Fungal Α-amylase Research Articles

Hypothesis for how linear glutenin holds gas in dough

A hypothesis is put forward to explain how dough resists uncontrolled expansion of gas cells. Hand kneading, mixer action or gas pressure shears swollen protein in dough. Shearing orients linear glutenin molecules parallel to the shearing forces. Molecules with little overlap are pulled apart in the shear gradient, giving them further chances of getting better overlap with other molecules. Mixing thus forms a well overlapped and coherent gluten. When starch granules are pressed together the protein phase between them gets squeezed out sideways and so undergoes shear at angles of up to 90° to the main direction of flow. A similar result is found if the swollen protein suspension is regarded as being pressed through the tortuous pores between granules: since there will be flow in every pore, the gluten is sheared locally parallel to the pore direction. The pores between starch granules form a continuous three-dimensional network; therefore the protein also forms such a network, which appears isotropic because the orientation is local. Unless a gluten is weak, bubbles can expand without bursting because this network of protein resists forces in all directions until gelling of starch sets the crumb structure; further expansion then tears the starch/protein matrix enough to release the pressure in the bubbles. Elasticity does not arise from stretching the bonds of polypeptide chains but because folded conformations of chains are incomparably more probable than unfolded ones. Brownian motion helps unfolded chains to refold rapidly. Relaxation during normal proof times, while letting unfolded chains refold, does not destroy all orientation and overlap. Part of the action of fungal α-amylase may be to widen the pores between starch granules, reduce the size of small granules that could block such pores and delay starch gelation by slowing the entry of water. Fat could ease the relative movement of granules and protein, and the work of expanding gas cells. The fibrils seen when flour particles are wetted may illustrate the effects on glutenin of shear, produced in this case by sudden release of osmotic pressure. Moulding by sheeting gives an overall direction of orientation (DO) lacking in newly mixed dough. For the surfaces of a rolled sheet this DO lies in the machine direction, but inside the sheet it is sideways. In one-piece bread the asymmetry of gas cells results from easier expansion in the main DO, i.e. along the loaf. In four-piece bread the lack of room to expand horizontally forces cells to elongate vertically.

A new procedure for the measurement of fungal and bacterial α-amylase

A procedure for the measurement of fungal and bacterial α-amylase in crude culture filtrates and commercial enzyme preparations is described. The procedure employs end-blocked (non-reducing end)p-nitrophenyl maltoheptaoside in the presence of amyloglucosidase and α-glucosidase, and is absolutely specific for α-amylase. The assay procedure is simple, reliable and accurate.

The bread improving effect of fungal α-amylase

Nine characteristics of 62 commercial flours have been measured and the volumes recorded of loaves baked from them by the Chorleywood Bread Process (a mechanical dough development procedure) in the absence and presence of three levels of added fungal α-amylase (FAA). A further 22 flours have been used to test the predictive powers of regressions, derived from the original 62, between loaf volume, flour characteristics and total α-amylase activity. The volumes of loaves from all 84 flours increased with increase in the FAA activity added. The loaf volume/FAA response curve had a negative exponential form but had poor predictive ability. The loaf volume improving effect of FAA did not correlate with measured flour characteristics. Fungal α-amylase prolonged dough expansion time during baking. Multiple regression analysis was used to show that loaf volume was correlated positively with flour protein and negatively with flour colour.

Some characteristics of a raw starch digestion inhibitory factor fromAspergillus niger

The effect of an inhibitory factor (IF) fromAspergillus niger 19 on raw starch digestion by pure glucoamylase I of blackAspergillus, pure glucoamylae ofRhizopus niveus, bacterial α-amylase, fungal α-amylase and various combination was investigated. The IF caused higher inhibition of raw starch hydrolysis by the combined action of glucoamylase and fungal α-amylase than of hydrolysis by the individual enzymes. A protein moiety of IF might play an active part in this inhibition phenomenon. The IF was bound to starch granules, preventing hydrolysis by the enzymes, and caused decreased raw starch hydrolysis yields.

Production of α-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

alpha-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55 degrees C and 5.5, respectively, and the apparent K(m) for starch was 0.8 mg ml. The products of alpha-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of alpha-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial alpha-amylase. Activities of alpha-amylase up to 4.4 U ml (1 U represents 1 mumol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml), alpha-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed alpha-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of alpha-amylase (initial concentration, 2.5 mg ml), they were found to be less effective than starch, but maltose was almost as effective. The fungal alpha-amylase was found to be stable at 60 degrees C in the presence of low concentrations of starch (</=5%), suggesting that it may be suitable for industrial application.

