The alpha-amylases as glycosylases, with wider catalytic capacities than envisioned or explained by their representation as hydrolases

In a study undertaken to illustrate the inadequacy of the familiar concept of carbohydrases as hydrolases, crystalline α-amylases from six different sources, as well as crude salivary amylase, were examined and found to catalyze the synthesis of maltose and maltosaccharides from α-d-glucopyranosyl fluoride, a stereoanalog of α-d-glucopyranose. These syntheses apparently involve initial formation of maltosyl fluoride and higher maltosaccharide 1-fluorides, traces of which were found in digests with certain α-amylases. That the reactions are due to the α-amylases themselves and not to some accompanying enzyme(s) appears certain from the purity and diversity of the preparations; their failure (with one exception) to attack α- or β-maltose; the correspondence of the synthesized products with the known specificity of α-amylases for α-1,4-d-glucosidic linkages (and capacity of different α-amylases to hydrolyze saccharides of different sizes). The “saccharifying” α-amylase of B. sublilis var amylosacchariticus was unique in producing maltosaccharides from both α- and β-maltose (i.e., by α-d-glucosyl transfer). However, 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 the catalytic range of α-amylase extends beyond hydrolysis and its reversal. Indeed, all transferred the glucosyl group of α-d-glycosyl fluoride preferentially to C₄-carbinols rather than water—a finding neither anticipated nor explained by the representation of α-amylases as hydrolases.     

The appearance of -1,3-glucanohydrolase activity during the differentiation of the gut of sand dollar plutei

The appearance and the increase of the activity of a β-1,3-glucanohydrolase (β-glucanase) was found to be coincident with the differentiation of the gut of the pluteus of the sand dollar Dendraster excentricus. The β-glucanase is extremely stable and is not inactivated by 2.5% sodium dodecyl sulfate (SDS) or trypsinization. The enzyme releases glucose from laminarin, a polysaccharide composed mainly of β-1,3-linked glucose. The β-glucanase activity can be eluted from acrylamide gels containing SDS. The enzyme is bound to membranes or particles that sediment readily, and SDS or sodium deoxycholate is needed to solubilize the activity.     

Two antigenically distinct forms of β-1,3-glucanase in sea urchin embryonic development

The enzyme β-1,3-glucanase is contained in the unfertilized eggs of most species of sea urchin. In some species, including Lytechinus variegatus, there is also substantial activity following gastrulation, and during remaining larval development. To determine if the same form of β-1,3-glucanase is present in both unfertilized eggs and after gut differentiation, an affinity purification procedure was utilized to isolate enzyme from unfertilized Lytechinus eggs. β-1,3-Glucanase is a 70,000-Da protein in this species, similar to the molecular weight of enzyme isolated from Strongylocentrotus purpuratus. Purified enzyme was used to generate an antibody that specifically recognized a 70,000-Da protein in unfertilized eggs by Western blot analysis, and stained the cortical granules of unfertilized eggs by immunofluorescence. The antibody also specifically immunoprecipitated β-1,3-glucanase activity from egg sonicates. The antibody was used to demonstrate that the form of β-1,3-glucanase present following gastrulation is antigenically distinct from the egg form. The 70,000-Da protein recognized by the antibody was no longer present by 24 hr, but embryos of this and later stages contained substantial amounts of activity, indicating the enzyme at these stages differs from the egg-specific form. In addition, the antibody was not capable of immunoprecipitating enzyme activity from pluteus sonicates. β-1,3-Glucanase has been partially purified from pluteus stage embryos, and appears to be a complex of approximately 200,000 Da. The enzyme is specific to endoderm and appears following differentiation of the gut, suggesting that it may function in larval digestion.     

Evolution of β-amylase: Patterns of variation and conservation in subfamily sequences in relation to parsimony mechanisms

Soybean and sweet potato β-amylases are structured as α/β barrels and the same kind of folding may account for all known β-amylases. We provide a comprehensive analysis of both protein and DNA (coding region) sequences of β-amylases. The aim of the study is to contribute to the knowledge of the evolutionary molecular relationships among all known β-amylases. Our approach combines the identification of the putative eightfold structural core formed by β-strands with a complete multi-alignment analysis of all known sequences. Comparing putative β-amylase (α/β)₈ cores from plants and microorganisms, two differentiated versions of residues at the packing sites, and a unique set of eight identical residues at the C-terminal catalytical site are observed, indicating early evolutionary divergence and absence of localized three-dimensional evolution, respectively. A new analytical approach has been developed in order to work out conserved motifs for β-amylases, mostly related with the enzyme activity. This approach appears useful as a new routine to find sets of motifs (each set being known as a fingerprint) in protein families. We demonstrate that the evolutionary mechanism for β-amylases is a combination of parsimonious divergence at three distinguishable rates in relation to the functional signatures, the barrel scaffold, and α-helix-containing loops. © 1996 Wiley-Liss, Inc.     

