Hiroshi Nishise

Production of raw cassava starch-digestive glucoamylase by a 2-deoxyglucose-resistant mutant of Rhizopus sp.

Abstract In order to improve the productivity of raw cassava starch-digestive glucoamylase of Rhizopus sp. MB46 in a liquid culture, a mutant strain, AF-1, which is resistant to 2-deoxyglucose, was derived. The mutant strain produced glucoamylase in the presence of 0.5% glucose though the parent strain did not. With a rice bran liquid medium the productivity was over 2-times that of the wild type strain. A rice bran liquid medium supplemented with β-cyclodextrin was also effective for glucoamylase production. Other maceration enzymes were also produced at a higher level with mutant strain AF-1 than with the wild type strain in a liquid culture as well as in a solid culture. The elution patterns of these enzymes on CM-cellulose column chromatography were principally the same with both strains except for glucoamylase. When 10% of raw cassava starch and cassava waste were digested with the culture filtrate of mutant strain AF-1, glucose was produced in 7% after 60-h incubation and 3.2% after 48-h incubation, respectively.

Production of raw cassava starch-digestive glucoamylase by Rhizopus sp. in liquid culture

Abstract Among about 200 Rhizopus strains isolated in Thailand, Rhizopus sp. MB46 was selected as a producer of raw cassava starch-digestive glucoamylase. Rice bran was effective for the enzyme production in a solid culture as well as wheat bran. Addition of turpentine oil into the rice bran solid culture increased the productivity. Rhizopus sp. MB46 was found to produce glucoamylase in a liquid culture containing 1% rice bran but not in one consisting of 10% raw cassava starch of 2% glucose. The productivity per 1 g solids in the medium in liquid culture was finally improved 6-times by utilization of n-hexane-treated rice bran, supplement of 0.1% meat extract and addition of gauze as a support. The activity was superior to that in turpentine oil-supplemented solid culture.

Glycerol dehydrogenase of a protoplast fusant of Cellulomonas sp. NT3060

Protoplast fusion of the glycerol dehydrogenase producing bacterium, Cellulomonas sp. NT3060, was carried out in the presence of polyethylene glycol 4000. Mutants able to dissimilate glycerol by means of substituting glycerol dehydrogenase for glycerol kinase were derived and used as parental strains. The fusant, F6-9, showed 1.7-fold specific activity (per extracted protein) and 2.7-fold total activity of glycerol dehydrogenase of the parental strains, respectively. These activities were 10- and 18-fold those of the wild-type strain. Glycerol dehydrogenase was purified from the wild-type strain, mutant GP1807 and fusant F6-9. The identity of the enzymes was determined by comparison of enzymatic characteristics, i.e., subunit structure, specific activity, substrate specificity, etc. This was also confirmed immunochemically by double-diffusion and neutralization analyses. The results indicated that the increase in glycerol dehydrogenase activity of the fusant could be attributed to quantitative alteration of the enzyme but not to a qualitative change.