of nukA, -M or -T resulted in the complete loss of the nukacin ISK-1 production phenotype. This finding suggested that nukAMT are indispensably associated with the biosynthesis of nukacin ISK-1. To our knowledge, this is the first report of the heterologous production of lantibiotic using the NICE system.     

Flux-Line Entanglement as the Mechanism of FLL-Melting Transition in Anisotropic HTSC

The mechanism of the flux-line-lattice (FLL) melting in anisotropic high-T _c superconductors in B is clarified by Monte Carlo simulations of the 3D frustrated XY model. The percentage of entangled flux lines abruptly changes at the melting temperature T _m , while no sharp change can be found in the number and size distribution of vortex loops around T _m . Therefore, the origin of this melting transition is the entanglement of flux lines. Scaling behaviors of the melting temperature T _m are consistent with this one-dimensional character of the entanglement mechanism of the FLL melting. That is, T _m does not depend on the system size in the ab plane, while it is scaled by the system size along the c axis, L _c , as T _m(L _c ) – T _m() L _c ^-d , d = 1.