Industrial enzyme production using a mold is usually carried out in a solid-state medium because a solid culture is superior to a liquid culture in terms of productivity. Alazard and Raimbault (1981) compared amylolytic enzyme production using Aspergillus niger in both liquid and solid cultures, and they concluded that the productivity of amylase in the solid culture was higher than in the liquid culture. Soccol et al. (1994) also showed some advantages of using a solid culture in comparison with a liquid culture for lactic acid and fumaric acid production. Pereira et al. (1993) compared pectinase production using Aspergillus niger in liquid and solid cultures and found that polygalacturonase and pectinesterase productivity is higher in a solid culture than in a liquid culture. However, the liquid culture is believed to allow easy control of culture conditions. Furthermore, the use of a liquid culture does not require that enzymes be extracted from a solid culture.In a previous paper, we attempted to control some specified metal-ion concentrations in a liquid medium and achieved a high level of glucoamylase (GA) production in a so-called metal-ion-regulated liquid medium. We achieved substantial results as the GA activity reached 710 m g-glucose/ml-enzyme solution/ min under 0.7 ppm of zinc ion, and a calcium ion concentration of 75 ppm (Fujio and Morita, 1996). We also characterized the GA properties from Rhizopus sp. A-11 and found that the GA showed a strong raw starchdigesting activity. The strain produced raw corn starch-digesting GA activity of 170 m g-glucose/ml-enzyme solution/min. Purification of the GA was very simple because of the high specific GA activity (based on GA activity) found in a liquid culture. As a result, the GA was purified about 5-fold with a 72.9% yield based on GA activity by using ammonium sulfate precipitation and successive column chromatography (Morita and Fujio, 1997). Although solid wheat bran medium is widely implemented for GA production by using Rhizopus strains (Elegado and Fujio, 1993), we concluded that GA production in the metal-ion-regulated liquid medium is superior to that of the wheat bran solid culture (Fujio and Morita, 1996). In this paper, we selected Rhizopus sp. A-11 for Maximum growth for Rhizopus sp. A-11 was obtained at a zinc ion concentration of 0.7 ppm in a liquid medium. Glucoamylase (GA, EC 3.2.1.3) production in Rhizopus sp. A-11 was maximized at 710 U/ml, at the presence of 75 ppm for calcium and 0.7 ppm of zinc ions in liquid medium. Zinc ion is known as an essential biometal for Rhizopus growth; however, growth was inhibited by the zinc ion concentration, not maximized. Although calcium ion was not necessary to Rhizopus growth, GA production using Rhizopus sp. A-11 was markedly stimulated by calcium ion concentration over 75 ppm in the liquid medium. The GA productivity of the present liquid culture was about 4.4 times higher than that of the solid state culture, based on the unit starch amount in the liquid and solid media carbon source. The charac...

In this study we attempted to control some specified metal-ion concentrations in a liquid medium and found a highly raw starch-digesting glucoamylase (RSDG) producing culture with protease-less activity, which we have called a metal-ion regulated liquid medium. Rhizopus sp. MKU 40 could not grow and produce glucoamylase (GA) in the liquid medium without metal ions (SLS medium). The addition of iron, magnesium, and zinc ions in the liquid medium was essential to growth and GA production of Rhizopus sp. MKU 40. Calcium ions also stimulated its growth and GA production. To summarize, the addition of 75 ppm calcium ions, 2 ppm iron ions , 49 ppm magnesium ions, and 0.7 ppm zinc ion to the SLS liquid medium was the best condition for producing GA and resulted in a high specific activity of GA (21.9 U/mg protein). Specific RSDG activity of the crude enzyme was 5 .7 U/ mg protein, and the ratio of RSDG activity to GA activity was 0 .26. Since the purification was easy, the purification of GA was done in only two steps (ammonium sulfate precipitation and column chromatography on CM-Sephadex C-50). The enzyme was purified about 4 .8-fold based on specific GA activity with an 86.0% yield based on GA activity from culture supernatant. The enzyme was proved to be homogeneous as judged by SDS-PAGE, and it has a molecular mass of about 80 .4 kDa by comparison of its relative mobility on SDS-PAGE with those of standard proteins. For purified enzymes, the ratio of RSDG activity to GA activity was 0.16.