Black Aspergilli produce two types of a-amylase, the acid-stable a-amylase (ASA) and the acid-unstable a-amylase (AUA). Each a-amylase was obtained as a homogeneous enzyme protein from the same culture broth of a strain of Aspergillus niger. The catalytic action and the products from starch of both a-amylases were quite similar, but the amylase activity permg enzyme protein of AUA was about 6 times as large as that of ASA (Table 2). Higher acid stability and higher heat stability of ASA than AUA were recognized (Figs. 1, 2 and 3). In order to elucidate the mechanism of the acid-stability of ASA, the chemical, physicochemical and enzymatic properties of both enzymes were compared. The molecular weights and the iso-electric points of ASA and AUA were 58, 000, 3.44 and 61, 000, 3.75 respectively (Table 3 and Fig. 4). The amino acid composition of ASA differed from that of AUA in the following features (Tables 4 and 5). (a) The lysine content was lower. (b) Although the totals of carboxyl and amide were almost equal, there were considerably more free carboxyl residues. (c) The serine content was higher. (d) The proline content was lower. One mole of amino-terminal leucine or isoleucine per mole of ASA and one mole of amino-terminal alanine per mole of AUA were detected. ASA contained 24 moles of mannose and 4 moles of hexosamine per mole of enzyme protein and AUA contained 7 moles of mannose and one mole of hexosamine (Table 6). Both a-amylases contained calcium without any detectable amount of other metals . By dialysis against acetate buffer calcium contents of both a-amylases were converged to one gram atom per mole of enzyme, but the activity and the acid stability of both enzymes did not change (Figs. 5 and 6). The last calcium could be removed by EDTA, being accompanied by the loss of activity. The activity could be recovered partially by the addition of calcium and this last one atom of calcium seemed to be essential for the maintenance of active structure of the a-amylases (Figs. 6 and 7). ORD in the visible region suggested the low content of a-helix in both enzymes and the rapid increase of levorotatory power of AUA was observed when it was acidified to pH 1.9 (Table 8). From ORD curves in the ultraviolet region, it was highly likely that both consisted of polypeptide folded in the same manner. Although the ORD curve of ASA at acidic pH was almost the same shape as that at neutral pH, that of AUA showed a marked change at pH 1.9. It should be noted, however, that the ORD curve suggested rather limited unfolding of the polypeptide chain of AUA in the acidic pH (Fig. 8). Spectrophotometric titration curve of tyrosine residues showed that ASA had abnormal tyrosines (11 residues of pK 11.2 and 7 residues of pK 12.9) besides normal 13 residues of pK 10.5, although the total of 31 tyrosine residues was normally titrated in 6 molar guanidine hydrochloride (Fig. 12). In AUA various types of tyrosine residues were also observed (5 of pK 10.7, 24 of pK 11.5 and of pK 13.0) (Fig. 13). In the hydrogen ion titration of ASA, 38 ionizable residues bound the proton between pH 5.5 and 1.5 and the remaining 34 ionizable residues bound the proton explosively between pH 1.5 and 1.2 along with acid denaturation (Fig. 14). In this pH region carboxyl residues were supposed to bind the proton and the total number of 72 was agreed well with the number of free carboxyl obtained from amino acid analysis of ASA. In the titration curve of AUA, rapid protonation at around pH 3.5 was observed (Fig. 15). The maximum number of the difference between the forward and the backward titration curves was 20 residues. These residues might be masked as carboxylate ions and the number coincided with the number of abnormal tyrosines. There were 34 carboxyl residues in ASA which were not titrated even at pH 1.5.

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The outline of the subsite theory was described which correlates the action patterns of amylases, i, e., the dependency of rate parameters on the degree of polymerization n of linea rsubstrates and the mode of cleavage of maltooligosaccharides, with the subsite structure of the enzyme. The Subsite structure is defined as the arrangement of a definite number of subsites constituting the active site of an amylase, each of which interacts specifically with a glucose residue of linear substrates with its own "subsite affinity" designated AZ (in units of kcal/mole). An n-mer substrate can be bound with the active site in a variety of "productive" and "nonproductive" binding modes, and the probability of a particular binding mode is determined by the subsite structure of the enzyme . Assuming that the subsite affinities are additive and that the intrinsic rate constant kint of substrate bond cleavage in a productive complex is independent of n and the binding mode, the action pattern of an amylase can quantitatively be described in terms of Ai's and kint ( the properties of the subsite structure), and vice versa. Subsite structures of four kinds of amylase, glucoamylase, Taka-amylase A, bacterial liquefying a-amylase and wheat bran j3-amylase, which have been evaluated from the ndependency of rate parameters, were displayed in histograms. For the three amylases other than glucoamylase, negative value of Subsite affinity is commonly seen at a subsite adjacent to the catalytic site, suggeting the "strain" in the enzyme-substrate complex . This explains the reason why maltose is hardly hydrolyzed by these enzymes . It was demonstrated that the subsite structure of glucoamylase can reasonably account fo rits substrate specificity towards synthetic substrates, i, e., the unexpectedly lower molecular activity for phenyl a-gulcoside compared with that for phenyl a -maltoside . The subsite theory is also useful to predict or interprete the change in action patte rns of amylases caused by altering one of the subsite affinities by chemical modificatio n. Calculation with Taka-amylase A showed that the molecular activity towards maltose could increase by afactor of several tens whereas that towards maltoheptaose decreases five fold, if a particular subsite affinity is decreased by 3 kcal/mole . An example of photooxidation of a histidine residue of bacterial liquefying a-amylase was described, in which the modified subsite was located by studying the effect of modification as a function of n . The feasibility of changing artificially the action patterns of amylases has thus been substantiated.

