α- and β-Amylases in seed germination

During the first 24 h of germination of wheat seeds, starch is hydrolysed by free β-amylase. In the next 24 h, some amount of inactive form of β-amylase is converted into active form and this together with α-amylase synthesizedde novo brings about the hydrolysis of starch. The amount of α-amylase is greater in seeds with embryo intact than with embryo excised after 24 h hydration. However, at later stages of seed germination α-amylase becomes predominant and the activity of β-amylase steadily diminishes.     

The effect of guar galactomannan and water availability during hydrothermal processing on the hydrolysis of starch catalysed by pancreatic α-amylase

The effects of water-soluble nonstarch polysaccharides (sNSP) on human metabolism are considered to be beneficial because they decrease postprandial glycaemia and insulinaemia following ingestion of starch-rich foods. The mechanisms by which sNSP attenuate the postprandial rise in blood glucose are not well understood but their presence increases the viscosity of gastrointestinal contents, which affects physiological functions, e.g. gastric emptying and peristalsis. Increased viscosity and decreased water activity during hydrothermal treatment of starch could influence α-amylase action.Using guar galactomannan as a representative of sNSP, we found that galactomannan has a direct noncompetitive inhibitory effect on α-amylase with a K_i value of approximately 0.5% (3.3 μM). The inhibition is not time dependent and studies suggest direct binding of the enzyme to galactomannan; the resulting galactomannan–amylase complex being inactive. Processing of starch at low water levels greatly affects the catalytic efficiency of α-amylase. The K_m value for starch heat treated in limited water is raised and k_cat is lowered relative to starch gelatinised in excess water. Since galactomannan has no effect on the K_m of α-amylase, we conclude that the inhibitory action of the polymer is not secondary to a decrease in available water. Neither does it seem to be a consequence of impaired diffusion of enzyme, substrate and products because of an increase in viscosity of the medium.Thus, the effects of sNSP in lowering postprandial glycaemia not only involve modifications of gut physiology, but also include direct inhibition of the first stage in the biochemical degradation of starch.     

Rice α-glucosidase isozymes and isoforms showing different starch granules-binding and -degrading ability

Insoluble starch granules stored in plant seeds have generally been considered to be degraded effectively by the combination of amylolytic enzymes following initial attack by de novo synthesized α-amylase at germination. We have shown that rice (Oryza sativa L., var Nipponbare) α-glucosidase isozymes (ONG1, ONG2, and ONG3) are also capable of binding to and degrading starch granules directly, indicating the direct liberation of glucose from starch granules by α-glucosidase at germination. ONG1 and ONG2 are encoded in a distinct locus of the rice genome, while ONG2 and ONG3 are generated by alternative splicing. Interestingly, each of the α-glucosidase isozymes showed different action toward starch granules. In addition, two ONG2 isoforms were found to be produced by post-translational proteolysis. The proteolysis induced changes in binding to and degradation of starch granules.     

High day–night transition temperature alters nocturnal starch metabolism in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Transitory starch plays a vital role in maintenance respiration as its degradation products provide substrate for the night respiration. A study was conducted with two contrasting rice cultivars: Vandana (high night temperature susceptible) and Nagina 22 (high night temperature tolerant) by subjecting them to increase in transition temperature from anthesis to physiological maturity. Night respiration on plant area basis increased by 35% in Vandana at 5 days after anthesis but was unaffected in tolerant cultivar. A simultaneous 18% decrease in starch content was observed in the susceptible cultivar. An analysis of the starch-metabolizing enzymes showed that activity of β-amylase increased markedly in Vandana whereas both β and α-amylase decreased in Nagina 22 following high day to night transition temperature exposure. The level of starch breakdown product, maltose, increased in the susceptible cultivar but glucose levels declined in both the cultivars. Concurrently, expression of chloroplastic isoforms α-amylase OsAMY1, OsAMY2 and β-amylase OsBAM2 increased in Vandana. A lower accumulation of dry matter was recorded in the susceptible than the tolerant cultivar. Our study elucidated the regulatory role of transitory starch in supporting the high day to night transition temperature-induced night-time respiration which is mediated by the increased activity of β-amylase through enhanced expression of OsBAM2 in flag leaves of susceptible cultivar.     

The surface structure of a complex substrate revealed by enzyme kinetics and Freundlich constants for α-amylase interaction with the surface of starch

Background

Starch is a main source of carbohydrate in human diets, but differences are observed in postprandial glycaemia following ingestion of different foods containing identical starch contents. Such differences reflect variations in rates at which different starches are digested in the intestine. In seeking explanations for these differences, we have studied the interaction of α-amylase with starch granules. Understanding this key step in digestion should help with a molecular understanding for observed differences in starch digestion rates.

