The conventional Sake brewing requires a large amount of heat especially in the rice steaming process. We have attempted to make Sake from unsteamed white rice (75% polishing yield) for the economy of energy. We made Sake according to the conventional Sake brewing method, except for the omission of rice steaming and the use of commercial enzymes for saccharification instead of rice koji. Activity of glucoamylase used was 5 to 10 times as high as that used for the conventional procedure. The resulting Sake contained more than 18% of alcohol after 15 days' fermentation, and had an excellent quality and was rich in variety. Proteins in raw rice grains were easily decomposed by proteolitic enzymes and more amino acids were produced than from cooked rice grains. The process of fermentation with raw rice grains was different from that with steamed rice in the following points: 1) At the early stage of moromi fermentation, reducing sugars were scarcely accumulated . 2) Amino acids contents in the final products (Sake) differed from each other depending on the activities of acid carboxypeptidase in the enzyme preparation used. 3) After cessation of the alcoholic fermentation, the amount of reducing sugars and amino acids in the moromi linearly increased. By controlling the water content of raw rice grains or wheat bran at the adequate level, Rhizopus and Aspergillus could grow very actively and produced various enzymes at the same level as steamed rice koji and wheat bran koji.

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In Brazil self-sufficiency in petroleum is extremely low and the main cause of the foreign exchange shortage is due to the import of oil. National alcohol project started in 1975 and production of about 10 million kl alcohol perannum will be achieved by 1985 with material of sugar cane etc. Also Brazil has made a lot of efforts steadily so as to use alcohol as fuel directly or to mix it into gasoline. Kansai Chemical Engineering Co., Ltd. established the joint corporation, producing 120 kl per day alcohol from sugar cane as a raw material, which has been operating successfully at the state of Parana in Brazil. Alcohol has drawn attention as one of substitute for petroleum in future. In several countries the production of alcohol from various raw materials has been widely trying.

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Starch granules of roots and tubers were, in general, more resistant to the action of amylases than those of cereals. However, taro starch granules were highly susceptible and starch granules of banana, high-amylose (amylose-extender, ae) maize and gingko were rather resistant to the action of pancreatin compared with those of normal maize. The susceptibility of starch granules to degradation by amylases depends not only on the source of the starch but also of the amylase. Three different types of α-amylase, namely, purified α-amylase of Streptonnyces precox NA-237 (SE-1), porcine pancreatic α-amylase (PPA) and crystalline α-amylase of Bacillus subtilis(BLA), degraded starch granules of sugary-2 (su₂), waxy (wx), normal and ae maize in decreasing order. However, the amounts of enzymes necessary for 10% hydrolysis of starch granules in 1 hr were SE-1: PPA : BLA =1:5:6 for sue, 1:30:930 for wx, 1:28:1340 for normal and 1:110:1046 for ae, respectively. For starch-granule susceptibility to glucoamylase of Rhizopus amagasakiens (crude preparation); 1) Starch granules of waxy-type cereals were, in general, more susceptible than those of their normal counterparts. This was shown for rice, barley, proso millet, maize and job's tears starch granules. 2) Fine starch granules of taro and grain amaranths were highly susceptible. Among the same species, smaller starch granules were more susceptible than larger ones. This was shown for potato and barley starch granules. 3) Negative correlation between starchgranule susceptibility to glucoamylase and absorptivity at 680 nm of iodine complex ("blue value") was obtained for various starches. Exceptions were dull(du) and sue maize starches. Also results indicated that 1) in general, X-ray diffractograms of starch granules relatively susceptible to amylases were A type, those of starch granules insusceptible to amylases were B type and those of starch granules intermediately susceptible to amylases were C type. 2) Photopastegrams showed two-step increase of transmittance for starch granules relatively susceptible to amylases and one-step increase of transmittance for starch granules insusceptible to amylases. 3) By scanning electron micrography (SEM) of various starch granules, we confirmed that starch granules susceptible to amylases showed polygonal shapes but starch granules insusceptible to amylases had smooth surfaces and round or oval shapes.

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Bacillus circulars F-2 can digest potato starch granules added to the medium as a sole carbon source. Scanning electron microscopic observation of potato starch granules recovered from cultured medium revealed that the granules were degraded gradually from their surface resulting in elongated granules with layered structure on their surface. The bacterium produces extracellular amylase when potato starch granules are used as a carbon source. Soluble starch or maltose, used as a carbon source instead of potato starch granules, induced little amylase. The amylase was purified from the culture supernatant to show a single protein band on SDS polyacrylamide gel electrophoresis and its general properties were studied. When acted on soluble starch, the purified enzyme produced maltohexaose alone in the early stage of hydrolysis. On further incubation, maltotetraose and maltose were produced with concomitant decrease of maltohexaose. The enzyme seems to act on amylaceous polysaccharides exowisely, by liberating maltohexaose successively from their nonreducing ends. Although the enzyme showed almost the same digestive activity as porcine pancreatic a-amylase and Streptococcus bovis α-amylase toward corn starch granules, it exhibited far higher digestive activity than those amylases toward potato starch granules.

