Yamane Tsuneo

l-serine production from methanol and glycine with an immobilized methylotroph

Abstract l -Serine production from methanol and glycine was attempted using immobilized resting cells of a methylotroph, Protomonas extorquens NR 1, under automatically controlled conditions. A Ca-alginate system was selected. The conditions for l -serine formation were optimized at 30°C. A concentration of glycine 100 g· l −1 which was the optimum concentration for l -serine production by free resting cells was used in the reaction mixture. The optimum concentrations of methanol and dissolved oxygen were 20 g· l −1 and 5 ppm, respectively. Under the optimum conditions, 11.3 g· l −1 of l -serine was produced within 36 h. The selectivities (mole of l -serine/mole of substrate consumed) of l -serine from methanol and glycine were 4.5% and 95.1%, respectively. The size of gel beads affected the l -serine formation rate. The initial rate of l -serine formation decreased with an increase in the size of beads. However, the l -serine formation rate increased at elevated concentrations of dissolved oxygen, even with large sized beads. This result implies that the oxygen diffusion inside the gel beads limited the l -serine formation rate. The observed effectiveness factor of the immobilized cells could be estimated by the theoretical effectiveness factor of the zero-order reaction with respect to the dissolved oxygen. Repeated use was not feasible without reactivation of the immobilized cells. Reusability was examined by reactivation of the immobilized resting cells in appropriate media for 12 h. The reactivated immobilized resting cells were used again in the next cycle. By this procedure, several cycles of l -serine formation were made possible.

Purification and properties of phosphatidylinositol-specific phospholipase C from Streptomyces antibioticus.

Abstract A novel type of phosphatidylinositol-specific phospholipase C (PI-PLC) was purified from culture supernatant of a strain of Actinomycetales, Streptomyces antibioticus. The purified enzyme showed a single band on native polyacrylamide gel electrophoresis (native PAGE) with a molecular weight of 32 kDa, but showed two polypeptides, named α- and β-peptides, on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weights of 23 kDa and 15 kDa, respectively. From the results of both eletrophoretic analysis and N-terminal amino acid sequencing, it was estimated that the enzyme was composed of α- and β-peptides. The enzyme could hydrolyze phosphatidylinositol, but not any other glycerophospholipids. The enzyme had pH and temperature optima at around 7.0 and 30°C, respectively, and was stable up to 50°C when incubated at pH 8.0 for 30 min. The PI-PLC was strongly activated by SDS, sodium deoxycholate (SDC) and diethyl ether, but not by Triton X-100, and inhibited by cetylpyridinium chloride (CPC). The enzyme was activated a little by Ca2+ and was inhibited completely by a chelating agent such as ethylenediaminetetraacetic acid (EDTA) and glycoletherdiaminetetraacetic acid (EGTA). Their inhibitions were restored by the addition of Ca2+, suggesting that a certain amount of Ca2+ is essential for the enzymatic activity.