Methods

For enzymes acting upon solid substrates, a Freundlich equation relates reaction rate to enzyme adsorption at the surface. The Freundlich exponent (n) equals 2/3 for a liquid-smooth surface interface, 1/3 for adsorption to exposed edges of ordered structures and 1.0 for solution–solution interfaces. The topography of a number of different starch granules, revealed by Freundlich exponents, was compared with structural data obtained by differential scanning calorimetry and Fourier transform infrared spectroscopy with attenuated total internal reflectance (FTIR-ATR).

Results

Enzyme binding rate and FTIR-ATR peak ratio were directly proportional to n and Δ_gelH was inversely related to n. Amylase binds fastest to solubilised starch and to granules possessing smooth surfaces at the solid–liquid interface and slowest to granules possessing ordered crystalline surfaces.

Conclusions

Freundlich exponents provide information about surface blocklet structures of starch that supplements knowledge obtained from physical methods.

General Significance

Nanoscale structures at the surface of starch granules influence hydrolysis by α-amylase. This can be important in understanding how dietary starch is digested with relevance to diabetes, cardiovascular health and cancer.     

Development of new α-amylases for raw starch hydrolysis

This paper describes the discovery of a new 4 domain α-amylase from Anoxybacillus contaminans which very efficiently hydrolyses raw starch granules. Compared to traditional starch liquefying α-amylases, this new 4 domain α-amylase contains a starch binding domain. The presence of this starch-binding domain enables the enzyme to efficiently hydrolyse starch at a temperature below the gelatinisation temperature. At a reaction temperature of 60°C and in combination with a glucoamylase from Aspergillusniger it was possible to liquefy 99% of the starch obtaining a DX value of 95%.

Furthermore, we describe how the current HFCS process can be turned into a low temperature simultaneous liquefaction and saccharification process by using this new 4 domain α-amylase in combination with a glucoamylase.     

Preparation of maltotriitol-rich malto-oligosaccharide alcohol from starch

Malto-oligosaccharide alcohols (MOSA) are one of the most important sugar alcohols widely used as sweetener in food, cosmetic, and pharmaceutical industries in recent years, of which maltotriitol-rich MOSA is much more recognized. With the aim of preparing maltotriitol-rich MOSA from starch, a novel process was developed and optimized. Starch was first liquefied with thermostable Bacillus licheniformis α-amylase. The liquefied starch was then saccharified to yield maltotriose-rich malto-oligosaccharides under the cooperative actions of Bacillus naganoensis pullulanase, Bacillus amyloliquefaciens α-amylase, and barley bran β-amylase. The maltotriitol-rich MOSA was finally prepared by chemical hydrogenation. Under the optimized conditions, maltotriitol-rich MOSA containing 42.18% maltotriitol was obtained with a conversion rate of 104.57% from starch. The process can be employed for large-scale preparation of maltotriitol-rich MOSA, and a further modification of the process can lead to the formulation of various types of MOSA with different percentages of components of sugar alcohols.     

Hydrolysis of amylopectin by amylolytic enzymes: level of inner chain attack as an important analytical differentiation criterion

Differences in amylase action pattern on amylopectin were demonstrated by the relation between the decrease in potassium iodide-iodine binding of waxy maize starch and the increase in reducing value during hydrolysis, as expressed by the RV₈₀ value (i.e., the reducing value for a potassium iodide-iodine binding value of 80% of that of the starting material). In the initial stages of the hydrolysis, the ratio of the increase in the level of reducing polysaccharides to the increase in the total level of reducing sugars formed during amylolysis of amylopectin can be considered as a measure of the level of inner chain attack (LICA) in the overall hydrolysis of the amylopectin structure and correlated with the respective RV₈₀ value. Bacillus amyloliquefaciens α-amylase and Aspergillus oryzae α-amylase, with the lowest RV₈₀ and the highest LICA values, hydrolysed the inner chains of amylopectin to a greater extent than did porcine pancreatic α-amylase. In the initial stages of hydrolysis, Bacillus stearothermophilus maltogenic amylase, like the Bacillus cereus β-amylase, did not display any significant degree of internal hydrolysis of amylopectin, in line with the high RV₈₀ and very low LICA values for these enzymes. However, at the later stages of hydrolysis, the maltogenic amylase probably exhibited a significant degree of internal hydrolysis of amylopectin, which itself seems to depend on temperature. The temperature dependence of the hydrolysis pattern of this enzyme is relevant for interpretation of its action as antifirming enzyme in bread-making applications.     

