Extracellular Isoamylase Produced by the Yeast Lipomyces kononenkoae

A strain of the starch-converting yeast Lipomyces kononenkoae produced, when grown on starch, a debranching enzyme that proved to be an isoamylase (glycogen 6-glucanohydrolase; E.C. 3.2.1.68). So far, only bacteria have been found to produce extracellular isoamylases. The yeast isoamylase enhanced β-amylolysis of amylopectin and glycogen and completely hydrolyzed these substrates into maltose when combined with a β-amylase but had no action on dextran or pullulan. By isopropanol precipitation and carboxymethyl cellulose chromatography, L. kononenkoae isoamylase was partially purified from the supernatant of cultures grown on a mineral medium with soluble starch. Optimum temperature and pH for activity of the isoamylase were 30°C and 5.6. The molecular weight was around 65,000, and the pI was at pH 4.7 to 4.8. The K_m (30°C, pH 5.5) for soluble starch was 9 g liter⁻¹.     

Antisense inhibition of isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm

This is the first report on regulation of the isoamylase1 gene to modify the structure of amylopectin and properties of starch by using antisense technology in plants. The reduction of isoamylase1 protein by about 94% in rice endosperm changed amylopectin into a water-insoluble modified amylopectin and a water-soluble polyglucan (WSP). As compared with wild-type amylopectin, the modified amylopectin had more short chains with a degree of polymerization of 5-12, while their molecular sizes were similar. The WSP, which structurally resembled the phytoglycogen in isoamylase-deficient sugary-1 mutants, accounted for about 16% of the total alpha-polyglucans in antisense endosperm, and it was distributed throughout the whole endosperm unlike in sugary-1 mutant. The reduction of isoamylase activity markedly lowered the gelatinization temperature from 54 to 43 degrees C and the viscosity, and modified X-ray diffraction pattern and the granule morphology of the starch. The activity of pullulanase, the other type of starch debranching enzyme, in the antisense endosperm was similar to that in wild-type, whereas it is deficient in sugary-1 mutants. These results indicate that the isoamylase1 is essential for amylopectin biosynthesis in rice endosperm, and that alteration of the isoamylase activity is an effective means to modify the physicochemical properties and granular structure of starch.     

Multiple branching in glycogen and amylopectin

The β-amylase limit dextrins of glycogen and amylopectin are completely debranched by joint action of isoamylase and pullulanase. Action of isoamylase alone results in incomplete debranching as a consequence of the inability of this enzyme to hydrolyze those A-chains that are two glucose units in length (half the total number of A-chains). From the reducing powers released by isoamylase acting (a) alone and (b) in conjunction with pullulanase, the relative numbers of A- (unsubstituted) and B- (substituted) chains in the β-dextrins, and therefore in the native polysaccharides themselves, can be calculated. Examination of a series of glycogens and amylopectins in this way showed that the ratio of A-chains: B-chains is markedly higher in amylopectins (1.5–2.6:1) than in glycogens (0.6–1.2:1). Glycogen typically contains A-chains and B-chains in approximately equal numbers; amylopectin typically contains approximately twice as many A-chains as B-chains. These polysaccharides therefore differ in degree of multiple branching as well as in average chain length. A consequence of these findings is that amylopectin cannot be formed in vivo by debranching of a glycogen precursor, as proposed by Erlander, since it is impossible to increase the A:B chain ratio by action of a debranching enzyme.     

Analyses of isoamylase gene activity in wild-type barley indicate its involvement in starch synthesis

The notion of debranching enzyme activity as a participant in starch synthesis is gaining acceptance. Inconsistent reports from mutant analyses implicate either isoamylase or pullulanase as a determinant in amylopectin formation and whether wild-type plants utilize one or the other, or both, of these debranching enzymes in starch synthesis is unclear. Recent results on the su1 mutant in maize suggest that both forms of debranching enzymes might be involved in amylopectin formation. We wished to find out if isoamylase takes part in starch synthesis by comparing isoamylase gene activity under three conditions: (1) during starch accumulation in developing sink tissues; (2) during starch degradation in germinating seeds; (3) in ectopic expression after applying sucrose, a starch precursor. We isolated the gene for barley isoamylase, iso1, and analysed its expression and regulation in germinating seeds, developing endosperm and vegetative tissues, and compared the isoamylase gene expression in sink tissues from three different species. Our results indicate that isoamylase gene activity is involved in starch synthesis in wild-type plants and is modulated by sucrose.     