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Bacillus natto IAM 1212 produces a-amylase by about 5 times as much as Bacillus subtilis Marburg strain, α-amylase of which is more thermostable and moves more slowly to anode at pH 8.3 than that of B. natto. DNA of B, natto could induce transformation in B. subtilis cells of many genetic charactors including amylase productivity. Genetic analyses of the transformants that acquired high amylase productivity revealed that a gene regulating a-amylase synthesis participates in the a-amylase-producing system of B, subtilis and this regulator gene (amy R) is closely linked to the a-amylase structural gene. The amyR gene is linked also to the aronG gene which has been reported to be linked to α-amylase-structural gene by S. Yuki. α-Amylases produced by parents (B, subtilis 6160 and B. natto IAM 1212) and two transformants (NA64 and NA20) were purified, their properties were investigated and their molecular weights were estimated to be 55, 000, 34, 000, 55, 000 and 42, 000, respectively. Thus, it was suggested that the two transformants produced a-amylases with hybrid charactor of their parents amylases. Immunological properties were all alike, but their substrate specificities were different. a-Amylases of 6160 and NA64 could hydrolyse maltotriose while those of B. natto and NA20 could not. Mutants which had a genetic charactor to increase the production of both a-amylase and protease simultaneously, were isolated from a transformable strain of B, subtilis 6160 by NTG treatment. This mutation seems to have occurred at a single gene of the bacterial chromosome and was not linked to aro116. When this mutation and a-amylase regulator gene amyR_h, (amyR of B, natto) coexisted in one cell, their synergistic effect on extra cellular α-amylase production was observed. Thus the level of α-amylase in culture medium of B. subtilis was elevated from 11 unit/ml to 140 unit/ml.

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(1) Raw starch digestion by black-koji amylase system was studied. (a) The ability of the black-koji amylase system to digest raw starch may be associated with ability to adsorb on raw starch. (b) Alpha-amylase has an extremely weak activity to digest raw starch, but glucoamylase has a strong activity to digest raw starch. (c) Alpha-amylase and glucoamylase, when used jointly, interact with each other increasing digestion of raw starch to about 3 times the sum of their separate activities . (d) Glucoamylase consists of two kinds of glucoamylases, that is, glucoamylase I and glucoamylase II. The former can adsorb on raw starch and is the principle of raw starch digestion. This enzyme also can hydrolyze β-limit dextrin from glycogen very easily. On the other hand, the latter, glucoamylase II, can not adsorb on raw starch and has an extremely weak activity to digest raw starch. This enzyme can scarcely hydrolyze β-limit dextrin from glycogen. (2) Alpha-amylase adsorption on raw starch and its relation to raw starch digestion were investigated. (a) Pancreatic a-amylase digests raw starch most strongly and is adsorbed on raw starch most easily. Fungal α-amylase digests raw starch most weakly and is adsorbed on raw starch imperceptibly. In the case of α-amylases from bacteria and malt, α-amylase adsorption curves reverse positions from those shown for digestion. (b) The adsorption of pancreatic α-amylase on raw starch is rapid and the adsorbed amylase is desorbed at a decelerating rate with time and is similar to the digestion activity curve. (c) Glucose and maltose, especially the latter, inhibit both amylase adsorption on raw starch and raw starch digestion, and so the raw starch digestion is accelerated by dialysis . (3) Pseudomonas isoamylase assists the raw starch, especially waxy starch, digestion by the black-koji glucoamylase I.

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Generally, amylases have been classified in the following groups; 1) a-amylase, mainly originated from bacteria and animal organs, 2)α-amylase from plant sources, 3) Glucoamylase from mold, 4) isoamylase from yeast, bacteria and plant origin. Recently several new amylases which were unabled to be classified in the usual groups as mentioned, were discovered from microbial origins . These new amylases are reviewed in this paper mainly from the viewpoint of the action pattern of these enzymes and also the future aspects of the production of maltooligosaccharides using these starch hydrolyzing enzymes. The following three different maltose forming amylases of microbial origin have already been discovered; B, polymyxa amylase by ROBYT and FRENCH, ⁶) B, megaterium amylase by HIGASHIHARA and OKADA⁷) and Streptomyces amylase by HIDAKA et al .⁸) Maltotetraose forming amylase from Pseudomonas by ROBYT et al .¹²) and maltohexaose forming amylase form Aerobacter by KAINUMA et al.1315) were characterized as the third and the fourth exo amylases in 1971 and 1972 respectively. An anomalous reaction of the maltohexaose forming amylase on j3-limit dextrin of amy lopectin to produce branched oligosaccharides of DP6, 7 and 8 is discussed from the point of the stereo-chemistry of a-1, 4 and α-1, 6 glucosidic bond . This is the first amylase which mimics α-1, 6 glucosidic linkage as α-1, 4 glucosidic linkage which had been observed in the priming reaction of E. coli phosphorylase.18) Besides the two novel exo-amylases, B. macerans cyclodextrin forming enzyme is discussed relating with the action pattern of this enzyme and a possible utilization of cyclodextrin.

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