The α-amylase, α-amylase inhibitor and proteinase inhibitor activities of proteins extracted from wheat varieties and their influence on the baking quality

The grain of six winter and spring wheat varieties, harvested in 1987, differing in baking quality were included in the study. A number of technological analysis were performed. Albumins, globulins and gliadins were washed out. The location of inhibitory activities in extracted proteins, against α-amylases of various origin. were tested and compared to the baking quality of the wheat grain. Also, the location of antitryptic activity and endogenous amylase activity in extractable proteins was compared to baking quality factors. Among the studied inhibitory activities against mammalian α-amylases some correlation were found between the inhibition activity of hog pancreas α-amylase and sedimentation test (r = 0.88). Furthermore inhibitory activities of wheat proteins towards insects α-amylase shown significant correlation between the inhibitors of Anagasta kuehniella α-amylase and sedimentation test (r = 0.90). as well as crude protein content (r = 0.86). Also the inhibition activity against Sitophilus granarius α-amylase versus sedimentation test presented some correlation (r = 0.80). Endogenous amylase activities (α- and β-amylase) have an inversely proportional correlation to crude protein content (r = ?0.82) and sedimentation test (r = ?0.85).     

Purification and Characterization of a Maltotriogenic α-Amylase I and a Maltogenic α-Amylase II Capable of Cleaving α-1,6-Bonds in Amylopectin

Extracellular α-amylases I and II, produced by a facultative thermophile Bacillus thermoamyloliquefaciens KP 1071 capable of growing at 30–66°C, were purified to homogeneity. α-Amylase I consisted of a single polypeptide with methionine residue at the NH₂-terminus. α-Amylase II consisted of two equivalent polypeptides each comprising a methionine at the NH₂-terminus. α-Amylase I hydrolyzed endotypically α-1,4-bonds in glycogen, amylopectin and β-limit dextrin, but not their α-1,6-bonds. α-Amylase II degraded amylopectin and β-limit dextrin in exo-fashion by cleaving preferentially α-maltose units from the non-reducing ends and hydrolyzing their α-1,6-branch points. α-Amylase II hydrolyzed maltotriose, phenyl-α-maltoside, α- and β-cyclodextrins and pullulan, whereas α-amylase I had no activity for all these sugars. α-Amylases I and II hydrolyzed maltotetraose, maltopentaose, α-limit dextrin and amylose, but they were inactive for maltose, isomaltose and panose. It was suggested that α-amylase I is the most thermostable type of hitherto known maltotriogenic endo-acting α-amylases, and α-amylase II is the first maltogenic exo-acting α-amylase able to split α-1,6-bonds in amylopectin.     

Die Wirkung von α-Amylasen auf β-Cyclodextrin und der Nachweis von α-Amylasefremdaktivitäten in ausgewählten Enzympräparaten

The action of α-amylases on β-cyclodextrin and the evidence of foreign activity of α-amylase in selected preparations of enzymes The interaction between cyclodextrins and α-amylases taken from different sources is discribed contradictious in the literature. Some α-amylases e.g. isolated from Aspergillus oryzae, porcine pancreas and saliva hydrolized cyclodextrins to glucose. The hydrolysis of cyclodextrins catalysed by α-amylase from Bacillus species have been described conflicting. In this paper the action of hydrolysis of different preparations of α-amylases on β-cyclodextrin have been investigated. It has been shown that Rohalase M3 (α-amylase from Aspergillus niger) cleaves the ring of β-cyclodextrin. 2 α-amylases from Bacillus subtilis are not able to hydrolyse β-cyclodextrin. The reasons for the different actions of hydrolysis have been discussed with size and structure of the active centre of the enzymes. Moreover, different preparation of hydrolysis have been tested on secondary activity of α-amylase. 2 glucoamylases from Aspergillus oryzae have been shown secondary activity of α-amylase. With the hydrolases α-glucosidase from fungies, β-amylase from malt, saccharase from yeast, invertase from S. cerevisiae and pullulanase from Aerobacter aerogenes no cleavage of the ring of β-cyclodextrin could be detected.     

