Inter organ comparison of amylases and starch content in mungbean seedlings

During seedling growth of mungbean in dark, depletion of cotyledonary starch is reflected by an increase in starch content of root and shoot. With progress of seedling growth, amylolytic activity increases in all organs i.e. cotyledons, shoots and roots. A rapid turnover of starch in shoots and roots has been proposed. Amylase activity of seedlings was in the order of cotyledons>shoots>roots. Five days after germination (DAG) α-amylase from cotyledons of mungbean seedlings was purified using ammonium sulphate precipitation, DEAE cellulose and sephadex G-150 column chromatography. Phytic acid was a stronger inhibitor of α-amylase than EDTA. Phytic acid, Hg²⁺, Zn²⁺ and Mn²⁺ were non-competitive inhibitors and the corresponding Ki values were 5.0–5.7, 0.36–0.38, 2.6–3.8 and 0.7–0.8 mol·M⁻³. Elution patterns of α-amylases of cotyledons, shoots and roots on sephadex G-100 column showed that cotyledonary α-amylase had a higher molecular mass than that of shoot and root α-amylases which had identical molecular masses. All α-amylases showed the same optimum pH 5.0 whereas optimum temperature was 55 °C for cotyledonary and 45 °C for shoot and root α-amylases. In all these tissues α-amylases were stable to 30 min heat treatment at 50 °C however unlike cereal α-amylases they lost activity at 70 °C. Km for α-amylases from cotyledons, shoots and roots with starch was 1.9, 4.3 and 6.6 mg per cm³, respectively. α-amylase of cotyledons and roots showed activity in reactions with various substrates in the order of starch>amylose>dextrin-I=dextrin-IV>α-cyclodextrin=β-cyclodextrin>amylopectin>pullulan. The shoot α-amylase showed high activity with amylopectin, which was comparable with that obtained with amylose, and the activity with α and β-cyclodextrin was higher in comparison with dextrin-I and IV. The α-amylases from these tissues liberated maltose, maltotriose and higher oligosaccharides from starch. It could be concluded that amylases from different organs of a seedling could have different physical and kinetic properties.     

Myoinositol hexaphosphate as a potential inhibitor of α-amylases

Myoinositol hexaphosphate (MHP) strongly inhibited α-amylases of different origins. The inhibition of wheat α-amylase is noncompetitive with an apparent K_i value of 1 mM, pH dependent and markedly increased by the preincubation of enzyme with MHP before the addition of substrate. Addition of Ca²⁺ did not reverse the inhibition of α-amylase indicating that its inhibition was not due to the binding of Ca²⁺ by MHP.     

Bifunctional inhibitor of α-amylase/trypsin from wheat grain

A trypsin inhibitor, isolated from whole-wheat grain (Triticum aestivum L.) by the method of biospecific chromatography on trypsin-Sepharose, was potent in inhibiting human salivary α-amylase. The bifunctional α-amylase/trypsin inhibitor was characterized by a narrow specificity for other α-amylases and proteinases. The high thermostability of the inhibitor was lost in the presence of SH group-reducing agents. The inhibitor-trypsin complex retained its activity against α-amylase. The inhibitor—α-amylase complex was active against trypsin. Studies of the enzyme kinetics demonstrated that the inhibition of α-amylase and trypsin was noncompetitive. Our results suggest the existence of two independent active sites responsible for the interaction with the enzymes.     

Characterization of Active Barley α-Amylase 1 Expressed and Secreted by Saccharomyces cerevisiae

Recombinant barley α-amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified α-amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k_cat/K_m was 2.7 × 10² mM^?1.s^?1, consistent with those of α-amylases from plants and other sources.     

Purification by Affinity Chromatography of {alpha}-Amylase--A Main Amylase in Cotyledons of Germinating Vigna mungo Seeds

In Vigna mungo cotyledons, the -amylase activity increased markedlyduring germination at 27°C in the dark, while the activityof other amylases was very low. The -amylase was purified from4-day-old cotyledons by affinity chromatography on epoxyactivatedSepharose 6B substituted with rß-cyclodextrin andby column chromatography on Bio-Gel P-200. Gel filtration andpolyacrylamide gel electrophoresis showed that the enzyme existsmostly as a monomer (43,000 daltons), but partially aggregatesto form dimer, trimer and further multimers. Ca²⁺ protectedthe -amylase against heat inactivation. Incubation of the enzymewith 5 mM EDTA or dialysis against 10 mM EDTA resulted in a50–90% loss of activity. The inactivation was partiallyreversed by the addition of Ca²⁺. Other properties, such asthe amino acid composition, K_m value, pH optimum and activationenergy were similar to those of other plant -amylases. (Received May 6, 1981; Accepted June 22, 1981)     

