|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1990 : ¥100,000 (Direct Cost : ¥100,000)
Fiscal Year 1989 : ¥1,700,000 (Direct Cost : ¥1,700,000)
1. The leavening bacterium Enterobacter cloacae GAO (hereafter referred to as GAO) produced H_2 and CO_2 gas when the GAO was cultured with G-medium which contained glucose and asparagine. The amount of gas increased adding at 0.1% casaminoacid for G-medium produced roughly 2.5-fold and H_2/CO_2 ratio was 0.60.
2. The formation of H_2 gas was accounted for the GAO growth with 0.1% casaminoacid G-media supplemented with 0.5% citric acid, alhpa -ketoglutaric acid, succinic acid, fumaric acid, malic acid and pyruvic acid respectively. The GAO produced total gas, in order of its abundace, fumaric acid, pyruvic acid, alpha -ketoglutaric acid, citric acid, malic acid and succinic acid. The ratio of H_2 to total gas were showen that alpha -ketoglutaric aicd was 85.1%, citric acid was 82.1% and succinic acid was 81.5%.
3. Two- beta -galactosidaese were purified from the cell extract of the GAO by ammonium sulfate fractionation, Sephacryl S-200 gel filtration, DEAE-Toyopearl column chromatography, affinity chromatography on 1-thio- beta -galactopyranoside-Agarose and mono-Q column chromatography. The enzuyme preparations homogeneous on polyacrylamide electrophresis. The enzyme was most active at pH 7.2 and 37^ﾟ C. The enzyme was stable between pH 6.0 to 7.0 at 30 min incubation at 37^ﾟ C. The molecular weights of the enzyme was estimated to be 220,000 by gel filtration on Sepharose CL-6B column, and 120,000 by SDS polyacrylamide gelelectrophoresis. This indicated that the enzymes might be of 2 subunits. The isoelectric point of the enzymes was pH 4.5. Bivalent metals with Mg, Zn, Mn, Co, Ni are strong activators when assayed at 1mM concentration. The beta -galactosidase from E. Cloacae GAO had high content of glycine and serine compared with Aerobacter cloacae.
4. In an attempt to isolation from the starter prepared with potato filling, we obtained four isolates which were rod shape, oxydase positive and Gram negative bacteria.