Hydrolysis of amylopectin by amylolytic enzymes: structural analysis of the residual amylopectin population

Amylopectin fine structures were studied following limited hydrolysis of gelatinised waxy maize starch by amylases with a different level of inner chain attack (LICA). This was done by size exclusion chromatography as well as by debranching the (partially hydrolysed) amylopectin samples and studying the size distributions of the released chains. α-Amylases from Bacillus amyloliquefaciens and Aspergillus oryzae, with a relatively high LICA, drastically altered amylopectin chain length distribution and reduced the amylopectin molecular size (MS) significantly even at a low to moderate degree of hydrolysis (DH). Porcine pancreatic α-amylase (PPA), with a rather low LICA but a high multiple attack action on amylose, reduced the amylopectin MS much slower. Following hydrolysis by PPA to a DH of 10% and enzymic debranching of the amylopectin residue, several subpopulations of chains consisting of 2-12 glucose units were detected, indicating a multiple attack action on the amylopectin side chains. During the early stages of hydrolysis, the maltogenic Bacillus stearothermophilus α-amylase (BStA) preferentially hydrolysed the exterior chains of amylopectin. However, during the later phases, BStA also hydrolysed inner chains, presumably with a high multiple attack action. The present results clearly show that different enzymes can be used for (limited) conversion of amylopectin into structures differing in molecular weight and chain length distributions.     

Bacillus stearothermophilus Neopullulanase Selective Hydrolysis of Amylose to Maltose in the Presence of Amylopectin

The specificity of Bacillus stearothermophilus TRS40 neopullulanase toward amylose and amylopectin was analyzed. Although this neopullulanase completely hydrolyzed amylose to produce maltose as the main product, it scarcely hydrolyzed amylopectin. The molecular mass of amylopectin was decreased by only one order of magnitude, from approximately 10⁸ to 10⁷ Da. Furthermore, this neopullulanase selectively hydrolyzed amylose when starch was used as a substrate. This phenomenon, efficient hydrolysis of amylose but not amylopectin, was also observed with cyclomaltodextrinase from alkaliphilic Bacillus sp. strain A2-5a and maltogenic amylase from Bacillus licheniformis ATCC 27811. These three enzymes hydrolyzed cyclomaltodextrins and amylose much faster than pullulan. Other amylolytic enzymes, such as bacterial saccharifying α-amylase, bacterial liquefying α-amylase, β-amylase, and neopullulanase from Bacillus megaterium, did not exhibit this distinct substrate specificity at all, i.e., the preference of amylose to amylopectin.     

Amylolytically-resistant tapioca starch modified by combined treatment of branching enzyme and maltogenic amylase

Tapioca starch was modified using branching enzyme (BE) isolated from Bacillus subtilis 168 and Bacillus stearothermophilus maltogenic amylase (BSMA), and their molecular fine structure and susceptibility to amylolytic enzymes were investigated. By BE treatment, the molecular weight decreased from 3.1 × 10⁸ to 1.7 × 10⁶, the number of shorter branch chains (DP 6–12) increased, the number of longer branch chains (DP >25) decreased, and amylose content decreased from 18.9% to 0.75%. This indicated that α–1,4 linkages of amylose and amylopectin were cleaved, and moiety of glycosyl residues were transferred to another amylose and amylopectin to produce branched glucan and BE-treated tapioca starch by forming α–1,6 branch linkages. The product was further modified with BSMA to produce highly-branched tapioca starch with 9.7% of extra branch points. When subject to digestion with human pancreatic α-amylase (HPA), porcine pancreatic α-amylase (PPA) and glucoamylase, highly-branched tapioca starch gave significantly lowered α-amylase susceptibility (7.5 times, 14.4 times and 3.9 times, respectively), compared to native tapioca starch.     

Hydrolysis of starches by the action of an α-amylase from Bacillus subtilis

A moderately thermophilic Bacillus subtilis strain, isolated from fresh sheep’s milk, produced extracellular thermostable α-amylase. Maximum amylase production was obtained at 40 °C in a medium containing low starch concentrations. The enzyme displayed maximal activity at 135 °C and pH 6.5 and its thermostability was enhanced in the presence of either calcium or starch. This thermostable α-amylase was used for the hydrolysis of various starches. An ammonium sulphate crude enzyme preparation as well as the cell-free supernatant efficiently degraded the starches tested. The use of the clear supernatant as enzyme source is highly advantageous mainly because it decreases the cost of the hydrolysis. Upon increase of reaction temperature to 70 °C, all substrates exhibited higher hydrolysis rates. Potato starch hydrolysis resulted in a higher yield of reducing sugars in comparison to the other starches at all temperatures tested. Soluble and rice starch took, respectively, the second and third position regarding reducing sugars liberation, while the α-amylase studied showed slightly lower affinity for corn starch and oat starch.     