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From a commercial enzyme from Aspergillus niger on marketing as a maceration agent of various plant tissues, several enzyme preparations were made which had relatively high activity of pectin depolymerase(poly(l, 4-α-galacturonide)glycanohydrolase, EC 3.2. 1.15), xylanase(1, 3-β-D-xylane xylanohydrolase EC 3.2.1.32) or cellulase (1, 4(1, 3; 1, 4)-β-D-glucan 4-hydrolase), and examined for maceration effect on sliced or disrupted sweet potato. The preparation which was strong in the activity of pectin depolymerase caused a rapid maceration at pH 4.5 and 40°C while the enzyme preparation which was high in xylanase and cellulase activities but slight in pectin depolymerase was almost sluggish in the effect. Sweet potato was steeped in 0.15% H₂SO₄ overnight to sterilize. Then, 0.1% each of maceration enzyme and glucoamylase (5000 units of pectin depolymerase and 3000 units of glucoamylase) was added to the grated potato and the mixture was incubated at pH 4.5 and 45°C. Two hours later, the mash was cooled at 30°C and yeast was added in a proportion of 10 g as dry weight per kg potato for alcohol fermentation. The alcohol produced in 6-day incubation was 14.5-15.5% (v/v). In order to shorten the fermentation period, the macerated mixture with pectin depolymerase was adjusted to pH 5.2-5.4 with sodium hydroxide, added with bacterial α-amylase, and the mixture was pumped through a chamber heated at 88-90°C with a retention time of 5 min. The dextrinized mash was acidified at pH 4.5 with sulfuric acid and subjected to alcohol fermentation after addition of glucoamylase and yeast. The fermentation by this method was completed in 42-44 hr and the alcohol produced was 17% (v/v).

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1. Ethanol fermentation of rice without cooking was performed by using black-koji amylase. From 14 to 15 vol% of ethanol was successfully produced in rice powder broth at 30°C and pH 3.5 for 5 to 10 days. 2. Semi-continuous ethanol fermentation of corn starch or cassava starch without cooking was carried out by using black Aspergillus glucoamylase preparation. Raw starch was mixed with Asp, niger glucoamylase preparation, water and yeast at pH 3.5 and 30°C. After one day fermentation, about 10 vol% of ethanol produced in the broth was distilled out in vacuum and the residue was reused as a source of amylase arid yeast for the next fermentation. Such fermentation process could be repeated forr twenty days, during which one dialysis was performed. 3. Ethanol fermentation of cassava roots or sweet potato roots without cooking was performed by using Asp. niger glucoamylase preparation or black Asp. mold bran. From 12 to 13 vol% of ethanol was produced in cassava broth at pH 3.5 and 30°C for five days. About 10 vol% ethanol was produced in sweet potato broth at pH 3.5 and 38°C for 3-4 days. 4. Rhizopus glucoamylase preparation could be used in ethanol fermentation of sweet potato without cooking at pH 4.5 and 38°C for 3-4 days, in which 0.02% of potassium pyrosulfite must be added to protect the contamination.

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A maltotetraose-forming amylase from Pseudomonas stutzeri was highly purified by adsorption on starch granules and by chromatographies on Sephadex G-100 and DEAE-cellulose. The purified enzyme showed a single band in polyacrylamide gel electrophoreses with or without sodium dodecylsulfate. The optimum pH for enzyme action on starch was 6.0-6.5, and the optimum temperature was 45°C. The purified enzyme attacked starch from the non-reducing end to produce α-anomer oligosaccharides. This indicated that the enzyme was an exo-α-amylase which had not hitherto been found. The enzyme activity was markedly inhibited by the addition of Cu²⁺, Hg²⁺, N-hromosuccinimide and 2, 3-butanedione. The molecular weight of the enzyme determined by the method of Weber and Osborn was about 5.7×10⁴. The isoelectric point of the enzyme was estimated to be 5.3 by polyacrylamide gel electrofocusing. The Km and ko values of this enzyme for starch, glycogen, short chain amylose and some maltooligosaccharides were calculated from Lineweaver-Burk plots.

