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.     

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.     

The limit dextrinases from ungerminated oats (Avena sativa L.) and ungerminated rice (Oryza sativa L.).

The limit dextrinases from ungerminated oats and rice have been purified, and their substrate specificity compared with a bacterial isoamylase preparation. Both cereal enzymes could hydrolyse (1 yields6)-alpha-D-glucosidic linkages in oligosaccharide alpha-dextrins, pullulan, amylopectin, and the beta-limit dextrins of amylopectin and glycogen. However, under comparable conditions, they were unable to attack glycogens.     

Amylopectin Synthase of Eimeria tenella: Identification and Kinetic Characterization

ABSTRACT. A soluble enzyme amylopectin synthase (UDP-glucose-α 1,4-glucan α-4-glucosyltransferase) which transfers glucose from uridine 5'-diphosphate glucose (UDP-glucose) to a primer to form α-I,4-glucosyl linkages has been identified in the extracts of unsporulated oocysts of Eimeria tenella . UDP-glucose and not ADP-glucose was the most active glucosyl donor. Corn amylopectin, rabbit liver glycogen, oyster glycogen and corn starch served as primers; the latter two were less efficient. The enzyme has an apparent pH optimum of 7.5 and exhibited typical Michaelis-Menten kinetics with dependence on both the primer and substrate concentrations. The Michaelis constants (Km). with respect to UDP-glucose, was 0.5 mM; and 0.25 mg/ml and 1.25 mg/ml with respect to amylopectin and rabbit liver glycogen. The product formed by the reaction was predominantly a glucan containing α-1,4 linkages. The specificity of the enzyme suggests that this enzyme is similar to glycogen synthase in eukaryotes and has been designated as amylopectin synthase (UDP-glucose-α-1,4-glucosetransferase EC 2.4.1.11).     

植物支链淀粉生物合成研究进展

植物支链淀粉占贮存淀粉的70%～80%,是决定植物果实或种子品质的关键成分.对植物支链淀粉生物合成途径及其代谢酶基因的研究,可大大推动支链淀粉结构的改造和在食品工业上的应用.该文介绍了植物支链淀粉的结构组成,详细阐述了参与支链淀粉生物合成的三类酶,即淀粉分支酶(starch branchingenzyme,SBE)、可溶性淀粉合酶(soluble starch synthase,SSS)和淀粉脱支酶(starch debranching enzyme,SDBE)的编码基因、酶学特性及其在支链淀粉合成中的作用,并就植物支链淀粉的合成模型加以探讨.同时提出了该研究领域尚待解决的问题,对其应用前景作了展望.     

The complexities of starch biosynthesis in cereal endosperms

Starch serves not only as an energy source for plants, animals, and humans but also as an environmentally friendly alternative for fossil fuels. Here, we describe recent findings concerning the synthesis of this important molecule in the cereal endosperm. Results from six separate transgenic reports point to the importance of adenosine diphosphate glucose pyrophosphorylase in controlling the amount of starch synthesized. The unexpected cause underlying the contrast in sequence divergence of its two subunits is also described. A major unresolved question concerning the synthesis of starch is the origin of nonrandom or clustered alpha-1,6 branch-points within the major component of starch, amylopectin. Developing evidence that several of the starch biosynthetic enzymes involved in amylopectin synthesis occur in complexes is reviewed. These complexes may provide the specificity for the formation of nonrandom branch-points.     

Effects of an added inclusion-amylose complex on the retrogradation of some starches and amylopectin

The effects of added cetyltrimethylammonium bromide (CTAB)-amylose complex on retrogradation of some starches (waxy-maize, maize, and potato starch) and on amylopectin from potato have been studied by differential scanning calorimetry (DSC). The starches and amylopectin samples with added CTAB-amylose complex received four different heat treatments prior to storage and DSC measurements that either melted the complex or left the complex intact. The calorimetry measurements showed that intact CTAB-amylose complex had much less effect on decreasing the retrogradation of the starches and the amylopectin than samples with melted complex prior to measurements. This is discussed in relation to possible complex formation of amylopectin and lipids and the effects of adding uncomplexed lipids on the retrogradation of waxy starches and amylopectin.     

Separation and Some Properties of Large and Small Amyloplasts throughout Development in Barley Endosperm

A method is described whereby amyloplasts from immature barley (Hordeum distichum L.) endosperm could be separated into two populations of large and small amyloplasts at all stages of development. The small amyloplasts had more amylopectin than the large at early stages, but by 60 days after anthesis, the large had the greater proportion of amylopectin. Starch synthetase activity was associated with both types of amyloplast. The nucleotide specificity of the starch synthetase associated with each population varied independently throughout development. At 25 days after anthesis, the large amyloplasts were more susceptible than the small to α-amylolysis; however, at 38 and 60 days, the small amyloplasts became more susceptible.     