Immunological comparison of bacterial α-amylases and multiple forms of Baci1lus licheniformis enzyme

A purified preparation of Bacillus licheniformis α-amylase was immunologeeally and electrophoretically compared with commercial crystalline α-amylase of Bacillus subtilis. The former enzyme reacted completely with rabbit antiserum to the same enzyme showing a single precipitin band, and moved toward the cathode in immuno-electrophoresis on agarose at pH 9.6. On the contrary, crystalline α-amylase of Bacillus subtilis migrated to the anode in immunoelectrophoresis at pH 8.6, though it weakly cross-reacted with the antiserum, suggesting that amylases of Bacillus licheniformis and Bacillus subtilis are not identical. In addition, the neutralization test of amylase activity showed that α-amylase of Bacillus licheniformis was much more susceptible to inhibition by the serum than was Bacillus subtilis α-amylase. Each of four species of Bacillus licheniformis α-amylase extracted from the sliced discs after disc electrophoresis on polyacrylamide gel was distinct from the others by showing individual migratory rate, but they were antigenically similar to each other and to the parent enzyme.     

Charakterisierung der Stärkehydrolysate nach Einwirkung einer thermostabilen α-Amylase

The action of thermostable α-amylase produced by Bacillus licheniformis 44MB82 strain on soluble and insoluble starch, amylose and amylopectin at temperatures 30°C and 90°C was studied. The hydrolysis of soluble starch proceeded rapidly for 10 to 15 minutes after which the maltodextrins thus formed were further dissociated. In the course of 60-minutes enzyme treatment mainly glucose, maltose and maltosugars (from G₃ to G₆) as low molecular weight products were found and the formation of maltcse and maltotriose was increased by the longer treatment. The hydrolysis of insoluble starch and amylopectin proceeded in the same way while the amylose was hydrolysed slowly.     

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t₅₀ of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K_m of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).     

Purification, partial characterization, and covalent immobilization–stabilization of an extracellular α-amylase from Aspergillus niveus

An extracellular amylase secreted by Aspergillus niveus was purified using DEAE fractogel ion exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5 % polyacrylamide gel electrophoresis (PAGE) and 10 % sodium dodecyl sulfate (SDS-PAGE). The enzyme exhibited 4.5 % carbohydrate content, 6.6 isoelectric point, and 60 and 52 kDa molar mass estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The amylase efficiently hydrolyzed glycogen, amylose, and amylopectin. The end-products formed after 24 h of starch hydrolysis, analyzed by thin layer chromatography, were maltose, maltotriose, maltotetraose, and maltopentaose, which classified the studied amylase as an α-amylase. Thermal stability of the α-amylase was improved by covalent immobilization on glyoxyl agarose (half-life of 169 min, at 70 °C). On the other hand, the free α-amylase showed a half-life of 20 min at the same temperature. The optima of pH and temperature were 6.0 and 65 °C for both free and immobilized forms.     

Purification and biochemical characterization of a novel SDS and surfactant stable,raw starch digesting,and halophilic α-amylase from a moderately halophilic bacterium,Nesterenkonia sp. strain F

An extracellular halophilic α-amylase from Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography, with a 10.8-fold increase in specific activity. The molecular mass of the amylase was estimated to be 100 kDa and 106 kDa by SDS–PAGE and gel filtration chromatography, respectively. The enzyme showed maximal activity at pH 7.5 and 45 °C. The amylase was active in a wide range of salt concentrations (0–4 M) with its maximum activity at 0.5 M NaCl or 1 M KCl and was stable at the salts concentrations between 1 M and 4 M. Fe³⁺, Cu²⁺, Zn²⁺ and Al³⁺ strongly inhibited the enzyme, whereas Ca²⁺ stimulated the amylase activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. The enzyme showed remarkable stability towards 0.5% SDS and sarcosyl, and 2% each of Triton X-100, Tween 80 and Tween 20. K_m value of the amylase for soluble starch was 4.5 mg/ml. The amylase hydrolyzed 38% of raw wheat starch and 20% of corn starch in a period of 48 h. The major products of soluble starch hydrolysis were maltose, maltotriose and maltotetraose, indicating an α-amylase activity.     

