1. A brief history of researches of amylase is described in regard to the development of production and application of enzymes.
2. The industrial production of several amylases in Japan is also described.
3. Specificities of several amylases are discussed from the point of view of molecular biology with a special reference to the facts such as-both α- and β-amylases found in animals and higher plants, respectively, exist in certain bacteria belonging to Bacillus. Generally, the bacteria produce one amylase of either α- or β-. Furthermore, the bacteria producing α-amylase produce only one type of α-amylase of either starch liquefying or saccharifying. The bacterial amylase is very similar in the specificity to that of the enzyme of higher plants or animals. Thus, it is presumed that the information gene for building amylase protein has been completed in an early old era of history of biology and that the diversity in specificity of amylase has occurred by gene mutation. This consideration may call the hypothesis again that the specificity of α-, β-, exo- or endohydrolysis type of amylase depends upon minor difference in the amino acid sequence of catalytic and binding sites of amylase protein.
4. The amylases to attack raw starch granules and several other enzymes to favor the saccharification of the starch in uncooked starchy material by amylase are also discussed in connection with improving the pretreating method of uncooked starchy material for alcohol fermentation.

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A review paper. Contents of amylose and distributions of α-1, 4-chains of amylopectin were determined by two enzyme-chromatographic methods using the following endosperm starches of maize (Zea mays L.) and rice (Oryza sativa L.) with definite genetic backgrounds: amylose-extender (ae), dull (du), sugary-1 (su₁), sugary-2 (su₂) and waxy (wx) single-mutants, ae wx double-mutant and their normal counterpart of the inbred Oh43 maize; low-amylose (cultivar: Norin 8), high-amylose and sugary (cultivar: Kinmaze) mutants of rice, and waxy (wx) and nonwaxy rice (cultivar: Taichung 65) grown under different temperature conditions after anthesis. References on starches other than maize and rice studied in the author's laboratories were also cited.

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The size and the shape of shellfish glycogen varied with the extraction procedure. The particles of glycogen prepared by extraction with dimethyl sulfoxide (DMSO) were relatively large (20-180nm, diam.) and showed rosette-like structure. The particles of glycogen prepared by trichloroacetic acid (TCA) extraction and the commercial glycogen were much smaller in size (10-130nm and 10-40nm, respectively) and did not show rosette-like structure. The general properties of these glycogen preparations were not significantly different from each other, except that the latter two preparations were more susceptible to pullulanase.
The size of the glycogen particles was decreased by treatment with TCA, phosphotungstic acid or 30% potassium hydroxide, but was not changed by treatment with 0.1M or 0.5M sodium hydroxide. Treatment with 2-mercaptoethanol did not disrupt large glycogen particles. Protease and β-amylase did not affect the size of glycogen particles. Large glycogen particles were partially split by treatment with α-amylase, suggesting that a large glycogen particle is composed of fundamental particles (20-30nm, diam.) linked through α-1, 4-glucan chains. On the basis of the foregoing results, a model of glycogen particle was proposed. The relation of the biosynthesis mechanism and the particle formation of glycogen was also discussed mainly from the viewpoint of the role of branching enzyme.

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A thermophilic fungus, Aspergillus K-27, produced extracellular amylolytic enzymes in a submerged culture with wheat starch as a carbon source at 45°C. By adding α-methyl-D-glucoside to the medium as an inducer, the enzyme production was doubled on five days cultivation. The enzymes strongly digested not only cereal but also tuber and root starches without gelatinization. The relative rates of hydrolysis of corn, tapioca, wheat, sweet potato and potato starches were 1.0, 0.91, 0.83, 0.75 and 0.72, respectively.
The crude enzyme fraction exhibited at least two different activities, glucoamylase and α-amylase, and the former was found to be the major one (70%) on treatment of the crude enzyme fraction at pH 3.5 and 45°C. Glucoamylase, which was purified to homogeneity as judged by disc-gel electrophoresis, showed considerable thermostability and resistance to heavy metal ions. The purified enzyme rapidly degraded rabbit liver glycogen and potato amylopectin to the limits of 98% and 81%, respectively, which were identical to those with the enzymes of Aspergillus niger and Rhizopus delemar. The K_m values of glucoamylase of K-27 for oligosaccharides were almost the same as those of A. niger and about half those of R. delemar, and the values for polysaccharides were about half those of GIII of R. delemar and 1/300-1/2700 of those of the enzyme of A. niger. The V_max values for all the substrates tested resembled those of the enzyme of R. delemar GIII and were 1.5-2 times as high as those of the enzyme of A. niger (Ref. 8).
The glucoamylase of K-27 showed higher digestibility of raw starch than the enzymes of A. niger and R. delemar. The α-amylase alone showed little raw starch digesting activity but it stimulated the activity of the glucoamylase greatly.

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A strain of black mold was isolated from a pith of sago palm (Metroxylon sagu) sampled at Papua New Guinea. The mold, which was identified as Chalara paradoxa, produced a strong amylase activity to hydrolyze starch granules. In this paper, the morphlogical description of the mold and the some characteristic properties of the crude enzyme was described.
The enzyme had extremely high activity to hydrolyze starch granules compared with the ones of conventionally studied raw starch-digesting amylase.