Sequence of archaealMethanococcus jannaschii α-amylase contains features of families 13 and 57 of glycosyl hydrolases: A trace of their common ancestor?

Two sequentially different, seemingly unrelated α-amylase families exist, known as family-13 and family-57 glycosyl hydrolases. Despite the common enzyme activity, it has as yet been impossible to detect any sequence similarity between the two families. The detailed analysis of the recently determined sequence of the α-amylase from methanogenic archaeonMethanococcus jannaschii using the sensitiveHydrophobic Cluster Analysis method revealed that this α-amylase contains features of both families of α-amylases. Thus theM. jannaschii α-amylase is similar to thePyrococcus furiosus α-amylase from family 57 while it obviously contains most of the sequence fingerprints characteristic for α-amylase family 13. Importantly, a glutamic acid residue equivalent with the family-13 catalytic glutamate positioned in the β5-strand segment was identified in members of family 57. The results presented in this report indicate that the two families, 13 and 57, are either the products of a very distant common ancestor or have evolved from each other, although at present they can represent two different α-amylase families with evolved different catalytic mechanisms, catalytic machinery and folds.     

Expression of α-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient

A rice suspension cell culture system has been established to study how sugar depletion regulates α-amylase expression, carbohydrate metabolism, and other physiological and cellular changes. It is shown here that a group of 44 kDa α-amylases are constitutively expressed whether or not the cells are starved of sucrose. However, expression of a new group of α-amylases of 46 kDa is dramatically induced when cells are starved of sucrose. Cellular sugar and starch were rapidly consumed and metabolic activity was decreased in the starved cells. Extensive autophagy also occurred in the starved cells, which caused an increase in vacuolar volume and degradation of cytoplasmic constituents including amyloplasts. Immunocytochemical studies revealed that α-amylases are localized in starch granules within amyloplasts, in cell walls, and in some of the vacuoles. The presence of putative signal sequences in the N-termini of nine rice α-amylases suggests hitherto unidentified pathways for import of α-amylases into amyloplasts. The studies show that differential α-amylase expression, carbohydrate metabolism, metabolic activity, and vacuolar autophagy are coordinately regulated by the sugar level in the medium. As the starved suspension cells exhibit some sugar-regulated characteristics of α-amylase expression in germinating rice embryos as well as physiological changes similar to those in senescing cells, this system represents an ideal tool for studying cellular, biochemical, and molecular biological aspects of α-amylase gene regulation, carbohydrate metabolism, senescence, and protein targeting in plants.     

Studies on extracellular and intracellular purified amylases from a thermophilic Bacillus stearothermophilus

Extracellular and intracellular amylases have been purified from a thermophilic Bacillus stearothermophilus and further studies have been made with the purified enzyme. The molecular weights for extra- and intracellular α- and β-amylases were found to be 47 000, 58 000, 39 000 and 67 000, respectively. α-Amylase (1,4-α-d-glucan glucanohydrolase, EC 3.2.1.1) and glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) were glycoproteins, whereas β-amylase (1,4-α-d-glucan maltohydrolase, EC 3.2.1.2) had little or no carbohydrate moiety. Extracellular FI (α-amylase), FIII (glucoamylase), FIV and FV (α-amylase) had carbohydrate moieties of 14.4, 27.0, 11.0 and 12.5%, respectively, whereas intracellular amylases FI (α-amylase), FII (β-amylase) and FIII (α-amylase) contained 15.2, 0.8 and 13.4% carbohydrate, respectively. The amino acid profile of the amylase protein digest showed a total number of 16 amino acids with aspartic acid showing the highest value followed by glutamic acid and leucine plus isoleucine. Compared to other thermostable amylases, proline and histidine contents were low. Both α- and β- amylase had the - SH group at their active site, which was essential for enzyme activity. EDTA and parachloromercuribenzoate exhibited dose dependent non-competitive inhibition of enzyme activity indicating the involvement of a divalent cation and the - SH group for activity.     

