Mikio Takaiwa

Purification and characterization of an alkaline amylopullulanase with both α-1,4 and α-1,6 hydrolytic activity from alkalophilic Bacillus sp. KSM-1378

Abstract The novel alkaline amylopullulanase produced by alkalophilic Bacillus sp. KSM-1378 was purified to an electrophoretically homogeneous state from culture medium. The purified enzyme was a glycoprotein with an apparent molecular mass of about 210 kDa and an isoelectric point of pH 4.8. The N-terminal amino acid sequence was Glu-Thr-Gly-Asp-Lys-Arg-Ile-Glu-Phe-Ser-Tyr-Glu-Arg-Pro and showed no homology to the N-terminal regions of other amylopullulanases reported to date. The enzyme was able to attack specifically the α-1,6 linkages in pullula.n to generate maltotriose as the major end product, as well as the α-1,4 linkages in amylose, amylopectin and glycogen to generate various oligosaccharides. The pH and temperature optima for the pullulanase and α-amylase activities were pH 9.5 and 50°C and pH 8.5 and 50°C respectively. Both activities were strongly inhibited by well characterized inhibitors, such as diethyl pyrocarbonate and N-bromosuccinimide. The pullulanase activity was specifically inactivated by Hg2+ ions, α-cyclodextrin and β-cyclodextrin while the amylase activity was strongly inhibited by EDTA and EGTA, although inhibition could be reversed by Ca2+ ions. It is suggested that the single alkaline amylopullulanase protein has two different active sites, one for the cleavage of α-1,4-linked substrates and one for the cleavage of α-1,6-linked substrates.

Enhanced Production of Extracellular Enzymes by Mutants of Bacillus That Have Acquired Resistance to Vancomycin and Ristocetin

A new mutagenic method for increasing the productivity of extracellular enzymes, such as cellulases, proteases, and amylases, has been developed, using vancomycin-and ristocetin-resistance as indicators. Among the mutants of Bacillus that were resistant to these atibiotics, strains with improved productivity of the extracellular enzymes were found at high frequency. The use of these antibiotics seems to provide a very effective method for the improvement of industrially important strains of Bacillus, regardless of the specific strains used and the particular extracellular enzymes produced by them.

A repeat-batch membrane bioreactor with a phase inversion for the desaturation of isopropyl palmitate by a mutant Rhodococcus strain

A repeat-batch membrane bioreactor was constructed for the novel bioconversion of isopropyl hexadecanoate to isopropyl cis-6-hexadecenoate by a Rhodococcus mutant. The addition of glutamate, thiamine, and MgSO(4) was very effective in improving not only the rate and yield of the bioconversion but also the maintenance of desaturation activity during cell recycling. An oil-in-water (O/W) type emulsion of the reaction medium was inverted to a water-in-oil (W/O) type by discharging the water phase from the reaction mixture. The continuous oil phase containing the product could effectively be recovered through a hydrophobic hollow-fiber module. By decreasing the oil-to-water ratio upon addition of fresh medium, the medium was spontaneously inverted again to an O/W type emulsion to proceed with the next conversion. The batch reaction coupled with the phase inversion could be repeated more than 13 times for over about 300 h operation. Finally, a highly purified product was obtained with high yield by the urea adduct procedure.