The influence of α-amylase supplementation, γ-irradiation (60Co) as well as long time of storage of wheat grain on flour technological properties

The varieties of winter wheat, Aria and Beta, were studied. The Aria variety was stored for the period of four years. The part of wheat grain from Beta variety was irradiated with γ rays (⁶⁰Co). In extracts from wheat kernels and flour protein content, total α- and β-amylolytic activity as well as α-amylolytic activity were determined. α-amylases of native and fungal origin were added to the flour obtained from samples of stored wheat kernels (Aria), irradiated and non-irradiated (Beta). Consequently native α-amylase activity of flour increased by 25 % and 50% respectively. Extensive technological estimation of grain and flour with amylase supplements was carried out. The study included: sedimentation analysis, falling number test, milling experiment, farinogram and extensogram analyses, measurement of the degree of damaged starch and flour colour, as well as baking experiment. The obtained experimental loaves of bread were tested for their ability to remain fresh. It was found out that the stored grain flour was characterized by the highest α -amylolytic activity and the lowest falling number value, whereas the irradiated grain flour showed the highest degree of starch damage and water absorption. When α-amylase supplementation to doughs wasn't accompanied either by irradiation or storage of grain, it definitely changed their physical properties for the worse. The negative influence of native α-amylases appeared to be less significant than that of fungal α-amylases. The positive influence of α-amylase supplementations, especially of those increasing by 25 % the native α-amylolytic activity of flour on volume, and freshness of loaves of bread was observed.     

Control of wheat α-amylase using inhibitors from cereals

A survey of 46 varieties of cereals and related species (including 27 different species from the Poaceae) indicated the presence of a strong inhibitor of wheat α-amylase in all seven Hordeum species tested. Rye contained a lower level of inhibitor activity, but the other species contained insignificant amounts of wheat α-amylase inhibitor activity. The partially purified barley inhibitor was most effective in inhibiting wheat α-amylase activity at high pH. The addition of chromosome 2 of barley to wheat (Chinese Spring addition line 2H) resulted in an apparent increase in the molecular weight of the α-amylase produced during germination. This was probably due to the formation of a complex between the inhibitor encoded by the asi gene on chromosome 2 of barley and wheat α-amylase 2. Breeding of wheat with the barley inhibitor gene may reduce the impact of the high α-amylase levels that result from pre-harvest sprouting in wheat.     

Effect of cereal,fungal and bacterial α-amylase on the rheological and breadmaking properties of medium-protein wheats

Optimal levels of cereal malt, fungal and bacterial α-amylase for baking of medium-protein flours were found to be 5 - 10. 20 and 0.35 SKB/100 g, respectively. The dough consistency decreased considerably with time, depending on the source and level of α-amylase. The paste viscosities of the flours were reduced considerably by the cereal malts even at 2.5 SKB/100 g than by the fungal and bacterial α-amylase used optimally. Satisfactory loaf volumes were obtained with optimised α-amylase supplements without extraneous sugar in the bread formula. With 0.35 SKB of bacterial α-amylase, a bread having distinguishably softer texture was obtained as compared to cereal and fungal α-amylases. Accumulated maltose in the crumb indicated the need for a strain of baker's yeast with an active maltase system to render in situ contribution of α-amylase supplements more effective.     

Natural plant enzyme inhibitors. α-amylase inhibitors in millets

Twelve varieties of sorghum (Sorghum bicolor), 14 varieties of pearl millet (Pennisetum typhoideum), 12 varieties of setaria (Setaria italica), four varieties of ragi (Eleucine coracana), 11 varieties of echinocloa millet (Echinocloa colona), 13 varieties of proso (Panicium miliaceum), 11 varieties of kodo (Paspalum scorbiculatum) and 11 varieties of miliare (Panicium miliare) were screened for inhibitory activity against human salivary amylase. Echinocloa, proso, kodo and miliare had no detectable activity. Two strains of sorghum and one strain of pearl millet did not show α-amylase inhibitory activity. All other seeds had activity, the highest being observed in sorghum. Setaria had no action on human, bovine and porcine pancreatic anylases. Sorghum inhibitor did not act on bovine and porcine pancreatic amylases. Pearl millet and ragi extracts inhibited all the four α-amylases. The inhibitors were non-dialysable and were inactivated by pepsin treatment. Setaria and sorghum inhibitors were relatively thermolabile compared to ragi and pearl millet inhibitors.     