Formation of O-Alkylhomoserines from Alcohols,by Yeasts Capable of Assimilating 1,2-Propanediol

The less reactive SH groups of soybean β-amylase, SH4, SH5, and SH6, were modified with p-chloromercuribenzoic acid or N-ethylmaleimide, after the reactive SH groups, SHI, SH2, and SH3, were blocked with 5,5′-dithiobis-(2-nitrobenzoic acid) and cyanide. The enzyme activity decreased, accompanied by the modification of SH4. α-Cyclodextrin protected SH4 from the modification more effectively than maltose. The SH4-modified enzyme still bound to glucose, maltose, and α-cyclodextrin. SH4 was concerned with neither the catalysis nor substrate binding but its large substituent affected the substrate binding site. The sequencing of the 5-(iodoacetoamidoethyl)-aminonaphthalene-1-sulfonate-labeled peptides showed that SH4, SH5, and SH6 are Cys343, Cys82, and Cys208, respectively. Comparison of the primary structure of β-amylases also showed that the sequence around SH4 (Cys343), as well as SH2 (Cys95), is strongly conserved between higher plant and bacterial β-amylases. These results agree with the structure model deduced from X-ray crystallography of soybean β-amylase.     

Biochemical characterization of digestive proteases and carbohydrases of the carob moth,Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae)

Digestive proteinases and carbohydrases of Ectomyelois ceratoniae (Zeller) larvae were investigated using appropriate substrates and inhibitors. Midgut pH in larvae was determined to be slightly alkaline. Midgut extracts showed optimum activity for proteolysis of hemoglobin at pH 9–12. Midgut proteinases also hydrolyzed the synthetic substrates of trypsin, chymotrypsin, and elastase at pH 8–11. Maximum digestive α-amylase activity was also observed at pH 8–11. However, optimum activity for α- and β-glucosidase occurred at pH 5–8. Alpha- and β-galactosidases optimum activities occurred at pH 5 and pH 6, respectively. Inhibitors of serine proteases were effective on midgut serine proteases (trypsin and chymotrypsin proteases). Zymogram analyses revealed at least five bands of total proteolytic activity in the larval midgut. Protease-specific zymogram analyses revealed at least four, two, and one isozymes for trypsin-, chymotrypsin-, and elastase-like activities respectively. Two α-amylase isozymes were found in the midgut of fifth instar larvae and in the whole bodies of 1st through 5th instar larvae. Zymogram studies also revealed the presence of one and two bands of activity for β- and α-glucosidase, respectively. Recycling of α-amylase and proteases in the larval midgut was not complete. At least one isozyme of trypsin, chymotrypsin, elastase, and α-amylase were not recycled and were observed in the larval hindgut.     

Molecular Cloning of α-Amylases from Cotton Boll Weevil, Anthonomus grandis and Structural Relations to Plant Inhibitors: An Approach to Insect Resistance

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of α-amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two α-amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5′ and 3′ RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7 kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several α-amylase inhibitors from plants were assayed against A. grandis α-amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and α-AI1 inhibitors on A. grandis α-amylase activity. This work suggests that genetic engineering of cotton to express α-amylase inhibitors may offer a novel route to A. grandis resistance.     

Polymorphism in Rice Amylases at an Early Stage of Seed Germination

A polymorphism in rice amylases at an early stage of seed germination is analyzed by zymogram. In nonglutinous cultivars of rice, α-amylase isozymes are mainly confirmed in germinating seeds. However, in glutinous cultivars, β-amylase isozymes, which are not confirmed in nonglutinous cultivars, make up the major part of the total amylase activity and the expression of α-amylases are repressed.     

Composition of α-amylase secreted by aleurone layers of grains of himalaya barley

α-Amylases secreted by the aleurone layer of whole barley grains were relatively rich in histidine and relatively poor in glutamate/glutamine and serine when compared to other eukaryotic proteins. The secreted α-amylases had an estimated 0.5 residues each of glucose, mannose and N-acetylglucosamine per molecule of protein (MW 41 400 daltons), and gave positive staining reactions for carbohydrate on sodium dodecylsulfate polyacrylamide gels. Because the average α-amylase molecule had less than one sugar residue per enzyme molecule, it was concluded that secreted α-amylases were heterogeneous with respect to glycosylation. A second protein co-purified with α-amylase, but the amino acid composition of this protein was different from that of barley or wheat α-amylase. This protein was composed of two 21 500 dalton polypeptides. No significant amounts of L-leucine (¹⁴C-U) were incorporated into this second protein in isolated aleurone tissue during incubation with gibberellic acid, perhaps because much of it was already present in the starchy endosperm at the time of hormone addition.     

