Four methods for the determination of human amylase activity in body fluids, amyloclastic, turbidimetric, chromogenic and saccharogenic methods, were reviewed. The sample size ordered from physician was markedly increased as it was possible to isolate isoenzymes of this enzyme. Owing to these circumstances, clinical laboratory needs a method practical for large numbers of samples. But above each procedure is not now perfect at this point . The so called glucosidase coupling method, a newly developed saccharogenic method, was noted. The standardization for expression of amylase activity was also discussed.

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The amylase activity found in serum and urine is composed of a heterogenous collection of isoenzymes. The amylase in human serum and urine was separated into 4 to 5 distinct isoenzymes by polyacrylamide gel electrophoresis, which have mobilities consistent with those of the pancreatic (Amylase-1, 2 and -4), and the salivary isoamylases (Amylase-3, 5 and -7). The normal isoamylase pattern was Amylase-1, 2, 3 and -5. Electrophoretic variants in normal persons are characterized by an additional isoenzyme band of slower anodal mobility than Amylase-1(=Amylase is) and an increased amylase activity of Amylase-2 (=Dominant Amylase-2), one of the minor components, up to the same levels as major bands. Pedigree studies confirmed an autosomal dominant mode of inheritance for these isoenzymes. Patients with pancreatitis had elevations of the pancreatic-type isoenzymes, while those with mumps had the salivary-type isoenzymes. In cases of clinically undiagnosed hyperamylasemia, elevations of isoenzyme activities were observed mostly in the salivary components; among these, pneumonia, primary lung cancer, diabetic coma, ovarian cancer and postoperative hyperamylasemia. These observations indicate that the isoenzyme pattern was not helpful in regard to etiology. However, in patients whose hyperamylasemia is of unknown etiology, amylase electrophoresis provides identification of the elevated isoenzyme type, thus providing the basis for the selection of further diagnostic procedures.

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Significantly elevated serum amylase activity may occur in some patients without elevation of urine amylase despite normal renal function. The cause lies in the presence in the serum of an amylase component having too large a molecular structure to be filtered through the kidneys into the urine. This disorder, termed "macroamylasemia, " has been observed in 30 patients over a period of five years. No distinct clinical syndrome consistently accompanies macroamylasemia. Even though abdominal pain is a common complaint, the condition is not characterized by any specific clinical syndrome. The prevalence of macroamylasemia in a series of 1, 052 h ospitalized patients was found to be 1.5%, while the frequency of the condition in 251 normal subjects was 0.4%. Studies concerned with the biochemical characteristics have indicated certain variations that point to heterogeneity: urea denaturation, ultracentrifugal characteristics, acidification tests, the effect of concanavalin A, nature of isozyme released from macroamylase complexes and affinity characteristics of amylase binding substances. Despite these additions to our knowlege, the precise genesis and composition of the macroamylase complex in case of macroamylasemia have yet to be fully elucidated.

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In the beer brewing industry throughout the world about 8 × 10⁶ tons of starch is annually converted into fermentable sugars and dextrins. Determining factors for the degradation of starch are the conditions of mashing and the action pattern of the amylases, α-amylase and β-amylase. Although present in germinating barley and in barley malt, α-glucosidase and debranching enzyme (plant pullulanase) exert little action in normal brewing procedures. Accordingly, the majority of the α-1, 6 linkages in amylopectin survive the brewing process. In a series of studies the formation and the composition of the dextrins present in beer wort have been examined by enzymic methods and quantitative gel filtration chromatography. The results show:-About 75% of the starch degradation products are glucose, maltose, and maltotriose. The remaining 25% constitutes the unf ermentable residue, generally known as dextrins.-About half of the dextrins consists of oligosaccharides with less than 10 glucose units, either linear or singly-branched.-About half of the dextrins contains 10 or more glucose units per molecule and may be termed megalosaccharides. These dextrins are multiply-branched.-The weight distribution of the dextrins versus their molecular weight exhibits a characteristic pattern for which the term "wavy distribution" has been coined, inter alia to indicate that the dextrins in wort and beer seem to fall into distinct groups. Subsequent structural analysis has revealed that these groups of dextrins are characterized by their number of α-1, 6 linkages.-The multiply-branched dextrins in wort and beer represent structural entities of dense branching in the original amylopectin (starch) molecules. Thus the study of dextrins in brewing also provides information of the fine structure of starch. Based on these findings a model for the degradation of starch in beer brewing by a-amylase and β-amylase will be proposed.