Secretion of mouse α-amylase from fission yeast Schizosaccharomyces pombe: Presence of chymostatin-sensitive protease activity in the culture medium

We have constructed two secretion vectors for Schizosaccharomyces pombe using an SV40 promoter and the secretion signals of the pGKL killer toxin complex derived from Kluyveromyces lactis. Although indigenous secretory glycoproteins tend to accumulate in the periplasmic space of S. pombe, we have succeeded in the secretion of mouse α-amylase into the culture medium. The efficiency of secretion, processing pattern, stability and culture conditions for mouse α-amylase were studied in S. pombe. The 128 kDa killer secretion signal was more effective in directing secretion of mouse α-amylase than the 28 kDa killer secretion signal. We detected a chymostatin-sensitive protease activity in the culture medium of S. pombe, which digests mouse α-amylase secreted into the culture medium. The addition of 5 μg/ml chymostatin was shown to protect mouse α-amylases from this degradation.     

Identification of α-amylase inhibitors in triticale grain

In this study three complete triticales, three substituted (one gene from rye has been replaced by one gene from wheat) triticales, and parental wheat and rye were analysed for α-amylase inhibitory activity to evaluate whether the genetic modification influenced triticale α-amylase inhibitory activity. Gel filtration chromatography and thermostability analyses were performed to partially isolate and characterize α-amylase inhibitors. Results demonstrated that substituted triticales and wheat had higher α-amylase inhibitory activities and higher water-soluble protein contents than complete triticales and rye. Sodium dodecylsulfate-PAGE-electrophoresis showed that all triticales, irrespective of their classification, inherited the water-soluble protein patterns from their parents: wheat and rye. In a substituted triticale (Pony ‘S’), two peaks with α-amylase inhibitory activity were resolved by gel filtration chromatography; they were designated T1 and T2 according to their order of elution. T1 showed a higher inhibitory activity but a lower thermostability at 70 °C than T2; T1 apparently comes from wheat, whereas T2 presumably comes from rye. © 1999 Society of Chemical Industry     

A simple gel-diffusion assay of α-amylase in ungerminated wheat grains

A rapid technique has been developed for non-destructive screening of half-grains of wheat for α-amylase content. The technique provides a sufficiently accurate estimation of the enzyme concentration to allow reliable selection for wide differences in activity and is suitable for screening large numbers of small samples, thus enabling entire F₂ generations to be assayed.     

Comparative immunological and chromatographic study of some plant β-Amylases

A comparative study has been made of the β-amylases of barley, wheat, rye, oats and sweet-potato by means of exclusion chromatography and immunochemical analysis. The reactivity of barley malt and wheat β-amylase was compared with different anti-barley and anti-wheat sera. In exclusion chromatography on Sephadex G100, barley β-amylase yielded four, and both wheat and rye, two active components, whereas oat and sweet-potato had only one active component. During the storage of barley, wheat and rye β-amylases the large-molecule components were split into smaller ones; no changes occurred in oat and sweet-potato β-amylases. On analysis against a specific barley β-amylase antiserum, wheat and rye β-amylase gave a reaction which indicated that they were immunologically partly identical with barley β-amylase, and identical with each other. This serum induced no reaction in β-amylases of sweet-potato and oats. The rye β-amylase precipitation line did not display enzymic activity after reaction with this antiserum. Analyses with different antisera of barley and wheat confirmed the partial immunological identity of barley malt and wheat β-amylase. With some barley antisera, partial inhibition of wheat β-amylase activity was observed. A similar phenomenon was apparent when barley malt β-amylase was precipitated with some wheat antisera.     

