An extracellular exo-maltohexaohydrolase [EC 3.2.1.98], which had been obtained up to now only as a cell-bound enzyme, was obtained in the culture liquor of an Aerobacter aerogenes (Klebsiella pneumoniae) mutant. The mutation was carried out by ultraviolet irradiation to achieve 1% survival. The culture conditions for the production of the extracellular exo-maltohexaohydrolase were investigated. The optimum culture medium consisted of 1.00% tryptone (Difco), 0.50% yeast extract (Difco), 0.28% K₂ HPO₄, 0.10% KH₂ PO₄, 0.50% CH₃ COONH₄, 0.02-0.03% MgSO₄ .7H₂ O and 1.00% starch hydrolyzate (DE 12). The optimum conditions of cultivation and the effects of various additives on enzyme production were also determined

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An extracellular exo-maltohexaohydrolase [EC 3.2.1.98] was obtained from the culture medium of a mutant from Aerobacter aerogenes (Klebsiella pneumoniae). This enzyme was purified by ammonium sulfate precipitation, DEAE-cellulose column chromatography and Sephadex G-100 gel filtration to 1, 100 fold of the activity of the original cuture liquor. It gave a single band on disc gel electrophoresis, and the molecular weight by SDS disc gel electrophoresis and Sephadex G-100 gel filtration were 65, 000 and 48, 000, respectively. In isoelectric focusing, the extracellular enzyme showed three bands, at isoelectric points (p1) of 7. 20, 7.60 and 7.80. This amylase showed maximum activity at 52°C and pH 7.0. The pH-stability range was relatively wide, the enzyme retaining more than 80% of its initial activity in the range of pH 5.0 to 10.0. It was stable below 50°C, and thermostability was improved by the addition of calcium ion. The relative reaction rates of the enzyme on various substrates were also determined. The enzyme produced maltohexaose from starch, amylose and amylopectin by exo-attack, but did not act on a-, B- or r-cyclodextrin, pullulan and maltose. The enzyme acted on Q-limit dextrins of amylopectin and glycogen to form branched oligosaccharides. The characteristics of the enzyme were similar to those of the cell-bound enzyme reported in the previous paper.

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The original method for the preparation of inclusionn complex of various polar organic compounds in the granule of polysaccharides was improved. (1) The complex has been prepared with the system composed of anhydrous polysaccharide, methanol and guest compound, and in this system the inclusion was always inhibited by the moisture. On the other hand, it was found that on the system in which ethanol was used as a solvent, the adequate amount of moisture promoted the inclusion and that the moisture in the interior of polysaccharide granule was more effective than that in the solvent. (2) In the original method the air-drying procedure was initiated under the condition in which large excess of solvent was present. On the basis of the elucidation of initiation conditions of the actual inclusion, the amount of solvent was reduced to 1/3 its original one. The improvement offered saving solvent, reducing air-drying period and increasing included amount of guests with lower boiling point. (3) For the preparation of complex with high content of guest the air-drying procedure was essential. (4) At least, with regard to benzaldehyde, the considerable amount (60 mg/g) could be included ever with water as a carrier solvent.

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To elucidate the effect of the conditions of liquefaction on the formation of maltulose, a maltose solution was treated under the conditions of varying temperature, duration and pH. The amounts of maltulose formed under these conditions were determined by the anthrone method after separation by paper chromatography with a solvent system of ethyl acetate/acetic acid/water (3:3: 1, v/v). More maltulose was produced at higher pH's, higher temperature and longer durations in the treatment. Maltulose was formed even at a low pH, if maltose was treated at a high temperature for a long period of time. The rate of maltulose formation was approximately doubled on raising a temperature of 10°C. A pronounced effect of pH was observed at higher than 6.5. The formation of maltulose during the enzymatic liquefaction of starch was measured on both laboratory and factory scales. Starch was liquefied first at 97-98°C for 120 min, autoclaved at 135°C for 15 min, and then liquefied again at 97-98°C for 30 min. The amount of maltulose formed under these conditions at pH 6.5 was 2.8 times as much as at pH 5.5. At a constant pH of 6. 5, the amount of maltulose formed at 97-98°C was 2.4 times as much as at 90°C.

