Glutamate decarboxylase (GAD) [EC 4.1.1.15] was purified from a cell-free extract of Lactobacillus brevis IFO 12005 by chromatographies on Sephadex G-100, DEAE-Sepharose CL-6B, and Mono Q. About 9 mg of purified GAD was obtained from 90.2 g of wet cells. The purified preparation showed a single protein band on SDS-PAGE. The molecular weights of purified GAD by SDS-PAGE and gel filtration on Superdex 200 were 60,000 and 120,000, respectively, indicating that GAD from L. brevis exists as a dimer. The N-terminal amino acid sequence of the purified GAD was NH2-Met-Asn-Lys-Asn-Asp-Gln-Glu-Gln-Thr-. The optimum pH and temperature of GAD were at pH 4.2 and at 30 degrees C. The GAD activity was increased by the addition of sulfate ions in a dose-dependent manner. The order of effects was as follows: ammonium sulfate > sodium sulfate > magnesium sulfate, indicating that the increase of hydrophobic interaction between subunits causes the increase of GAD activity. The purified GAD reacted only with L-glutamic acid as a substrate and the K(m), kcat, and kcat/K(m) values were 9.3 mM, 6.5 S-1, and 7 x 10(2) M-1 S-1, respectively.

In this study, the glutamate decarboxylase (GAD) gene from Lactobacillus brevis IFO12005 (Biosci. Biotechnol. Biochem., 61, 1168-1171 (1997)), was cloned and expressed. The deduced amino acid sequence showed 99.6% and 53.1% identity with GAD of L. brevis ATCC367 and L. lactis respectively. The His-tagged recombinant GAD showed an optimum pH of 4.5-5.0, and 54 kDa on SDS-PAGE. The GAD activity and stability was significantly dependent on the ammonium sulfate concentration, as observed in authentic GAD. Gel filtration showed that the inactive form of the GAD was a dimer. In contrast, the ammonium sulfate-activated form was a tetramer. CD spectral analyses at pH 5.5 revealed that the structures of the tetramer and the dimer were similar. Treatment of the GAD with high concentrations of ammonium sulfate and subsequent dilution with sodium glutamate was essential for tetramer formation and its activation. Thus the biochemical properties of the GAD from L. brevis IFO12005 were significantly different from those from other sources.

A cationic Au(I) complex catalyzed the cycloisomerization of aromatic enynes that possess a substituent on their alkyne terminus. Cyclization of the 6-endo-dig type proceeded dominantly to give 1,3-di-and 1,2,3-trisubstituted naphthalenes.

Triynes having a phenylene‐bridged 1,5‐diyne moiety were transformed into substituted tetraphenylenes by the title sequence. A cationic Rh–ligand species catalyzed this highly enantioselective reaction. This protocol is a new and easy approach to the construction of the tetraphenylene skeleton and enables an efficient asymmetric synthesis (see scheme; R=H; Z=NTs, C(CO2Me)2, O).