Interaction of amyloglucosidase and α-amylase supplements in breadmaking

Amyloglucosidase (Novo 150 L, 5 ml/100 kg) used with optimum α-amylase supplements [MSKB/100 g flour] of wheat malt, 7.5, fungal, 20, and bacterial, 0.4, increased the reducing sugars of sour doughs (pH 4.55, 3 h/30 °C) by 71.7 to 72.2, 94.0 to 103.1 and 71.7 to 77.0% as compared to 48.3 to 58.0, 61.1 to 71.3 and 7.3 to 12.0% of initial pH 5.8 doughs, respectively. Amyloglucosidase alone or with α-amylase supplements had negligible effect on mixing properties of dough, falling number values and paste viscosities which were affected more by the wheat malt α-amylase. The combined enzymes had a beneficial effect on bread quality of the medium-protein flours without extraneous sugar in the formula.     

Inhibition of wheat α-amylase by bran phytic acid

A proportion of the wheat samples tested for α-amylase activity by the Hagberg penetrometer method during the 1961 New Zealand harvest gave higher results on flours than on wheatmeals milled from the same wheats. This effect is due to the lower level of calcium available as enzyme cofactor in the meals, caused by combination with phytic acid. Because of this effect and because the internal distribution of α-amylase in wheat grains is variable, testing for sprout damage in milling wheat is best carried out on flours rather than wheatmeals.     

BACTERIAL EXPRESSION OF THE BIFUNCTIONAL α-AMYLASE/SUBTILISIN INHIBITOR FROM BARLEY

The bifunctional α-amylase/subtilisin Inhibitor (BASI) is an endogenous inhibitor of the high pl cereal α-amylases encoded by the amyl genes. Evaluation of the potential role of this protein in malting and brewing would be greatly assisted by the availability of large quantities of the protein. We have produced the protein by expression of the barley gene in bacteria. The barley gene was cloned into a pMAL vector and expressed as a fusion protein. The purified fusion protein was successfully cleaved with a specific protease to release the native BASI protein. The BASI produced by bacterial expression will be a useful source of the protein for studies of interactions with barley α-amylases and studies of the influence of this protein on malting and brewing.     

Screening of α-Amylase Suitable for Evaluating the Degree of Starch Retrogradation

To estimate the degrees of starch retrogradation in the complex foods, an enzymatic method using α-amylase from Bacillus subtilis was proposed in the previous report (Tsuge, H. et al.: Starch/Stärke 42 (1990), 213–216). However, actual digestibility of the enzyme for the native starch granules was not checked at that time. A comparative study to see the digestibility of native starch granules was carried out using four different α-amylase preparations and digestion of retrograded wheat starch was tested by two α-amylase preparations. Pancreas α-amylase preparation digested some native starch granules to a great extent, while Aspergillus oryzae enzyme did not digest native starch granules virtually. In conclusion, α-amylase preparation from A. oryzae was an ideal enzyme as the tool to distinguish between raw and gelatinized starches. It was justified for the use of A. oryzae enzyme as well as B. subtilis α-amylase to evaluate the retrograded starch contents in the complex foods.     

IMMUNOCHEMICAL. STUDY ON BARLEY α-AMYLASES*

Polymorphism of barley α-amylase was studied using immuno-electrophoresis and immuno-absorption in a gel medium with an anti-barley malt α-amylase immune serum: α-amylase from germinated seeds is antigenically heterogeneous. The two antigens which were demonstrated evolved differently upon germination. The bulk of the enzyme activity extracted from the seeds at different stages of germination differed antigenically from α-amylases found in developing barley seeds.     