Calcium regulation of the secretion of α-amylase isoenzymes and other proteins from barley aleurone layers

The effect of calcium on the secretion of α-amylase (EC 3.2.1.1) and other hydrolases from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was studied. Withdrawal of Ca²⁺ from the incubation medium of aleurone layers preincubated in 5 μM gibberellic acid (GA₃) and 5 mM CaCl₂ results in a 70–80% reduction in the secretion of α-amylase activity to the incubation medium. Agar-gel electrophoresis shows that the reduction in α-amylase activity following Ca²⁺ withdrawal is correlated with the disappearance of group B isoenzymes from the incubation medium. The secretion of isoenzymes of group A is unaffected by Ca²⁺. The addition of Ca²⁺ stimulates the secretion of group-B isoenzymes but has no measurable effect on either the α-amylase activity or the isoenzyme pattern of aleurone-layer extracts. Pulse-labelling experiments with [³⁵S]methionine show that Ca²⁺ withdrawal results in a reduction in the secretion of labelled polypeptides into the incubation medium. Immunochemical studies also show that, in the absence of Ca²⁺, α-amylase isoenzymes of group B are not secreted into the incubation medium. In addition to its effect on α-amylase, Ca²⁺ influences the secretion of other proteins including several acid hydrolases. The secretion of these other proteins shows the same dependence on Ca²⁺ concentration as does that of α-amylase. Other cations can promote the secretion of α-amylase to less and varying extents. Strontium is 85% as effective as Ca²⁺ while Ba²⁺ is only 10% as effective. We conclude that Ca²⁺ regulates the secretion of enzymes and other proteins from the aleurone layer of barley.     

Microbial α-amylase: A biomolecular overview

α-Amylase is an important amylolytic enzyme participating in hydrolysis of starch, the most common carbohydrate in nature. Compared to plant and animal origins, microbial α-amylase is the most popular source of industrial α-amylase. As such, high productive and favourable α-amylases for wider range of applications are highly sought after demands. The expression of α-amylase is regulated by its structural gene, amyR, DegU-P, PrsA lipoprotein, cutinase and other similar flanking genes, components of gene expression regulatory systems, molecular chaperones and enzymes. Moreover, the characteristics of α-amylase are closely related to the structures of constitutive domains and conserved regions, particularly the functional regions such as Ca²⁺-binding sites, non-catalytic carbohydrate-binding modules and surface-binding sites. Recent production of α-amylase based on genetic engineering and academic researches focused on mechanisms of catalysis greatly benefit from these biomolecular studies. Despite rapid developments, no reviews have systematically summarized these fundamental biomolecular studies. This review outlines microbial α-amylase at gene and structure levels by covering these significant aspects. The computer analytical tools are also reported, especially frequently used databases. A deeper understanding of the biomolecular basis of microbial α-amylase will significantly pave greater opportunities for industrial α-amylase and open our minds towards its related or even other enzymes.     

A permeability transition in liver mitochondria and liposomes induced by α,ω-dioic acids and Ca2+

The article examines the molecular mechanism of the Ca²⁺-dependent cyclosporin A (CsA)-insensitive permeability transition in rat liver mitochondria induced by α,ω-dioic acids. The addition of α,ω-hexadecanedioic acid (HDA) to Ca²⁺-loaded liver mitochondria was shown to induce a high-amplitude swelling of the organelles, a drop of membrane potential and the release of Ca²⁺ from the matrix, the effects being insensitive to CsA. The experiments with liposomes loaded with sulforhodamine B (SRB) revealed that, like palmitic acid (PA), HDA was able to cause permeabilization of liposomal membranes. However, the kinetics of HDA- and PA-induced release of SRB from liposomes was different, and HDA was less effective than PA in the induction of SRB release. Using the method of ultrasound interferometry, we also showed that the addition of Ca²⁺ to HDA-containing liposomes did not change the phase state of liposomal membranes—in contrast to what was observed when Ca²⁺ was added to PA-containing vesicles. It was suggested that HDA/Ca²⁺- and PA/Ca²⁺-induced permeability transition occurs by different mechanisms. Using the method of dynamic light scattering, we further revealed that the addition of Ca²⁺ to HDA-containing liposomes induced their aggregation/fusion. Apparently, these processes result in a partial release of SRB due to the formation of fusion pores. The possibility that this mechanism underlies the HDA/Ca²⁺-induced permeability transition of the mitochondrial membrane is discussed.     