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Conventionally named "Maltase" which hydrolyze maltose can be roughly classified into two main groups being represented by α-glucosidase and glucoamylase from a view point of their action patterns. The former can be further divided into three groups as follows. The first group includes the enzymes which hydrolyze not only maltose but also some heterosaccharides such as sucrose or arylglucosides. The second group includes the enzymes which hydrolyze preferentially maltooligosaccharides rather than the higher molecular glucan. The enzymes which belong to the third group hydrolyze either oligosaccharide or α-glucan such as starch or glycogen. Although α-glucosidase belonging to this group may be regarded as glucoamylase, this enzyme may be distinguished from glucoamylase by means of comparing its reaction velocities on maltose and glucan, that is, reaction velocity of α-glucosidase on maltose is generally higher than that on α-glucan while the activity of glucoamylase on maltose is muchless than that on glucan.

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The diversity of α-glucosidase was discussed from the viewpoint of substrate specificity. The substrate specificities of α-glucosidases from various origins were compared with one another on the hydrolysis velocity of α-glucosidic bond, calculated, from Vmax for the hydrolysis of substrate. It seems that many of α-glucosidases can be divided into the following three types of their substrate specificities, except an enzyme such as honey bee α-glucosidase having unusual properties. The first group (“Type I”) is the typical α-glucosidase which hydrolyzes heterogeneous substrates such as phenyl-α-glucoside and sucrose more rapidly than maltose. The second group (“Type II”) is the type of so-called maltase, showing especially high activity to homogeneous substrates such as maltooligosaccharides, but feeble or no activity to α-glucoside and sucrose. The third group (“Type III”) is characterized as α-glucosidases possessing glucoamylase activity. However, the substrate specificity of the third group may be classified into category of the second group, except that this type of a-glucosidases are capable of attacking α-glucans. The active site of α-glucosidase, like glucoamylase, was also shown to be made up by the subsite structure. The subsite affinities in the active site of buckwheat α-glucosidase, evaluated in accordance with the subsite theory, were compared with those of Rh, delemar glucoamylase. The difference in the substrate specificities between α-glucosidase and glucoamylase was interpreted on the basis of their subsite affinities.

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By using the pure anomeric forms of substrates separately under conditions to limit mutarotation, it was found that the condensation reactions by amylases require donor substrates of specific configuration. That is, crystalline glucoamylase from Rhizopus niveus was found to catalyze the rapid synthesis of maltose and a slower synthesis of isomaltose specifically from β-D-glucopyranose. Crystalline sweet potato β-amylase, likewise, was found to catalyze the rapid synthesis of maltotetraose specifically from β-maltose, and crystalline hog pancreatic β-amylase the rapid synthesis of maltotetraose specifically from α-maltose. A rapid approach to equilibrium was found both in maltose synthesis from β-D-glucopyranose by glucoamylase, and in maltotetraose synthesis from β-maltose by β-amylase. Moreover, essentially the same equilibrium level (K_eq =ca.0.13) was obtained. The configurational inversion accompanying both condensations reveals their mechanism as one of glycosyl transfer. Crystalline α-amylases from six different sources, as well as crude salivary amylase, were found to catalyze the synthesis of maltose and maltosaccharides from α-D-glucopyranosyl fluoride, a stereoanalog of α-D-glucopyranose. The entire group of α-amylases had the capacity to promote α-D-glucosyl transfer from α-D-glucosyl fluoride to C₄-carbinol sites, demonstrating for the first time that α-amylases possess in common the capacity to catalyze glycosylation(i.e., glycosyl-hydrogen interchange) reaction extending beyond hydrolysis and its reversal. Similar de novo syntheses of maltosaccharides from α-maltosyl fluoride by α-amylases were also discussed.

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1. Fungal amylases 1) Aspergillus awamori : Alpha-amylase has an extremely weak activity to digest raw starch, but glucoamylase has strong activity to digest raw starch. Glucoamylase I, can adsorb on raw starch, is the principle of raw starch digestion. Glucoamylase II, can not adsorb on raw starch, has an extremely weak activity to digest raw starch. 2) Rhizopus sp. and Aspergillus oryzae: These two fungi have the similar amylase system with those of Asp, awamori on raw starch digestion and raw starch adsorption. 2. Comparison of four kinds of α-amylase 1) Pancreactic α-amylase digests raw starch most strongly and can adsorb on raw starch most easily. Asp, oryzae α-amylase digests raw starch most weakly and adsorbs on raw starch imperceptibly. In the case of α-amylases from bacteria and malt, α-amylase adsorption curves give reverse position from those shown for digestion. Apparently the ability to adsorb on raw starch does not necessarily indicate ability to digest the raw starch. 2) 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. Debranehing enzymes 1) Pseudomonas isoamylase: This enzyme, can adsorb on raw starch, assists the raw starch, especially waxy starch, digestion by Aspi awamori glucoamylase I or II. 2) Aerobacter pullulanase: This enzyme, can not adsorb on raw starch, assists the raw starch digestion by Rhizopus glucoamylase I. 4. Beta-amylases 1) Plant β-amylase can not adsorb on raw starch and has no activity to digest raw starch. 2) Bacterial β-amylase can adsorb on raw starch and has a strong activity to digest raw starch.