Bacillus stearothermophilus neopullulanase selective hydrolysis of amylose to maltose in the presence of amylopectin

The specificity of Bacillus stearothermophilus TRS40 neopullulanase toward amylose and amylopectin was analyzed. Although this neopullulanase completely hydrolyzed amylose to produce maltose as the main product, it scarcely hydrolyzed amylopectin. The molecular mass of amylopectin was decreased by only one order of magnitude, from approximately 10(8) to 10(7) Da. Furthermore, this neopullulanase selectively hydrolyzed amylose when starch was used as a substrate. This phenomenon, efficient hydrolysis of amylose but not amylopectin, was also observed with cyclomaltodextrinase from alkaliphilic Bacillus sp. strain A2-5a and maltogenic amylase from Bacillus licheniformis ATCC 27811. These three enzymes hydrolyzed cyclomaltodextrins and amylose much faster than pullulan. Other amylolytic enzymes, such as bacterial saccharifying alpha-amylase, bacterial liquefying alpha-amylase, beta-amylase, and neopullulanase from Bacillus megaterium, did not exhibit this distinct substrate specificity at all, i.e., the preference of amylose to amylopectin.     

The carbohydrases of Bacillus stearothermophilus NCIB 11412 and NCIB 10814

Summary Two strains (NCIB 11412 and NCIB 10814) of the thermophilic organism Bacillus stearothermophilus were found to produce complex carbohydrase systems. The enzyme activities in each system include -amylase as the major component, maltase, pullulanase, a minor amylase and cyclodextrinase. The latter three activities are produced in low yield in both strains. A crude enzyme preparation from each strain possessed maltogenic properties on hydrolysis of soluble starch. Following rigorous purification procedures, the purified major -amylase from either strain did not produce maltose as a major end-product of starch hydrolysis. However, a partially purified mixture of pullulanase, minor amylase and cyclodextrinase activities from NCIB 11412 and NCIB 10814 produced 56.4% and 62.0% maltose, respectively, from soluble starch.     

The Characterisation of an Alkali-Stable Maltogenic Amylase from Bacillus lehensis G1 and Improved Malto-Oligosaccharide Production by Hydrolysis Suppression

A maltogenic amylase (MAG1) from alkaliphilic Bacillus lehensis G1 was cloned, expressed in Escherichia coli, purified and characterised for its hydrolysis and transglycosylation properties. The enzyme exhibited high stability at pH values from 7.0 to 10.0. The hydrolysis of β-cyclodextrin (β-CD) produced malto-oligosaccharides of various lengths. In addition to hydrolysis, MAG1 also demonstrated transglycosylation activity for the synthesis of longer malto-oligosaccharides. The thermodynamic equilibrium of the multiple reactions was shifted towards synthesis when the reaction conditions were optimised and the water activity was suppressed, which resulted in a yield of 38% transglycosylation products consisting of malto-oligosaccharides of various lengths. Thin layer chromatography and high-performance liquid chromatography analyses revealed the presence of malto-oligosaccharides with a higher degree of polymerisation than maltoheptaose, which has never been reported for other maltogenic amylases. The addition of organic solvents into the reaction further suppressed the water activity. The increase in the transglycosylation-to-hydrolysis ratio from 1.29 to 2.15 and the increased specificity toward maltopentaose production demonstrated the enhanced synthetic property of the enzyme. The high transglycosylation activity of maltogenic amylase offers a great advantage for synthesising malto-oligosaccharides and rare carbohydrates.     

Modulation of Substrate Preference of Thermus Maltogenic Amylase by Mutation of the Residues at the Interface of a Dimer

To elucidate the relationship between the substrate size and geometric shape of the catalytic site of Thermus maltogenic amylase, Gly50, Asp109, and Val431, located at the interface of the dimer, were replaced with bulky amino acids. The k _cat/K _m value of the mutant for amylose increased significantly, whereas that for amylopectin decreased as compared to that of the wild-type enzyme. Thus, the substituted bulky amino acid residues modified the shape of the catalytic site, such that the ability of the enzyme to distinguish between small and large molecules like amylose and amylopectin was enhanced.     

Beta-Amylases from Alfalfa (Medicago sativa L.) Roots

Amylase was found in high activity (193 international units per milligram protein) in the tap root of alfalfa (Medicago sativa L. cv. Sonora). The activity was separated by gel filtration chromatography into two fractions with molecular weights of 65,700 (heavy amylase) and 41,700 (light amylase). Activity staining of electrophoretic gels indicated the presence of one isozyme in the heavy amylase fraction and two in the light amylase fraction. Three amylase isozymes with electrophoretic mobilities identical to those in the heavy and the light amylase fractions were the only amylases identified in crude root preparations. Both heavy and light amylases hydrolyzed amylopectin, soluble starch, and amylose but did not hydrolyze pullulan or β-limit dextrin. The ratio of viscosity change to reducing power production during starch hydrolysis was identical for both alfalfa amylase fractions and sweet potato β-amylase, while that of bacterial α-amylase was considerably higher. The identification of maltose and β-limit dextrin as hydrolytic end-products confirmed that these alfalfa root amylases are all β-amylases.     