An investigation of the action of porcine pancreatic α-amylase on native and gelatinised starches

The action of pancreatic α-amylase (EC 3.2.1.1) on various starches has been studied in order to achieve better understanding of how starch structural properties influence enzyme kinetic parameters. Such studies are important in seeking explanations for the wide differences reported in postprandial glycaemic and insulinaemic indices associated with different starchy foodstuffs. Using starches from a number of different sources, in both native and gelatinised forms, as substrates for porcine α-amylase, we showed by enzyme kinetic studies that adsorption of amylase to starch is of kinetic importance in the reaction mechanism, so that the relationship between reaction velocity and enzyme concentration [E₀] is logarithmic and described by the Freundlich equation. Estimations of catalytic efficiencies were derived from measurements of k_cat/K_m performed with constant enzyme concentration so that comparisons between different starches were not complicated by the logarithmic relationship between E₀ and reaction velocity. Such studies reveal that native starches from normal and waxy rice are slightly better substrates than those from wheat and potato. After gelatinisation at 100°C, k_cat/K_m values increased by 13-fold (waxy rice) to 239-fold (potato). Phosphate present in potato starch may aid the swelling process during heating of suspensions; this seems to produce a very favourable substrate for the enzyme. Investigation of pre-heat treatment effects on wheat starch shows that the relationship between treatment and k_cat/K_m is not a simple one. The value of k_cat/K_m rises to reach a maximum at a pre-treatment temperature of 75°C and then falls sharply if the treatment is conducted at higher temperatures. It is known that amylose is leached from starch granules during heating and dissolves. On cooling, the dissolved starch is likely to retrograde and become resistant to amylolysis. Thus the catalytic efficiency tends to fall. In addition, we find that the catalytic efficiency on the different starches varies inversely with their solubility and we interpret this finding on the assumption that the greater the solubility, the greater is the likelihood of retrogradation. We conclude that although α-amylase is present in high activity in digestive fluid, the enzymic hydrolysis of starch may be a limiting factor in carbohydrate digestion because of factors related to the physico-chemical properties of starchy foods.     

Cloning of α-amylase gene fromSchwanniomyces occidentalis and expression inSaccharomyces cerevisiae

The cloning of α-amylase gene ofS. occidentalis and the construction of starch digestible strain of yeast,S. cerevisiae AS. 2. 1364 with ethanol-tolerance and without auxotrophic markers used in fermentation industry were studied. The yeast/E.coli shuttle plasmid YCEp1 partial library ofS. occidentalis DNA was constructed and α-amylase gene was screened in S.cerevisiae by amylolytic activity. Several transformants with amylolysis were obtained and one of the fusion plasmids had an about 5.0 kb inserted DNA fragment, containing the upstream and downstream sequences of α-amylase gene fromS. occidentalis. It was further confirmed by PCR and sequence determination that this 5.0 kb DNA fragment contains the whole coding sequence of α-amylase. The amylolytic test showed that when this transformant was incubated on plate of YPDS medium containing 1 % glum and 1 % starch at 30°C for 48 h starch degradation zones could be visualized by staining with iodine vapour. α-amylase activity of the culture filtratate is 740–780 mU/mL and PAGE shows that the yeast harboring fusion plasmids efficiently secreted α-amylase into the medium, and the amount of the recombinant α-amylase is more than 12% of the total proteins in the culture filtrate. These results showed that α-amylase gene can be highly expressed and efficiently secreted inS. cerevisiae AS. 2.1364, and the promotor and the terminator of α-amylase gene fromS. occidentalis work well inS. cercvisiac AS. 2.1364.     

Transient responses of <Emphasis Type="Italic">Wickerhamia</Emphasis> sp. yeast continuous cultures to qualitative changes in carbon source supply: induction and catabolite repression of α-amylase synthesis

The purpose of this study was to evaluate the inductive effect of starch and maltose, and the repressive/inhibitory effect of glucose, on amy-1 gene expression and α-amylase production by Wickerhamia sp., using continuous culture under transient-state conditions at a dilution rate (D) of 0.083 h^?1. Induction and repression kinetics of α-amylase were studied by changing the medium feed from glucose to maltose or starch in the induction experiments and vice versa in the repression experiments. Expression levels of amy-1 gene were measured by RT-qPCR. Results showed that starch was a more efficient inducer of α-amylase synthesis compared to maltose, with maximum accumulation rate constants of 0.424 and 0.191 h^?1, respectively. In contrast, α-amylase synthesis in starch and maltose cultures was partially repressed by glucose as indicated by a specific activity close to basal levels and a decay constant rate (??0.065 and ??0.069 h^?1, respectively) higher than ??D. A linear dependence of the specific rate of α-amylase production on mRNA relative abundance of amy-1 gene was observed. An inhibitory effect of glucose was not observed even at a concentration of 30 g L^?1. In conclusion, the transient continuous culture is a useful tool to determine the qualitative and quantitative effects of maltose and starch on α-amylase induction and of glucose on enzyme repression, as well as to obtain a detailed understanding of the dynamic behavior of the yeast culture. Furthermore, results showed that amylaceous substrates can be very effective carbon sources for the production of α-amylase without being inhibited by glucose.     