Purification and characterization of a novel α-amylase from a newly isolated Bacillus methylotrophicus strain P11-2

An aerobic bacterial strain P11-2 with high amylolytic activity was isolated from soil sample collected from wheat field of Jiyuan, China. The strain was identified as Bacillus methylotrophicus by morphological and physiological characteristics as well as by analysis of the gene encoding the 16S rRNA. The α-amylase was purified to homogeneity by a combination of 80% (NH₄)₂SO₄ precipitation, DEAE FF anion exchange, and superdex 75 10/300 GL gel filtration chromatography. The purified α-amylase exhibited specific activity of 330.7 U/mg protein that corresponds to 13.1 fold purification. The relative molecular mass of the α-amylase was 44.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for enzyme activity were 7.0 and 70 °C, respectively. The α-amylase activity was stimulated by Mg²⁺, Ba²⁺, Al³⁺ and dl-dithiothreitol (DTT), however, Ca²⁺ almost had no activation or inhibition on the α-amylase. After 4 h of reaction toward soluble starch, the end products were glucose, maltose and maltotriose. The 10 residues of the N-terminal sequence of the purified α-amylase were SVKNGQILHA, which showed no homology to other reported α-amylases from Bacillus strain.     

Purification and Some Properties of Acid-stable α-Amylases from Shochu koji (Aspergillus kawachii)

Aspergillus kawachii α-amylase [EC 3.2.1.1] I and II were purified from shochu koji extract by DEAE Bio-Gel A ion exchange chromatography, Sephacryl S-300 gel chromatography (pH 3.6), coamino dodecyl agarose column chromatography and Sephacryl S-200 gel chromatography. By gel chromatography on a Sephacryl S-300 column, the molecular weights of the purified α-amylase I and II were estimated to be 104,000 and 66,000, respectively. The isoelectric points of α-amylase I and II were 4.25 and 4.20, respectively. The optimal pH range of α-amylase I was 4.0 to 5.0, and the optimum pH of α-amylase II was 5.0. The optimum temperatures of both α-amylases were around 70°C at pH 5.0. Both α-amylases were stable from pH 2.5 to 6.0 and up to 55°C, retaining more than 90% of the original activities. Heavy metal ions such as Hg^{2 +} and Pb^{2 +} were potent inhibitors for both α-amylases.     

Interactions of barley α-amylase isozymes with Ca2 + , substrates and proteinaceous inhibitors

α-Amylases are endo-acting retaining enzymes of glycoside hydrolase family 13 with a catalytic (β/α)₈-domain containing an inserted loop referred to as domain B and a C-terminal anti-parallel β-sheet termed domain C. New insights integrate the roles of Ca^2?+?, different substrates, and proteinaceous inhibitors for α-amylases. Isozyme specific effects of Ca^2?+? on the 80% sequence identical barley α-amylases AMY1 and AMY2 are not obvious from the two crystal structures, containing three superimposable Ca^2?+? with identical ligands. A fully hydrated fourth Ca^2?+? at the interface of the AMY2/barley α-amylase/subtilisin inhibitor (BASI) complex interacts with catalytic groups in AMY2, and Ca^2?+? occupies an identical position in AMY1 with thiomaltotetraose bound at two surface sites. EDTA-treatment, DSC, and activity assays indicate that AMY1 has the highest affinity for Ca^2?+?. Subsite mapping has revealed that AMY1 has ten functional subsites which can be modified by means protein engineering to modulate the substrate specificity. Other mutational analyses show that surface carbohydrate binding sites are critical for interaction with polysaccharides. The conserved Tyr380 in the newly discovered ‘sugar tongs’ site in domain C of AMY1 is thus critical for binding to starch granules. Furthermore, mutations of binding sites mostly reduced the degree of multiple attack in amylose hydrolysis. AMY1 has higher substrate affinity than AMY2, but isozyme chimeras with AMY2 domain C and other regions from AMY1 have higher substrate affinity than both parent isozymes. The latest revelations addressing various structural and functional aspects that govern the mode of action of barley α-amylases are reported in this review.     