Electrophoretic studies of α-amylase in wheat. II

The lack of correlation between the degree of sprouting and the α-amylase activity in wheat and rye, as well as the apparent variation in the falling number during ripening, can be explained as the result of two amylase systems acting during different stages of the development of the grain. During the early stages of the development of the grain, α-amylase is continuously inactivated. This process is reversible, however, and when the evaporation of moisture is retarded the α-amylase activity increases as a consequence of the higher amount of dissolved enzyme. This results in an occasional decrease in the falling number, which might amount to more than 50 sec. During germination, a new kind of α-amylase develops. The synthesis of the new form of α-amylase is irreversible and causes a much greater and permanent reduction in the falling number. During the initial stages of germination, the amylase activity thus increases owing to the combined action of both the original amylase and the new form. The two kinds of amylases show different electrophoretic patterns. Drying the grain after harvesting reduces the activity of ‘green’ amylase (amylase in unripened grain), which might explain the frequent observations of increasing falling number during storage.     

An Examination of α-Glucanase Activity from Thermoactinomyces putidus

Extracellular extracts from a strain of Thermoactinomyces putidus were shown to possess two α-amylases but there was no limit dextrinase (pullulanase) activity. This was demonstrated despite the fact that initial tests suggested that there may be some action towards pullulan on agar plates containing this substrate and in enzyme digests. The difficulties in using pullulan as a substrate in testing for limit dextrinase are discussed. The α-amylase was optimal at pH 5.8 and was inactivated by EDTA. It retained 84% of its activity after treatment for 30 min at 60°C in the absence of substrate. These results are considered with respect to the classification of this and other related microorganisms.     

Interrelations of Starch and Fungal α-Amylase in Breadmaking

The interrelation of starch in flour and fungal α-amylase in breadmaking was investigated using a low protein and a high protein flour. Loaf volume and bread quality were improved by the fungal α-amylase supplements. Gel chromatography for elution of DMSO soluble starch fraction and the results of amylose and amylopectin contents revealed somewhat similar situation in the fresh bread which differed from the elution profiles of soluble fractions of 3 and 5 day old loaves prepared with and without α-amylase supplements. Decreased recovery of starch in the DMSO soluble fractions indicated some degree of retrogradation. The higher amylose contents of the 5 day old bread crumb soluble starch fraction indicated a change in amylose and amylopectin ratio.     

Differential biochemical and kinetic properties of α-amylases from Rhyzopertha dominica (F.) progenies reared on wheat varieties differing in α-amylase inhibitory activity

The present study was accomplished to gain insights into the biochemical and kinetic properties of Rhyzopertha dominica’s α-amylase isoforms (named RdA70, RdA79, and RdA90) expressed in progenies reared in wheat varieties differing in α-amylase inhibitory activity. An inverse relationship was observed between the progenies’ amylase activity and the wheat inhibitory activity. Wheat samples with a high and low infestation (named HI-Borlaug and LI-Villa Juarez, respectively) were chosen to simplify the study. The progenies amylases were isolated by hydrophobic interaction chromatography, while the wheat samples were analyzed in α-amylase inhibitory activity by size exclusion chromatography. The isoforms RdA70 and RdA90 from LI-Villa Juarez progeny showed higher enzyme activities (73.8 and 43.4%, respectively) than those from HI-Borlaug. When the amylase isoforms were tested in susceptibility to inhibition by the inhibitory albumins, those from LI-Villa Juarez were more susceptible than those of the HI-Borlaug. Determination of the kinetic parameters revealed that RdA70 from the HI-Borlaug progeny showed 3.0-fold less starch affinity than that from the LI-Villa Juarez (K_m of 12.3 ± 1.8 versus 4.0 ± 0.3). The rest of the α-amylases did not show the same pattern as RdA70 in the HI-Bourlag as that their starch affinity was further reduced (RdA79 K_m = 23.1 ± 4.3, RdA90 K_m = 17.1 ± 2.9). Estimation of IC₅₀ values confirmed the high sensitivity of the three α-amylases of the LI-Villa Juarez progeny to wheat α-amylase inhibitors. The inhibitor constant K_i was the lowest for RdA70 in the LI-Villa Juarez progeny indicating the inhibitor’ tight binding to that isoenzyme. These results suggest that R. dominica uses RdA70 to bind large amounts of wheat α-amylase inhibitors than RdA79 and RdA90 as a physiological defense mechanism.