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A strain of Streptococcus bovis isolated from bovine rumen has a strong fermentability of raw starch and produces a single extra-cellular α-amylase, and the activity of this amylase on raw starch is remarkable when compared with ordinary bacterial amylase. Authors have studied the crystallization and some characteristics of this amylase. It was known that this amylase came under the category of bacterial saccharif ying α-amylase from the mutarotation of reducing sugar produced and the mode of action on soluble starch. This crystallized amylase was adsorbed on raw starch completely, and showed acceleration of the starch digestion under the presence of Ca²⁺. Especially, various raw grain starches were dissolve completely producing chiefly glucose and maltose. The remarkable activity for raw starch suggests that this amylase should be specially called as “raw starch hydrolase.”

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Many pullulan-hydrolyzing enzymes have been purified from several microorganisms and higher plants since 1961 when Bender and Wallenf els found pullulanase (EC 3.2.1.41 pullulan 6-glucanohydrolase) in culture filtrate of Aerobacter aerogenes. They are classified into four groups according to their substrate specificities, that is, the types of glucoamylase (EC 3.2 .1.3 1, 4-α-glucan glucanohydrolase), pullulanase, isopullulanase (EC 3.2. 1.54 pullulan 4-glucanohydrolase) and the Thermoactinontyces vulgaris α-amylase that produces panose from pullulan. Three kinds of enzymes described above, except glucoamylase, are considered to be the specific enzymes acting on α-1, 6-glucosidic linkages or α-1, 4-glucosidic linkages adjacent to α-1, 6-glucosidic linkages of starch, glycogen and oligosaccharides. In this paper, the progress of researches concerning pullulanase, isopullulanase and the Thernmactinomyces α-amylase is summarily described and, in particular, rough estimation of subsites of two pullulanases and isopullulanase was performed on the basis of data that have hitherto been reported. It is suggested that Streptococcus mitis pullulanase, A. aerogenes pullulanase and Aspergillus niger isopullulanase have at least seven, six and four subsites, respectively.

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An amylase from Streptomyces hygroscopicus SF-1084 was isolated and some properties were studied. The purification of the amylase was carried by the method of ammonium sulfate precipitation, DEAE-Sephadex A-50 column chromatography and Sephadex G-150 gel filtration to get a single band on a polyacrylamide disc gel electrophoresis. It is shown that the amylase is a kind of α-amylase and produces more than 75% maltose from starch. The amylase was fairly heat-stable and Ca²⁺ was essential for the heat-stability. The action pattern of the amylase on the starch will be summarized as follows; at the earlier stage of hydrolysis of starch, the amylase produces much maltotriose and maltotetraose. Then maltotetraose is hydrolyzed to maltose and after 30 of DE, maltotriose is hydrolyzed to maltose and glucose. But in this case much maltose is produced by the action of transglucosidation. As the result of these actions, the α-amylase from Streptomyces hygroscopicus SF-1084 produces more than 75% of maltose from starch.

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Two amylases produced by Streptomyces were purified, and some properties were studied. The one, named NA-468 amylase, was produced by a strain of Streptomyces griseus and formed about 55% of maltotriose from starch. NA-468 amylase was purified to almost 100-fold of the culture broth, and showed its maximal activity at 45°C and pH 5.6-6.0. Amylose was hydrolyzed to almost 100%, but waxy starch Q-limit dextrin was not acted. It was found that NA-468 amylase cleaved the third glucosidic bond from the end of the maltooligosaccharide. The other one, named NA-273 amylase, was produced by a strain of Streptomyces praecox, and formed more than 80% of maltose from starch. NA-273 amylase was purified to about 170-fold, and showed its maximal activity at 47°C and pH 6.0. Acting on amylose, soluble starch and waxy starch β-limit dextrin, the amylase produced a large amount of α-maltose. The amylase also hydrolyzed maltotriose to form maltose without the formation of glucose. The action mechanism of it was considered that glucose formed by hydrolysis was transferred to the non-reducing end of other maltotriose, and that maltotetraose formed was again hydrolyzed to maltose.

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