Characterization of an exo-acting intracellular α-amylase from the hyperthermophilic bacterium Thermotoga neapolitana

We cloned and expressed the gene for an intracellular α-amylase, designated AmyB, from the hyperthermophilic bacterium Thermotoga neapolitana in Escherichia coli. The putative intracellular amylolytic enzyme contained four regions that are highly conserved among glycoside hydrolase family (GH) 13 α-amylases. AmyB exhibited maximum activity at pH 6.5 and 75°C, and its thermostability was slightly enhanced by Ca²⁺. However, Ca²⁺ was not required for the activity of AmyB as EDTA had no effect on enzyme activity. AmyB hydrolyzed the typical substrates for α-amylase, including soluble starch, amylose, amylopectin, and glycogen, to liberate maltose and minor amount of glucose. The hydrolytic pattern of AmyB is most similar to those of maltogenic amylases (EC 3.2.1.133) among GH 13 α-amylases; however, it can be distinguished by its inability to hydrolyze pullulan and β-cyclodextrin. AmyB enzymatic activity was negligible when acarbose, a maltotetraose analog in which a maltose residue at the nonreducing end was replaced by acarviosine, was present, indicating that AmyB cleaves maltose units from the nonreducing end of maltooligosaccharides. These results indicate that AmyB is a new type exo-acting intracellular α-amylase possessing distinct characteristics that distinguish it from typical α-amylase and cyclodextrin-/pullulan-hydrolyzing enzymes.     

Properties and synthesis de novo of auxin-induced α-amylase in pea cotyledons

Analysis of starch-degrading enzymes in a crude extract of detached cotyledons of Pisum sativum L. by polyacrylamide gel electrophoresis (PAGE) demonstrated the presence of one band of -amylase (EC 3.2.1.1) activity. The activity of only this amylase was promoted in cotyledons incubated with 2,4-dichlorophenoxyacetic acid (2,4-D). The auxin-induced -amylase from pea cotyledons was purified to homogeneity, as judged by the criterion of a single band after PAGE. The relative molecular mass (M_r), estimated by gel filtration, was approx. 42 000 and the enzyme contained no carbohydrate moiety. Sodium dodecylsulfate-PAGE yielded a single band that corresponded to an M_r of 41 000. The isoelectric point was 5.85 and the aminoacid composition was similar to that of -amylase from other plants. When [³H]leucine was fed to detached dry cotyledons prior to incubation, the radioactivity in -amylase from cotyledons incubated in the presence of 2,4-D was found to be approx. 10-fold higher than that from cotyledons incubated in distilled water. When -amylase from cotyledons incubated with ²H₂O that contained 2,4-D and the tritiated amylase were centrifuged together in a CsCl density gradient, the peak of enzymatic activity of deuterated -amylase was shifted to a denser fraction than the peak of radioactivity of the tritiated enzyme. These results show that auxin-induced -amylase in pea cotyledons is synthesized de novo.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - pI isoelectric point - SDS sodium dodecyl sulfate We are very grateful to Mr. Kazuo Itoh and Mrs. Matsumi Doe for carrying out the analysis of amino-acid composition.     

Regulation of the accumulation of mRNA for alpha-amylase in barley aleurone

The effect of gibberellic acid and Ca²⁺ on the accumulation of α-amylase mRNAs in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) was studied using cDNA clones containing sequences of mRNAs for the high and low isoelectric point (pI) α-amylases. There is no significant hybridization between the two α-amylase cDNA clones under the hybridization and washing conditions employed. These clones were therefore used to monitor levels of mRNAs for high and low pI α-amylases. It is shown that although the synthesis of the high pI α-amylase proteins depends on the presence of Ca²⁺ in the incubation medium, the accumulation of mRNA for this group occurs to the same degree in the presence or the absence of Ca²⁺. The accumulation of low pI α-amylase mRNA is also not affected by the presence or absence of Ca²⁺ in the incubation medium. These results establish gibberellic acid, not Ca²⁺, as the principal regulator of α-amylase mRNA accumulation in barley aleurone, while Ca²⁺ controls high pI α-amylase synthesis at a later step in the biosynthetic pathway.     

Effect of trophic conditions on asparagine transamination in wheat plants

In dialyzed extracts from winter wheat plants transamination reactions occurred between asparagine and α-ketoglutaric acid (L-asparagine+2-oxoacid=2-oxosuccinamate+ +amino acid; 2. 6. 1. 14). Reactions with pyruvate exhibited a very low activity. Besides transamination products,i. e. glutamate and alanine, aspartic acid was formed in both reactions. Deamidation was more intensive in the weak reaction asparagine-alanine and less intensive in the asparagine-glutamate reaction. When calculated per dry weight unit the activity was the same in plants of all variants (three experimental variants—Knop, potassium humate, water). A higher, activity was found in root dialysates; however, a highly significant difference could be observed only between shoots and roots of Knop variant. When evaluating results in terms of protein content we found a significant difference between mineral variant (Knop—the lowest activity) and both deficient variants (potassium humate, water—the highest activity). Thus the highest growth activity was in connection with the lowest transamination activity and vice versa, which was the same as in transaminations of aspartic acid. In the case of asparagine, too, one can consider the possibility of its utilization via transamination for biosynthesis of glutamic acid in plants which have, for reasons of nutrition, a low level of this metabolically important amino acid.     