The hyperthermophilic α-amylase from <Emphasis Type="Italic">Thermococcus</Emphasis> sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium ions for thermostability, and is a promising alternative to commercially available α-amylases to increase the efficiency of industrial processes like the liquefaction of starch. We analyzed the amino acid sequence of this α-amylase by sequence alignments and structural modeling, and found that this α-amylase closely resembles the α-amylase from Pyrococcus woesei. The gene of this α-amylase was cloned in Escherichia coli and the recombinant α-amylase was overexpressed and purified with a combined renaturation-purification procedure. We confirmed thermostability and exogenous calcium ion independency of the recombinant α-amylase and further investigated the mechanism of the independency using biochemical approaches. The results suggested that the α-amylase has a high calcium ion binding affinity that traps a calcium ion that would not dissociate at high temperatures, providing a direct explanation as to why the addition of calcium ions is not required for thermostability. Understanding of the mechanism offers a strong base on which to further engineer properties of this α-amylase for better potential applications in industrial processes.     

Effect of adrenergic agents on α-amylase release and adenosine 3′,5′-monophosphate accumulation in rat parotid tissue slices

The role of cyclic AMP in stimulus-secretion coupling was investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20–30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both α-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effective. A parallel order of potency and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fraction. The results suggest that these drugs are acting on the parotid acinar cell through a β₁-adrenergic mechanism.At the lowest concentrations tested, each of the adrenergic agonists stimulated significant α-amylase release with no detectable stimulation of cyclic AMP accumulation. Even in the presence of theophylline, phenylephrine at several concentrations increased α-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intracellular concentration of cyclic AMP may not be necessary for stimulation of α-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of α-amylase release by isoproterenol.Stimulation of α-amylase release by phenylephrine was only partially blocked by either α- or β-adrenerg blocking agents, whereas stimulation of adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolamine. Phenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and α-amylase release. However, phenoxybenzamine also potentiated the stimulation of α-amylase release by N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate. These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using α-adrenergic blocking agents as tools for investigation of α- and β-adrenergic antagonism.     

Production,Purification, and Characterization of α-Amylase from Solventogenic Clostridium sp. BOH3

A solventogenic strain of Clostridium sp. BOH3 produces extracellular α-amylase (7.15 U/mg protein) in reinforced clostridial medium supplemented with sugarcane bagasse hydrolysate (1 % w/v) and a small amount of starch (0.1 % w/v), which is essential for the expression of α-amylase. In the presence of α-amylase, BOH3 utilizes starch directly without any pretreatment and produces butanol almost equivalent (～90 %) to the production of butanol from glucose. α-Amylase can be purified from culture supernatant by using one-step weak anion exchange chromatography with a yield of 43 %. In peptide fingerprinting analysis, this enzyme shows homology with α-amylase produced by Clostridium acetobutylicum ATCC824. However, the molecular weight is 54 kDa, which is smaller than α-amylase of ATCC824 (84 kDa). This enzyme has optimum temperature at 45–50 °C and optimum pH at 4.5–5.5. Under this condition, the enzyme activity is 91.32 U/mg protein, and its K _m and V _max values are 1.71?±?0.02 mg/ml and 96.13?±?0.15 μmol/min/mg protein, respectively. Activity of this α-amylase can be enhanced (>1.5 times) by addition of Ca²⁺ and Co²⁺ and its activity can be maintained at an acidic pH (pH 3–5) for about 24 h. These unique characteristics suggest that this enzyme can be used for saccharification of starch for production of biofuel in one pot.     

Modified α-amylase activity among insecticide-resistant and -susceptible strains of the maize weevil, Sitophilus zeamais

Fitness cost is usually associated with insecticide resistance and may be mitigated by increased energy accumulation and mobilization. Preliminary evidence in the maize weevil (Coleoptera: Curculionidae) suggested possible involvement of amylases in such phenomenon. Therefore, α-amylases were purified from an insecticide-susceptible and two insecticide-resistant strains (one with fitness cost [resistant cost strain], and the other without it [resistant no-cost strain]). The main α-amylase of each strain was purified by glycogen precipitation and ion-exchange chromatography (≥70-fold purification, ≤19% yield). Single α-amylase bands with the same molecular mass (53.7 kDa) were revealed for each insect strain. Higher activity was obtained at 35-40 °C and at pH 5.0-7.0 for all of the strains. The α-amylase from the resistant no-cost strain exhibited higher activity towards starch and lower inhibition by acarbose and wheat amylase inhibitors. Opposite results were observed for the α-amylase from the resistant cost strain. Although the α-amylase from the resistant cost strain exhibited higher affinity to starch (i.e., lower K_m), its V_max-value was the lowest among the strains, particularly the resistant no-cost strain. Such results provide support for the hypothesis that enhanced α-amylase activity may be playing a major role in mitigating fitness costs associated with insecticide resistance.