Corn grain was found to be readily fermented with a reasonable alcohol yield without any process of steaming or sterilizing the grain, if provided that the grain was finely pulverized and incubated as a slurry keeping the pH at 4.5 and the temperature between from 25 to 28°C after the addition of Rhizopus glucoamylase preparation and pressed baker's yeast. For the slurry consisted of one part of pulverized corn grain in two point seven weights of water, the addition of twenty units of glucoamylase and zero point seven mg of pressed baker's yeast per g starch of the slurry was enough for nearly completion of alcohol fermentation in four days under above conditions. Unlike the case of alcohol fermentation of polished rice grain powder by a similar method, no distinct effect of proteinase was observed for alcohol fermentation of corn grain powder. Also, no particular effect of pectin depolymerase in the glucoamylase preparation was observed for production of alcohol from corn grain. However, this enzyme was effective to reduce viscosity of the fermented beer. Buckwheat grain powder was also readily fermented by using the commercial enzyme. However, barley grain was not fermented so readily as corn or buckwheat grains.

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The raw-starch-digesting activities of five glucoamylases, one each from Aspergillus niger, and Aspergillus sp. K-27, and three forms from Rhizopus delemar, GI, GII and GIII, were discussed as to the kinetic parameters (K_m and V_max) for maltooligosaccharides, amylopectin and glycogen, and their molecular structures. The R. delemar GIII and Aspergillus sp. K-27 enzymes, which showed strong raw starch digesting activity, showed much lower K_m values for amylopectin and glycogen than the A. niger and R. delemar GI and GII enzymes, which lack raw-starchdigesting activity. The R. delemar GI and GII enzymes were suggested to be the products of limited proteolysis of the GIII enzyme after biosynthesis from the results of amino acid and carbohydrate analyses of these three forms and the partial proteolysis of the GIII enzyme with chymotrypsin. From these results and other lines of evidence, “starch binding site” other than the active site on the raw-starch-digesting enzyme was postulated. It is considered that this site shows high affinity to high molecular substrates and raw starch, and is involved in the hydrolyses of raw starch granules, amylopectin and glycogen, and the absorption on starch granules. The amino acid composition of this site of the GIII enzyme was suggested to be rich in serine, tyrosine and aspartic acid (including asparagine).
All glucoamylases hydrolyze starch from its non-reducing end and their action stops at the C-6-phosphorylated residues and the residue just before the C-3-phosphorylated residue. Therefore, they are unable to completely hydrolyze potato starch, -20%, depending on the amount of the phosphate, remaining as limit dextrin. It is concluded that the cooperative action of α-amylase and phosphatase is essential for the complete degradation of starch.
Aspergillus sp. K-27 produces a strong raw starch degrading enzyme, which is a mixture of glucoamylase and α-amylase, with an activity ratio of 7:3. The glucoamylase alone showed strong activity toward various raw starches, including potato starch, but the activity was enhanced synergistically with the addition of a small amount (1%) of the α-amylase. However, this synergistic action was observed also more or less with other α-amylases, Taka-amylase, and Bacillus subtilis liquefying and saccharogenic α-amylases.
The relationship between the structure and function of the “starch binding site” and the mechanism of the synergistic action of glucoamylase and α-amylase as to raw starch degradation is an interesting problem for future studies.

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Action of bacterial β-amylase of three strains isolated from soils on raw starches were studied in comparison with β-amylase of soy-bean. Three bacterial β-amylases have been strongly adsorbed on raw starch granules and digest it. On the other hand, soy-bean β-amylase is adsorbed on raw starch imperceptibly, but digests it a little.
The β-amylase of a strain Bacillus sp. No. 2718 has a strong activity to digest raw starch.
The effects of temperature on raw starches digestion of corn, wheat by No. 2718 β-amylase and soy-bean enzyme were as follows; 1) The bacterial β-amylase easily digested every raw starch below its gelatinization temperature but soy-bean β-amylase digested it a little. 2) Raw starch digestion by No. 2718 β-amylase is accelerated by heating.
Starch granules digested by No. 2718 β-amylase and soy-bean enzyme were observed by scanning electron microscopy.

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Since our discovery of a strong raw starch digestion activity of black-koji amylase compared with yellow-koji amylase or malt amylase, we have investigated the reason why black-koji amylase has such a strong activity and researched the application of this activity to direct ethanol fermentation of starch materials without cooking.
The reason was explained by the following facts.
1) Black-koji amylase contains a larger amount of glucoamylase than yellow-koji amylase does.
2) Glucoamylase is the principal amylase on raw starch digestion.
3) Glucoamylase action on raw starch is stimulated by adding α-amylase.
Ethanol fermentation of starch materials without cooking was developed as follows.
1) Ethanol fermentation of sweet potato without cooking was carried out by using mold bran of Aspergillus awamori.
2) Ethanol fermentation of rice and corn without cooking was carried out by using submerged culture of Asp. awamori as amylase preparation.
3) Semi-continuous ethanol fermentation of corn starch without cooking was performed by using glucoamylase preparation of Asp. niger.
4) Ethanol fermentation of sweet potato without cooking was performed by using Rhizopus glucoamylase preparation.
5) Ethanol fermentation of cassava pellet without cooking was performed by using Rhizopus mold bran without adding yeast.