Amylo-1,6-glucosidase/1,4-α-glucan: 1,4-α-glucan 4-α-glycosyltransferase: Specificity toward polysaccharide substrates

The substrate specificities of the glucosidase-transferase debranching enzyme systems from yeast and rabbit muscle were examined by the use of polysaccharide substrates of defined outer chain lengths. The results were consistent with the specificities ascribed to the transferase portion of the debranching enzyme system by previous studies using maltosaccharide substrates. The specificities of the two enzyme systems were also examined in the reversion reaction. The results showed that both systems displayed an inverse specificity to that observed in the hydrolytic reaction. This suggested that the reversion reaction reflects the specificity of the glucosidase portion of the debranching system. The major differences between the specificities of the yeast and rabbit muscle systems were found to lie in the specificity of the transferase and in the ability of the yeast system to debranch native glycogen and amylopectin.     

The influence of amylose on starch granule structure

Starch granules are principally composed of the two glucose polymers amylose and amylopectin. Native starch granules typically contain around 20% amylose and 80% amylopectin. However, it is possible to breed plants that produce starch with very different amylose and amylopectin contents. At present, the precise structural roles played by these two polymers are incompletely understood. In this study, small-angle X-ray scattering techniques have been applied to investigate the effect of varying amylose content on the internal structure of maize, barley and pea starch species. The results suggest that amylose disrupts the structural order within the amylopectin crystallites.     

The action of starch synthase II on 6"'-alpha-maltotriosyl-maltohexaose comprising the branch point of amylopectin.

The principle of using a chemically synthesized, well-defined branched oligosaccharide to provide a more detailed knowledge of the substrate specificity of starch synthase II (SSII) is demonstrated. The branched nonasaccharide, 6"'-alpha-maltotriosyl-maltohexaose, was investigated as a primer for particulate SSII using starch granules prepared from the low-amylose pea mutant lam as the enzyme source. The starch granule preparation from the lam pea mutant contains no starch synthases other than SSII and is devoid of alpha-amylase, beta-amylase and phosphorylase activity. SSII was demonstrated to catalyse a specific nonprocessive elongation of the nonreducing end of the shortest unit chain of 6"'-alpha-maltotriosyl-maltohexaose, i.e. the maltotriose chain. Maltotriose and maltohexaose, representing the two linear building units of the branched nonasaccharide, were also tested as primers for SSII. Maltotriose was elongated more efficiently than 6"'-alpha-maltotriosyl-maltohexaose and maltohexaose was used less efficiently. Compared to the surface exposed alpha-glucan chains of the granule bound amylopectin molecules, all three soluble oligosaccharides tested were poor primers for SSII. This indicates that in vivo, the soluble oligosaccharides supposedly released as result of amylopectin trimming reactions are not re-introduced into starch biosynthetic reactions via the action of the granule bound fraction of SSII.     

The effects of amylose content on the molecular size of amylose, and on the distribution of amylopectin chain length in maize starches

The amylose to amylopectin ratios in six maize starch samples of differing amylose contents were measured by enzymatic debranching, followed by high performance size exclusion chromatography (HPSEC). The molecular size of amyloses, estimated by -log K_wav, shows progressive decrease with the increase in amylose content in maize starches. The gel permeation chromatographs of the corresponding amylopectins, debranched with isoamylase, showed bimodal distributions containing long and short chains. The average chain length of amylopectin has a correlation with amylose content. The correlation coefficients between amylose content and average chain length, long chain length, weight ratio and the mole ratio of long and short chain length, were 0.97, 0.92, 0.96, 0.94 respectively. The maize starch with the highest amylose content has the lowest amylose molecular size and the longest chains, with a high ratio of long to short chains in its amylopectin fraction. Comparing the values of amylose content determined by HPSEC of starch or debranched starch with those of the iodinecomplex method, we conclude that long chains of amylopectin in high amylose starches contribute significantly to apparent amylose content.     

The mechanism of Q-enzyme action and its influence on the structure of amylopectin

Q-Enzyme is responsible for the synthesis of the 1,6-branch linkages in amylopectin. Its action on a model amylodextrin containing a single branch linkage has been studied. It is concluded that the enzymic process whereby the branch linkages of amylopectin are synthesized is a random action of the branching enzyme on a complex—possibly a double helix—formed between two 1,4-α-glucan chains. This action pattern predicts a novel arrangement of the units chains in amylopectin.     

Starch Degradation in Spinach Leaves: ISOLATION AND CHARACTERIZATION OF THE AMYLASES AND R-ENZYME OF SPINACH LEAVES

The properties of two amylase activities which differ in their substrate specificity and subcellular location as well as a chloroplast-associated R-enzyme (debranching activity) are reported. An extrachloroplastic amylase is resolved by gel filtration chromatography into two activities of 80,000 and 40,000 daltons. Both extrachloroplastic activities hydrolyze amylopectin and shellfish glycogen and only slowly hydrolyze rabbit liver glycogen, β-limit amylopectin, and amylose. In contrast, the major chloroplastic amylase attacks all of these glucans at comparable rates. Glucan hydrolysis by both the extrachloroplastic and chloroplastic amylase generates not only maltose but appreciable amounts of other oligosaccharides, whereas maltotetraose hydrolysis produces glucose, maltose, and maltotriose. The action patterns displayed by the amylase activities indicate that both are endoamylases, although they lack the typical Ca²⁺ requirement or heat stability of seed endosperm α-amylases. Dithiothreitol, glutathione (oxidized or reduced), ascorbate, dehydroascorbate, and dithiothreitol plus thioredoxin have no effect on either the chloroplastic or extrachloroplastic amylase activities.     