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Action of Bacillus circulans F-2 amylase on raw starches of various origins was studied in comparison with those of porcine pancreatic α-amylase and Streptococcus bovis α-amylase. Three amylases showed the highest digestive activity against mochi rice starch and the lowest activity against potato starch. Three amylases digest cereal starches such as corn starch at approximately the same rate. However, only B. circulans F-2 amylase could digest potato starch at a substantial rate.
Starch granules digested by B. circulans F-2 amylase were observed by scanning electron microscopy. Differences in digestion patterns observed among various starches were discussed in relation to the differences in environment of granule development.
Crosslinked starches prepared by the reaction between epichlorohydrin and raw starches were found to be effective carbon source for the production of B. circulans F-2 amylase. Different from raw potato starch, these starches can be autoclaved and so greatly facilitate the cultivation of this bacterium. Crosslinked potato starch is superior to crosslinked corn starch as, in the presence of the former, the bacterium produces higher amount of amylase with shorter cultivation period than in the presence of the latter.

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Alcoholic fermentation from grains with a noncooking system was successfully carried out on an industrial scale. It was found that an enzyme preparation from Rhizopus sp. was suitable for alcoholic fermentation with a noncooking system on an industrial scale. It was suggested that some enzymes, other than glucoamylase, (such as α-amylase and cellulase) were essential for alcoholic fermentation with noncooking.

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For construction of starch-fermentable yeasts which are practically useful for ethanol production from raw starch, we have made some attempts to isolate the secretive glucoamylase-genes from microorganisms. Recently, we achieved the molecular cloning of a glucoamylase-producing gene from the cells of Saccharomyces diastaticus. The isolated gene included 2764 bases encoding 778 amino acid residues, and it was found that the gene in vector-plasmid is expressible in both Saccharomyces cerevisiae and Schizosaccharomyces pombe to give an extracellular glycoamylase. Molecular size of peptidyl part of the purified secretive-glucoamylase was calculated to be approximately 44.5 kilodalton (kd) consisting of 41-kd large and 3.4-kd small subunits, however, an expected molecular-size of the peptidyl part from the gene structure is 80 or more kilo-dalton. The isolated mRNA from the gene donor yeast had a molecular weight corresponding to approximately 2600 bases. Thus, maturation of the enzyme might take place at posttranslational level.

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1. The saccharification of various pulverized and uncooked cereal grain by a commercial preparation of Rhizopus glucoamylase at pH 4.0 and 30°C was not particularly effected by the addition of bacterial α-amylase, bacterial proteinase or fungal acidic proteinase. However, the addition of bacterial α-amylase was found to improve the adhesiveness of the mash prepared by mixing pulverized cereal grains and water for saccharification or alcohol fermentation.
2. Both the crude enzymes prepared by extracting the wheat bran culture of Rhizopus niveus and Aspergillus kawachii with water, respectively, were more active at acidic sides than the commercial Rhiz. glucoamylase.
3. The best result of saccharification and alcohol fermentation of uncooked cereal grains was obtained with the mash incubated with a mixture of commercial Rhiz. glucoamylase and Asp. kawachii or Rhiz. niveus cultured on wheat bran. This fact may indicates that the saccharification amylase in the bran culture is not only stable but also, certain enzymes such as hemicellulolytic enzyme produced on the bran act to favor the saccharification of starch in the cereal grains.

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A single strain of Aspergillus awamori var. kawachi produced three glucoamylases, raw starch-digesting GAI, raw starch-indigesting GAI′ and II, which were classified respectively into type A, B, C based on the difference in hydrolysis curves for glycogen. The raw starch-digesting amylase was selectively obtained in the submerged Culture A. Limited stepwise degradations of glucoamylase with co-existent proteases and glycosidases resulted in the occurrence of multiple glucoamylases. GAI (MW 90000, N-terminal Ala, C-terminal Arg) was converted to GAI′ (MW 83000, N-terminal Ala, Ile, C-terminal Arg, Thr) and an inactive glycopeptide Gp-I (MW 7000, N-terminal Ala, C-terminal Ser) with proteases. The liberated Gp-I abundant in hydroxy amino acids, preserved the adsorbability onto raw starch observed in GAI. By the characteristics of Gp-I, “Raw starch-affinity site” essential for raw starch-digestions was proposed in the part corresponding to Gp-I on GAI molecule. A protease-, glycosidase-negative mutant HF-15 produced a highly raw starch-digesting GAO (MW 250000) under all tested cultural conditions. The mutant α-amylase and GAO were bound to chitin at “Chitin-binding site” so tight that a “Chitin-immobilized amylase” was obtained without the aid of a cross linking glutaraldehyde. This preparation digested even raw starch to the high extent as glucose but lacked transglucosidase activity. Experiments using the immobilized amylase for production of glucose syrup and alcohol fermentation demonstrated the possibilities of recycling the enzyme. An ethanol tolerant Philippine yeast Saccharomyces uvarum inulyticum var. nov. w-y-2 and the mutant HF-15 liquid koji were applied for the high concentration-ethanol fermentation up to 20%v/v of uncooked corn starch materials.

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