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Contamination of maltulose in high-fructose syrup is undesirable. To reduce this contamination in the final product, the conditions of enzymatic liquefaction of starch should be controlled to a minimum formation of this oligosaccharide. For selection of the suitable conditions, a reliable analytical method of maltulose and other oligosaccharides is necessary. We have tested paper chromatography to separate these oligosaccharides in two samples of the isomerized sugar syrup, which were obtained commercially and produced by treating maltose with strongly basic anion-exchange resin. Maltose (R_g =0.68) could be separated from maltulose (R_g =0.79) by using a solvent system of ethyl acetate/acetic acid/water (3 : 3 : 1, v/v), while isomaltose (R_g =0.64) was hardly separable from maltose under these conditions. By using a solvent system of n-butanol/pyridine/water (6:4:3, v/v), isomaltose (R_g =0.64) could be separated from maltose (R_g =0.73) and maltulose (R_g =0.73), both of which were unseparable each other. Therefore, a combination of the two paper chromatographic systems gives the complete separation of maltose, maltulose and isomaltose. The amounts of each sugar was measured by the anthrone method after extraction of appropriate area of paper with water. The complete separation of these oligosaccharides is also possible on either a liquid chromatography or a high-performance liquid chromatography. These methods are, however, impractical for our purpose, because it takes too long (11 hr) for the separation with the former apparatus and the column of the latter is easily deteriorated.

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Potato starch production in Japan is exclusively limited to Hokkaido district, where 30 starch factories produce 250, 000 tons of starch a year. Up to the present, waste water (WW), except flume water, is mainly applied to arable land or treated with long term airation. In this report, agricultural use and some effects of WW are reviewed. 1. The irrigation of WW has been practiced to grass land from 1969. The spray irrigation of WW deepened the green color of grasses and improved the growth and yield of orchard grass and timothy in the following spring. However, a large quantity of irrigation caused a reduction in the population of red clover in the mixture of grasses, or orchard grass in the mixture of ladino clover. The grasses fell down after heading time due to their thick growth, and were high in water content, giving rise to the difficulties in making hay. In such case, T-N, NO3-N, P and K contents of grasses were high, while Ca and Mg contents were low. A proper quantity of spray irrigation of WW, containing about 400 ppm of N and 800 ppm of K2O, was concluded to be 30 mm to mixed grassland and 60 mm to gramineous grassland a year, respectively. 2. In an optimal amount of WW, the ratio of removal to effluence was about 9:1 in N or K, and the differences between plots of fertilizers or WW were very little. When a large amount of WW was applied, the effluence of N and Ca increased temporarily. The K accumulated markedly in the soil. But, the growth and yield of the upland crops were improved. The measurement of three-element in the plots applied with WW suggested the disuse of K fertilizer, and reduction of N and P fertilizers. The amount of WW applied should be decided first by the optimal amount of K for each crop and then, N and P should be supplemented as fertilizer in consideration of the application standard of fertilizers and their utilization efficiencies of WW. The application of WW with wheat straw are considered to bring about synergistic effects, but that with beet-top (leaf and crown root) may evoke the surplus of fertilizer. 3. The WW transportation is carried out by a pipeline-sprinkler or a motor-sprinkler. The former is effective to treat a large amount of dilute WW, but it is inadequate for its application to beet field after harvest due to excess nutrients or short period before winter. Therefore, the pipeline-sprinkling method needs to cover a large area and costs a great deal. The latter is effective for the application of only fruit water to wheat field or potato field after early harvest. It makes possible to use widely, but needs the airation system for this dilute WW.

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