Kazuhiro Yamade

Continuous alcohol production from starchy materials with a novel immobilized cell/enzyme bioreactor

Abstract In a novel bioreactor system that was packed with both Al alginate biocatalyst A (entrapment of adsorbed-glucoamylase on γ-alumina) and biocatalyst B (entrapment of a mixture of different strains of yeast cells), 60–95 kg/m 3 of alcohol solution were continuously produced through parallel operation of saccharifying and fermenting liquefied starchy materials of potato and grains without contamination. This continuous process was anaerobically performed at a pH of 2.8–3.2 and a temperature of 30°C.

Purification and Properties of an Ice-Nucleating Protein from an Ice-Nucleating Bacterium, Pantoea ananatis KUIN-3

An ice-nucleating protein (INP) from the extracellular ice-nucleating matter (EIM) of Pantoea ananatis (Erwinia uredovora) KUIN-3 was purified and characterized. The EIM produced by the strain KUIN-3 was purified by ultrafiltration, sucrose density-gradient ultracentrifugation and gel filtration. The INP was purified using of column chromatography on hydroxyapatite and Superdex 200 in the nondenaturing detergent of 0.1% (w/v) Triton X-100. The purified INP was composed of one subunit of 117 kDa according to SDS-PAGE. It has become apparent that the INP was the ice-nucleating lipoglycoprotein based on the reaction of carbohydrate stain and lipid stain with the INP. It was inhibited by p-mercuribenzoate and N-bromosuccinimide. The activity of the INP gradually decreased from 65 degrees C. The pH stability was held between pH 7.0 and pH 11.0. The INP had a lower ice-nucleating temperature below pH 6.0. It has become apparent that the INP consisted of the class C structure in the EIM based on its freezing difference spectrum in D2O versus H2O.

Production of D-Glutamate from L-Glutamate with Glutamate Racemase and L-Glutamate Oxidase

We studied production of D-glutamate from L-glutamate using a bioreactor consisting of two columns of sequentially connected immobilized glutamate racemase (EC 5.1.1.3, from Bacillus subtilis IFO 3336) and L-glutamate oxidase (EC 1.4.3.11, from Streptomyces sp. X119-6): L-glutamate was racemized by the glutamate racemase column, and then L-glutamate was oxidized by the L-glutamate oxidase column. Consequently only D-glutamate remained, and was easily separated from the α-ketoglutarate formed by anion-exchange chromatography. Both enzymes were highly stabilized by immobilization. The pH and temperature optima of immobilized glutamate racemase (pH 8, 40°C) were similar to those of immobilized L-glutamate oxidase (pH 7, 50°C). Accordingly, we connected the two columns tandemly to do both enzyme reactions under the same conditions. Actually 4.5 μmol of D-glutamate was produced and isolated from 10 μmol of L-glutamate, about 90% of the theoretical yield.

A novel process of ethanol production accompanied by extraction of sugar in cane chips

Abstract By connecting a rhomboid unit for fermentation and an exchangeable tubular unit for extraction, a novel bioreactor was designed to produce high concentration of ethanol solution from non-peeled sugar cane chips by means of a cyclic system for exchanging old chips with new ones. The rhomboid and tubular units were packed with 1.0–1.5 mm biocatalyst entrapped yeast-cells with Al alginateand cane chips of 2.0–3.0 mm in width, respectively. The volume ratio of the two units was 1.0. At the start of the first cycle, 2 g/ l of Al 2 (SO 4 ) 3 14–18 H 2 O solution was added to the bioreactor, where the ratio of the solution to the working volume was 0.85. Both sugar extraction and fermentation were anaerobically performed at pH 2.5 and a temperature of 30°C by circulating the solution through the two units. After each cycle, the tubular unit was exchanged for a new unit packed with new chips. The combined solution of free ethanol and the ethanol obtained by pressing the old chips was re-used as the circulating solution. When the volume ratio of the biocatalyst to the total working volume was 0.1 and the amount of dried cane chips in the tubular unit was 200 g/ l , 16% (w/v) ethanol was produced after 7 cycles. Each cycle was established at about 20 h. The number of free cells in the circulating solution was only 2×10 7 /ml after 7 cycles.