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Various kinds of enzymes are known to be secreted into human urine as normal components. However, the enzymatic and physicochemical properties, as well as the tissue sources of the urine enzymes, remains to be clarified. Human urine may be the sole limitless source of certain human enzymes. Among these enzymes, current results which are concerned with the purification methods, the enzymatic and physicochemical properties and the origins of human urine α-amylases and α-glucosidases are presented, and also the discrimination between pancreatic and salivary α-amylase isozymes using by the specific monoclonal antibodies is described and discussed in this review article.

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CGTase (EC 2.4.1.19) genes from B. stearothermophilus and B. macerans were cloned and expressed in E. coli and B. subtilis. Enzymes from recombinants have the same enzymatic properties as those from donor strains, respectively. Amino acid sequences of the enzymes were deduced from DNA sequences of the two genes. B. stearothermophilus enzyme is consisted of 711 amino acids which is contained 31 residues as a signal peptide. On the other hand, B. macerans enzyme is consisted of 713 amino acids which is contained 27 residues as a signal peptide. Amino acid homology between two enzymes is 60%, and those between CGTases and α-amylases from various origins are 20-30%. Amino acid homology between each CGTase and A. oryzae α-amylase is the highest, 30%.

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Rhizopus glucoamylase gene was cloned, sequenced and expressed in yeast. After improvement of the promotors, yeast strains, vectors and carbon sources, upto 13U/ml of glucoamylase was secreted. This means that about 8% of the total yeast protein was secreted as glucoamylase. The study of the structure and function relationship of this glucoamylase suggested that the glucoamylase consisted of two domains; adsorptive domain and catalytic domain. By using the resultant recombinant yeast strain, alcoholic fermentation of starchy materials was also achieved.

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We have isolated and sequenced a gene (amy) coding for α-amylase [EC. 3.2.1.1] from the Streptomyces hygroscopicus genome. Subcloning experiments indicated that amy could be expressed from the lac promoter in E. coli or its own promoter in S. lividans. The amy nucleotide sequence indicated that it coded for a protein of 52 kilodaltons (478 amino acids). The conserved amino acid sequences of other α-amylase were found in three separate regions of the S. hygroscopicus α-amylase. The 30-residue leader sequence showed similarities to those found in other prokaryotes. The DNA sequence 5′ to the amy structural gene contained a sequence complementary to the 3′-terminal sequence of 16S rRNA of S. lividans. The transcriptional start points of amy were determined, but the promoter of amy was not similar to the consensus sequence found in other prokaryotes. The recombinant strain, S. hygroscopicus containing amy on a high copy plasmid, produced α-amylase 4 times excess than the host strain.

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A Bacillus subtilis secretion vector was constructed from its α-amylase gene (amyE), in which a putative signal peptide was composed of 41 amino acids. A thermostable α-amylase gene (amyT631) from B. stearothermophilus A631 was cloned in a B. subtilis plasmid pUB110. The isolated plasmid was designated as pTUB607. Then amyT631 was introduced into the B. subtilis secretion vector, after amyT631 was partially digested by a exonuclease BAL 31. Chimeric plasmids pTUB613, pTUB616 and pTUB617 were isolated. pTUB613 and pTUB616 encoded Cys residue in the NH₂-terminal regions of the fused amyT631. B. subtilis strains containing pTUB613, pTUB616 and pTUB617 produced fused extracellular thermostable α-amylases whose molecular weight was estimated to be approximately 63, 000, because the putative signal peptide of amyE was cleaved between 31 and 32 amino acid position from the translation initiator Met. The molecular weight of the parental pTUB607-α-amylase was estimated to be 61, 000. Fused extracellular thermostable α-amylase from pTUB613 and pTUB616 showed an increased thermostability at 90°C, while pTUB607-α-amylase did not. The increased thermostability was suppressed when the pTUB613 and pTUB616-α-amylase were heated in the presence of 100mM mercaptoethanol. The extracellular parental thermostable α-amylase from pTUB607 contained sole Cys residue at position 363. Thus it is suggested that the increase of the thermostability in pTUB613-and pTUB616-α-amylases is closely related to the formation of intramolecular disulfide bonds.

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