Production and Characteristics of Raw-Starch-Digesting α-Amylase from a Protease-Negative Aspergillus ficum Mutant

Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N'-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher alpha-amylaseactivity than the parent strain under submerged culture at 30 degrees C for 24 h. About 70% of the total alpha-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable alpha-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable alpha-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal alpha-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.

Studies on screening and isolation of α-amylase inhibitors of soil microorganisms (I)

To find amylase inhibitors produced by microorganisms from soil, a strain which had a strong inhibitory activity against bacterial α-amylase was isolated from the soil sample collected in Seoul. The morphological and physiological characteristics of this strain on several media and its utilization of carbon sources showed that it was one ofStreptomyces species according to the International Streptomyces Project method. The amylase inhibitor of this strain was purified by means of acetone precipitation, adsorption on Amberlite XAD-2, and column chromatography on Amberlite CG-50 and SP-Sephadex C-25. The inhibitor was stable at the pH range of 1∼10 and at 100°C for half an hour, and had inhibitory activities against other amylases such as salivary α-amylase, pancreatic α-amylase, fungal α-amylase and glucoamylase. The kinetic studies of the inhibitor showed that its inhibitory effect on starch hydrolysis by α-amylase was non-competitive.

New Enzymes for Brewing: Promozyme, a Debranching Enzyme and SP-249, a Glucanase

Two newly developed polysaccharide-degrading enzymes were evaluated. One, a pullulanase, Promozyme™, can be used alone during mashing, wort treatment, or fermentation to increase the fermentability of wort. When used during fermentation in combination with a fungal α-amylase, Fungamyl®, apparent attenuations above 100% can be reached. The other enzyme, a multicomponent carbohydrase, SP-249, degrades the nonstarch polysaccharides of barley and malt, resulting in reduced wort viscosity and improved runoff. SP-249 is more efficient than commercial β-glucanases.

New Enzymes for Brewing: Promozyme, a Debranching Enzyme and SP-249, a Glucanase

Two newly developed polysaccharide-degrading enzymes were evaluated. One, a pullulanase, Promozyme™, can be used alone during mashing, wort treatment, or fermentation to increase the fermentability of wort. When used during fermentation in combination with a fungal α-amylase, Fungamyl®, apparent attenuations above 100% can be reached. The other enzyme, a multicomponent carbohydrase, SP-249, degrades the nonstarch polysaccharides of barley and malt, resulting in reduced wort viscosity and improved runoff. SP-249 is more efficient than commercial β-glucanases.

Determination of fungal α-amylase by flow injection analysis

A manual iodine-starch method is adapted to analyse broths from Aspergillus oryzae fermentations. The method is base on degradation of starch by the enzyme, and measurement of the residual starch/iodine complex at 570 nm. Calibration graphs are linear for the range 0.01–0.1 amylase units ml −1. Eighty samples per hour can be processed.

Immobilization of biocatalysts on new route transition metal-activated inorganic supports

Controlled pore glass was activated by transition metal salts, FeCl 2, FeCl 3, VCl 3 and ZrCl 4, and derivatized by the method of Cabral et al. 1 Glucoamylase (exo-1,4-α- d-glucosidase, EC 3.2.3.1) was coupled to the aldehyde derivative, the resulting initial activities being 61.7, 71.8, 57.6 and 63.2 U g −1 of matrix, respectively. The immobilized glucoamylase preparations in which the carrier was activated with zirconium (IV) chloride displayed a very stable behaviour, with a half-life of 1709 h, in contrast with the preparations activated with iron(II) chloride ( t 1 2 = 43 7 h , iron(III) chloride ( t 1 2 = 518 h ) and vanadium(III) chloride ( t 1 2 = 893 h ). Several biocatalysts, fungal α-amylase (EC 3.2.1.1), bacterial α-amylase (EC 3.2.1.1), β- d-fructofuranosidase (invertase, EC 3.2.1.26), papain (EC 3.4.22.2) and yeast cells of Saccharomyces cerevisiae with β- d-fructofuranosidase activity were immobilized on aldehyde derivatives of titanium(IV)-activated porous silica (Spherosil), half-lives of 140, 157, 980, 236 and 413 h being obtained, respectively.

アミラーゼによる生でんぷん分解

(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.