Specificity of starch synthase isoforms from potato.

In higher plants several isoforms of starch synthase contribute to the extension of glucan chains in the synthesis of starch. Different isoforms are responsible for the synthesis of essentially linear amylose chains and branched, amylopectin chains. The activity of granule-bound starch synthase I from potato has been compared with that of starch synthase II from potato following expression of both isoforms in Escherichia coli. Significant differences in their activities are apparent which may be important in determining their specificities in vivo. These differences include affinities for ADPglucose and glucan substrates, activation by amylopectin, response to citrate, thermosensitivity and the processivity of glucan chain extension. To define regions of the isoforms determining these characteristic traits, chimeric proteins have been produced by expression in E. coli. These experiments reveal that the C-terminal region of granule-bound starch synthase I confers most of the specific properties of this isoform, except its processive elongation of glucan chains. This region of granule-bound starch synthase I is distinct from the C-terminal region of other starch synthases. The specific properties it confers may be important in defining the specificity of granule-bound starch synthase I in producing amylose in vivo.     

Structural and thermodynamic properties of rice starches with different genetic background Part 1. Differentiation of amylopectin and amylose defects

A combined DSC - HPAEC-PAD approach, gel permeation chromatography and mild long-term acidic hydrolysis were employed to study the effects of amylopectin chain-length distributional and amylose defects on the assembly structures of amylopectin (crystalline lamellae, amylopectin clusters) in A-type polymorphic starches extracted from 11 Thai cultivars of rice with different amylose level. Joint analysis of the data allowed determining the contributions of different populations of amylopectin chains to the thermodynamic melting parameters of crystalline lamellae. It was shown that amylopectin chains with DP 6-12 and 25or=37 could be related to chains stabilizing these structures. The total effect of amylose and amylopectin defects can be described by means of Thomson-Gibbs' equation. The increase of defects in the assembly structures is accompanied by rise of the rates of acidic hydrolysis of both amorphous and crystalline parts in starches.     

Observations on the crystallization of amylopectin from aqueous solution

If concentrated aqueous solutions of amylopectin are stored at 2°C aggregation occurs with the eventual formation of an opaque elastic gel. The gel can be melted by heating to 100°C. During gel formation the amylopectin crystallizes into the B-type crystalline modification of starch and the crystalline order approaches that of the native starch granule.     

Effect of carbon source and dissolved oxygen level on cell growth and pullulanase production byBacillus stearothermophilus G-82

Cell growth and extracellular pullulanase production ofBacillus stearothermophilus G-82 were investigated in batch culture using a defined medium with glucose, maltose, pullulan or amylopectin as carbon source. Maximum enzyme activity was with pullulan or amylopectin. Cell growth in batch culture was better under oxygen unlimited conditions, while higher total and specific enzyme activities, using pullulan or amylopectin, were obtained in oxygen-limited conditions. Enzyme accumulation took place in the late growth phase. The highest enzyme production of 300 U/I was reached when pullulan was used as carbon source in conditions of oxygen limitation.     

Studies on carbohydrate-metabolising enzymes Part XXVII. A comparison of the action of yeast isoamylase and bacterial pullulanase on amylopectin

The action of purified yeast isoamylase on amylopectin, like that of bacterial pullulanase, results in the hydrolysis of the outermost inter-chain linkages with the liberation of linear maltosaccharides having an average degree of polymerisation of approximately 15 -glucose residues. This hydrolytic action distinguishes yeast isoamylase from yeast amylo-(1→6)-glucosidase, which acts by a combination of transferase and glucosidase activities. The products of enzyme action on amylopectin are discussed in relation to the probable molecular structure of the polysaccharide.     

A combined reduction in activity of starch synthases II and III of potato has novel effects on the starch of tubers

A chimeric antisense construct has been used to generate transgenic potatoes ( Solanum tuberosum L.) in which activities of both of the main starch synthases responsible for amylopectin synthesis in the tuber (SSII and SSIII) are reduced. The properties of starch from tubers of these plants have been compared with those of starches from transgenic plants in which activity of either SSII or SSIII has been reduced. Starches from the three types of transgenic plant are qualitatively different from each other and from the starch of control plants with unaltered starch synthase activities, with respect to granule morphology, the branch lengths of amylopectin, and the gelatinisation behaviour analysed by viscometry. The effects of reducing SSII and SSIII together cannot be predicted from consideration of the effects of reducing these two isoforms individually. These results indicate that different isoforms of starch synthase make distinct contributions to the synthesis of amylopectin, and that they act in a synergistic manner, rather than independently, during amylopectin synthesis.