Applications of a high maltose forming,thermo-stable α-amylase from an extremely alkalophilic Bacillus licheniformis strain AS08E in food and laundry detergent industries

An alkalophilic bacterial strain was isolated from the soil sample of Assam, North-East India. This strain was found capable of growing and producing α-amylase at extremely alkaline pH (12.5). By molecular characterization, this bacterium was identified as Bacillus licheniformis strain AS08E. Statistical optimization of media components resulted in 3-fold increase in the production of α-amylase from this bacterium. From this strain, a major extracellular α-amylase of ∼55 kDa was purified to homogeneity with a 14.5-fold increase in its specific activity. The N-terminal sequence of this enzyme showed extensive identity with α-amylases purified from thermostable bacteria. The purified enzyme showed optimum activity at pH 10.0 and 80 °C, and demonstrated stability toward various surfactants, organic solvents, and commercial laundry detergents. The spectroflurometric analysis suggests that the enzyme has a strong binding affinity toward soluble starch. TLC analysis of starch degradation product displays this α-amylase as a high maltose-forming enzyme. The future application of this enzyme in food and detergent industries is highly promising.     

Demonstration and preliminary characterization of α-amylase in the sweetpotato whitefly,Bemisia tabaci (Aleyrodidae: Homoptera)

An α-amylase that hydrolyzes unmodified starch or amylopectin azure was demonstrated in crude and partially purified extracts prepared from whole carcasses of sweetpotato whiteflies (SPW) (Bemisia tabaci Genn.).All nymphal instars and adult SPW, including newly eclosed crawlers that had not yet fed on plant materials, were found to have active α-amylase. α-Amylase activity per mg protein was greatest in 1st instars and decreased with age up to the “pupal” stage, with a very slight increase in activity in adults. However, activity per individual did not differ substantially as a function of age.The α-amylase had an apparent molecular weight of about 70 kDa, an isoelectric point of 6.32 and eluted with about 250 mM NaCl from a strongly basic anion-exchange column.The enzyme activity was inhibited by EDTA and not activated by either NaCl or KNO₃. CaCl₂ strongly enhanced activity.α-Amylase activity was greatest at pH 7.0, but there was considerable activity at pHs above 7.0.The K_m of the α-amylase was 1.47 Mm with p-nitrophenyl α-d-malto-heptaoside as substrate.The presence of an amylolytic enzyme in a phloem-feeding insect is unexpected and raises questions about current assumptions of feeding behavior of this species.     

Characterization of two coleopteran α-amylases and molecular insights into their differential inhibition by synthetic α-amylase inhibitor,acarbose

Post-harvest insect infestation of stored grains makes them unfit for human consumption and leads to severe economic loss. Here, we report functional and structural characterization of two coleopteran α-amylases viz. Callosobruchus chinensis α-amylase (CcAmy) and Tribolium castaneum α-amylase (TcAmy) along with their interactions with proteinaceous and non-proteinaceous α-amylase inhibitors. Secondary structural alignment of CcAmy and TcAmy with other coleopteran α-amylases revealed conserved motifs, active sites, di-sulfide bonds and two point mutations at spatially conserved substrate or inhibitor-binding sites. Homology modeling and molecular docking showed structural differences between these two enzymes. Both the enzymes had similar optimum pH values but differed in their optimum temperature. Overall, pattern of enzyme stabilities were similar under various temperature and pH conditions. Further, CcAmy and TcAmy differed in their substrate affinity and catalytic efficiency towards starch and amylopectin. HPLC analysis detected common amylolytic products like maltose and malto-triose while glucose and malto-tetrose were unique in CcAmy and TcAmy catalyzed reactions respectively. At very low concentrations, wheat α-amylase inhibitor was found to be superior over the acarbose as far as complete inhibition of amylolytic activities of CcAmy and TcAmy was concerned. Mechanism underlying differential amylolytic reaction inhibition by acarbose was discussed.     

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.