Microbial acid-stable α-amylases: Characteristics,genetic engineering and applications

Among the wide variety of amylolytic enzymes synthesized by microorganisms, α-amylases are the most widely used biocatalysts in starch saccharification, baking industries and textile desizing. These enzymes randomly cleave the α-1,4-glycosidic linkages in starch, generating maltose and malto-oligosaccharides. The commercially available α-amylases have certain limitations, such as limited activity at low pH and Ca²⁺-dependence, and therefore, the search for novel acid-stable and thermostable amylases from extremophilic microorganisms and the engineering of the already available enzymes have been the major areas of research in this field over the years. Several attempts have been made to find suitable microbial sources of acid-stable and thermostable α-amylases. Acid-stable α-amylases have been reported in fungi, bacteria and archaea. α-Amylases that are active at elevated temperatures have been reported in bacteria as well as in archaea. α-Amylases that possess both characteristics, to the extent required for their various applications are very scarce. The developments that have been made in molecular biology, directed evolution and structural conformation studies of α-amylases for improving their properties to suit various industrial applications are discussed in this review.     

A comparison of the effects of Ca2+ and gibberellic acid on enzyme synthesis and secretion in barley aleurone

The effects of the addition and withdrawal of gibberellic acid (GA₃) and Ca²⁺ on enzyme synthesis and secretion by barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. Incubation of layers in GA₃ plus Ca²⁺ affects the total amount of secreted α-amylase (EC 3.2.1.1) and acid phosphatase (EC 3.1.3.2) by promoting the appearance of different isoenzymic forms of these enzymes. The release of α-amylase isoenzymes 1–4 in response to GA₃ plus Ca²⁺ has a lag of 6 h. When layers are incubated in GA₃ alone for 6 h prior to the addition of Ca²⁺, isoenzymes 1–4 appear in the medium after only 30 min. When the addition of Ca²⁺ to layers pretreated in GA₃ is delayed beyond 12 h, its effectiveness in stimulating the synthesis and release of isoenzymes 3 and 4 is diminished. After 35 h of preincubation in GA₃, addition of Ca²⁺ will not stimulate synthesis of α-amylase isoenzymes 3 and 4. Aleurone layers preincubated for 6 h in GA₃ will respond to Ca²⁺ when the GA₃ is withdrawn from the incubation medium by producing α-amylase isoenzymes 1–4. The converse is not the case, however, since layers preincubated in Ca²⁺ for 6 h will not produce all isoenzymes of α-amylase when subsequently incubated in GA₃. The Ca²⁺-stimulated release of α-amylase from GA₃ pre-treated layers is dependent on the time of incubation in Ca²⁺ and the concentration of the ion. The response to Ca²⁺ is temperature-dependent, and other divalent cations such as Mg²⁺ cannot substitute for Ca²⁺. We conclude that Ca²⁺ influences α-amylase release by influencing events at the biochemical level.     

A chimeric α-amylase engineered from <Emphasis Type="Italic">Bacillus acidicola</Emphasis> and G<Emphasis Type="Italic">eobacillus thermoleovorans</Emphasis> with improved thermostability and catalytic efficiency

The α-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable α-amylase gene of Geobacillus thermoleovorans (Gt-amy). The chimeric enzyme (Ba-Gt-amy) expressed in Escherichia coli displays marked increase in catalytic efficiency [K _cat: 4 × 10⁴ s^?1 and K _cat/K _m: 5 × 10⁴ mL^?1 mg^?1 s^?1] and higher thermostability than Ba-amy. The melting temperature (T _m) of Ba-Gt-amy (73.8 °C) is also higher than Ba-amy (62 °C), and the CD spectrum analysis revealed the stability of the former, despite minor alteration in secondary structure. Langmuir–Hinshelwood kinetic analysis suggests that the adsorption of Ba-Gt-amy onto raw starch is more favourable than Ba-amy. Ba-Gt-amy is thus a suitable biocatalyst for raw starch saccharification at sub-gelatinization temperatures because of its acid stability, thermostability and Ca²⁺ independence, and better than the other known bacterial acidic α-amylases.