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Exo-maltohexaohydrolase [EC 3.2.1.98] was discovered in 1971 by K. Kainuma et al. as a cell-bound enzyme. Recently, we found that an ultraviolet-induced mutant from Aerobacter aerogenes (Klebsiella pneumoniae) produced an extracellular exo-maltohexaohydrolase. The mutation was carried out by ultraviolet irradiation to achieve 1% survival. The culture conditions for the production of the extracellular enzyme were investigated. The effects of various additives on the enzyme production were also studied. The enzyme was purified by ammonium sulfate precipitation, DEAE-cellulose column chromatography and Sephadex G-100 gel filtration to 1100 fold of the activity of the original culture liquor. It gave a single band on polyacrylamide disc gel electrophoresis and the molecularV weight by SDS polyacrylamide gel electrophoresis and Sephadex G-100 gel filtration were 65, 000 and 48, 000 respectively. The 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 calciumion. The enzyme acted on Q-limit dextrins of amylopectin and glycogen to form branched oligosaccharicles. The characteristics of the enzyme were similar to those of the cell-bound enzyme with a few exceptions.

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An α-amylase which produces maltohexaose as the main product from starch was found in the culture filtrate of Bacillus circulans G-6 which was isolated from soil and identified by the author. The enzyme was purified by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex G-200 column chromatography. The purified enzyme was homogeneous in disc electrophoresis. The optimum pH and temperature of the enzyme were around pH 8.0 and around 6°C, respectively. The enzyme was stable in the range of pH 5-10 . The metal ions such as Hg²⁺, Cue+, Zn²⁺, Fe²⁺ and Co²⁺ inhibited the enzyme activity. The molecular weight was about 76, 000. The yield of maltohexaose from soluble starch of DE (dextrose equivalent) 1.8-12.6 was about 30%, and the combined action of the enzyme and pullulanase or isoamylase increased the yield of maltohexaose.

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The kinetic studies on the reactions of human pancreatic and salivary α-amylases with several maltooligosaccharides (maltotetraose, maltopentaose, maltohexaose, and maltoheptaose) were carried out. The susceptibility to hydrolysis with human pancreatic α-amylase decreased in the order of maltopentaose, maltohexaose, maltotetraose, and maltoheptaose, while with human salivary α-amylase maltopentaose was hydrolysed slightly slower than maltohexaose but fairly faster than maltotetraose or maltoheptaose from a viewpoint of the rates of reactions based on the amount of substrate changed. The relative rages of production of substrates, utilized in the coupled yeast α-glucosidase reaction, increased in the order of maltoheptaose, maltohexaose, maltotetraose, and maltopentaose with human pancreatic α-amylase, while with human salivary α-amylase in the order of maltoheptaose, maltotetraose, maltohexaose, and maltopentaose. Thus, maltopentaose was considered to be the best substrate for the α-glucosidase coupled method of α-amylase determination.

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We developed a new method of the determination of human serum and urine amylase as a routine analysis for clinical purpose, using β-2, 4-dichlorophenylmaltopentaoside as substrate. The new method is based on the following reactions.β-2, 4-dichlorophenyl-G₅human→α-amylase β-2, 4-dichlorophenyl-G₂+G₃β-2, 4-dichlorophenyl-G₂+G₃α-glucosidase→β-2, 4-dichlorophenyl-G₁+4G₁β-2, 4-dichlorophenyl-G₁β-glucosidase→2, 4-dichlorophenol+G₁2, 4-dichlorophenol+4-aminoantipyrine KIO₄→Red Quinone Dye (505 nm)In this method, the amylase activity in human serum and urine is accurately determined without interference from endogeneous glucose.

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The determination of α-amylase activity in serum, urine or other body fluids is widely used in clinical laboratory for the diagnosis of pancreatic disorders. We review the various laboratory methods including the advocated ones as well as the advanced or proposed ones. We learned from a recent survey of clinical laboratories that half of the participants used the amyloclastic methods and 40% used the methods involving dye-labeled starches. Perhaps both methods are convenient, but the former is not a strictly linear reaction, and absorbance peak of starch-iodine color complex shifts as hydrolysis of starch substrate proceeds. The latter is rapid and precise, but the costs of the substrates which can be obtained from commercial sources are expensive and furthermore, the results do not correlate well with those from other major methods. Kinetic methods have been accepted increasingly. These methods involve several coupled enzyme systems or different defined substrates and are more sensitive and have better linearlity of response to enzymatic activity. More advanced techniques involving chromogenic substrates with or without coupled enzyme reaction have been proposed, in which amylase activity would be measured more directly and probably free from the interferences owing to endogeneous glucose or maltose. Many challenges have been given to standardize the assay method of a-amylase activity in clinical chemistry, and much efforts are being made to provide the reference method and the reference material as well.

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