Transglycosylation by barley α-amylase 1

The transglycosylation activity of barley α-amylase 1 (AMY1) and active site AMY1 subsite mutant enzymes was investigated. We report here the transferase ability of the V47A, V47F, V47D and S48Y single mutants and V47K/S48G and V47G/S48D double mutant AMY1 enzymes in which the replaced amino acids play important role in substrate binding at subsites at −3 through −5. Although mutation increases the transglycosylation activity of enzymes, in the presence of acceptors the difference between wild type and mutants is not so significant. Oligomer transfer reactions of AMY1 wild type and its mutants were studied using maltoheptaose and maltopentaose donors and different chromophore containing acceptors. The conditions for the chemoenzymatic synthesis of 4-methylumbelliferyl-α-d-maltooligosaccharides (MU-α-d-MOSs) were optimized using 4-methylumbelliferyl-β-d-glucoside as acceptor and maltoheptaose as donor. 4-Methylumbelliferyl-α-d-maltoside, -maltotrioside, -maltotetraoside and -maltopentaoside have been synthesized. Products were identified by MALDI-TOF MS. ¹H and ¹³C NMR analyses showed that AMY1 V47F preserved the stereo- and regioselectivity. The produced MU-α-d-MOSs of degree of polymerization DP 2, DP 3 and DP 5 were successfully applied to detect activity of Bacillus stearothermophilus maltogenic α-amylase, human salivary α-amylase and Bacillus licheniformis α-amylase, respectively in a fast and simple fluorometric assay.     

A thermophilic extracellular -amylase from Bacillus licheniformis

A strain of Bacillus licheniformis isolated from soil produced an extracellular α-amylase(s) with unusual characteristics. The enzyme was purified 126-fold by starch adsorption, DEAE-cellulose treatment, and CM-cellulose column chromatography. Four active protein bands were detected by disc electrophoresis in poly-acrylamide gel although the enzyme behaved as a single peak during both ultracen-trifugation and chromatography using CM-cellulose and Sephadex G-100. The enzyme showed a very broad pH-activity curve and had substantial activity in the alkaline range. The optimal temperature was 76 °C at pH 9.O. The enzyme was stable between pH 6 and 11 at 25 °C, and below 60 °C at pH 8.0. Using Sephadex G-100 gel filtration, a molecular weight of 22,500 was estimated for the enzyme. The action pattern on amylose and amylopectin is unique in that the predominant product during all stages of hydrolysis is maltopentaose.     

The DsbA signal peptide-mediated secretion of a highly efficient raw-starch-digesting,recombinant α-amylase from Bacillus licheniformis ATCC 9945a

In this study, a new approach for extracellular production of recombinant α-amylase in Escherichia coli was investigated. A gene encoding a highly efficient raw-starch-digesting α-amylase from Bacillus licheniformis ATCC 9945a was cloned and expressed in E. coli. The gene encoding mature α-amylase was cloned into the pDAss expression vector, and secretion of the gene product was regulated by fusion to the signal peptide of DsbA, a well-characterized E. coli periplasmic protein. E. coli BL21 (DE3) carrying pDAss vector containing amylase gene had approximately 2.5-fold higher volumetric enzyme productivity than the natural system. The recombinant enzyme showed higher efficiency for digesting diverse raw starches when compared with the native enzyme and was similar to commercial α-amylase in its ability to hydrolyze raw starches. The properties of the recombinant enzyme demonstrate the potential of the DsbA signal peptide approach for the secretory production of the fully active, industrially important recombinant enzyme.     

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.     

The cluster structure of barley amylopectins of different genetic backgrounds

The unit chains of amylopectin are organized into clusters. In this study, the cluster structure was analysed in detail in four different genotypes of barley, of which two possessed the amo1 genetic background. Amylose content of the barley starches differed from 0 to 32.6%. Isolated amylopectin was hydrolysed with α-amylase from Bacillus subtilis into domains, defined as groups of clusters, which were size-fractionated by methanol. The domain fractions were further treated with α-amylase to release single clusters. Amylopectin, domains and clusters were subsequently treated with phosphorylase and β-amylase to produce φ,β-limit dextrins and the detailed internal structures of these different structure levels were investigated. Analysis was performed with gel-permeation and anion-exchange chromatography. Equal amount of A-chains were detected in all barleys, but the distribution of B-chains differed. At least two types of domain structures were identified in all four barley varieties. Large domains were built up by large clusters and small domains by small clusters. In all four barley samples the number of long chains was small suggesting that shorter chains with a degree of polymerization of 25-35 also are involved in the interconnection of clusters. The cluster structure of the amylopectin correlated with the genetic background. The two barley samples with amo1 genetic background possessed a more dense structure. Internal chain lengths in these two barleys were shorter resulting in larger domains built up by larger clusters.