Action of Pseudomonas isoamylase on various branched oligo- and poly-saccharides

Pseudomonas isoamylase (EC 3.2.1.68) hydrolyzes (1 → 6)-α-D-glucosidic linkages of amylopectin, glycogen, and various branched dextrins and oligosaccharides. The detailed structural requirements for the substrate are examined qualitatively and quantitatively in this paper, in comparison with the pullulanase of Klebsiella aerogenes. As with pullulanase. Ps. isoamylase is unable to cleave D-glucosyl stubs from branched saccharides. Ps. isoamylase differs from pullulanase in the following characteristics: (1) The favored substrates for Ps. isoamylase are higher-molecular-weight polysaccharides. Most of the branched oligosaccharides examined were hydrolyzed at a lower rate, 10% or less of the rate of hydrolysis of amylopectin. (2) Maltosyl branches are hydrolyzed off by Ps. isoamylase very slowly in comparison with maltotriosyl branches. (3)Pr. isoamylase requires a minimum of three D-glucose residues in the B- or C-chain.     

The Starch-Debranching Enzymes Isoamylase and Pullulanase Are Both Involved in Amylopectin Biosynthesis in Rice Endosperm

The activities of the two types of starch debranching enzymes, isoamylase and pullulanase, were greatly reduced in endosperms of allelic sugary-1 mutants of rice (Oryza sativa), with the decrease more pronounced for isoamylase than for pullulanase. However, the decrease in isoamylase activity was not related to the magnitude of the sugary phenotype (the proportion of the phytoglycogen region of the endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and isoamylase activity was extremely low or completely lost in the whole endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice endosperm. We presume that isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that isoamylase was modified in some sugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that the Sugary-1 gene encodes the isoamylase gene of the rice genome.     

Effects of substrate branching characteristics on kinetics of enzymatic depolymerizaion of mixed linear and branched polysaccharides: I. Amylose/amylopectin alpha-amylolysis

Hydrolysis reactions of homopolysaccharides, which differ in their degree of branching, and mixtures of linear and branched polymers were carried out with alpha-amylase. The branching structures of both the original amylopectin substrate and the cluster domains of amylopectin, obtained by ethanol precipitation of the products of the action of alpha-amylase, were characterized via enzymatic digestion with debranching enzyme (i.e., isoamylase), followed by the fractions of the resulting products using gel filtration chromatography. The structural properties (i.e., molecular weight, molecular weight distribution, and branching characteristics) of the resulting products during depolymerization of amylose, amylopectin and their mixtures via alpha-amylase were characterized by size exclusion chromatography coupled with a low angle laser right scattering (SEC/LALLS) technique. It was determined that substrate branching characteristics strongly influence both the observed enzymatic activity as well as the enzyme's action pattern. A simplified kinetic model that represents the hydrolysis reactions of amylose and amylopectin mixtures via endo-acting alpha-amylase is proposed. We found that that reaction kinetics (i.e., enzyme affinity) was also governed by the substrate's conformation in solution. The relationships between the mass fraction of branched polymers and the kinetic parameters during alpha-amylolysis were compared with those predicted by the kinetic model. Excellent agreement was found between the model predictions and the experimental observations. The results reported here imply and interrelationship between enzyme action and polymeric substrate structural properties. (c) 1994 John Wiley & Sons, Inc.     

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.     

Formation of Isoamylase by Pseudomonas

We have isolated a Pseudomonas sp. (strain SB15) which produces an isoamylase (EC 3.2.1.9). Highest yields of this enzyme were obtained when the bacterium was grown in shaken culture in a medium containing maltose, dextrin, starch, or isomaltose. Specific carbon and nitrogen sources were required for growth. The most satisfactory medium consisted of 2% maltose, 0.4% sodium glutamate, 0.3% diammonium hydrogen phosphate, and other inorganic salts. The optimal pH for enzyme production was 5 to 6. The enzyme is stable between pH 3 and 6 but is extremely labile above pH 7. It splits amylopectin completely by combined action with beta-amylase but does not attack pullulan.     

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.     

TreX from Sulfolobus solfataricus ATCC 35092 displays isoamylase and 4-alpha-glucanotransferase activities

A treX in the trehalose biosynthesis gene cluster of Sulfolobus solfataricus ATCC 35092 has been reported to produce TreX, which hydrolyzes the alpha-1,6-branch portion of amylopectin and glycogen. TreX exhibited 4-alpha-D-glucan transferase activity, catalyzing the transfer of alpha-1,4-glucan oligosaccharides from one molecule to another in the case of linear maltooligosaccharides (G3-G7), and it produced cyclic glucans from amylopectin and amylose like 4-alpha-glucanotransferase. These results suggest that TreX is a novel isoamylase possessing the properties of 4-alpha-glucanotransferase.     

Biochemical and genetic analysis of the effects of amylose-extender mutation in rice endosperm

Biochemical analysis of amylose-extender (ae) mutant of rice (Oryza sativa) revealed that the mutation in the gene for starch-branching enzyme IIb (BEIIb) specifically altered the structure of amylopectin in the endosperm by reducing short chains with degree of polymerization of 17 or less, with the greatest decrease in chains with degree of polymerization of 8 to 12. The extent of such change was correlated with the gelatinization properties of the starch granules, as determined in terms of solubility in urea solution. The ae mutation caused a dramatic reduction in the activity of BEIIb. The activity of soluble starch synthase I (SSI) in the ae mutant was significantly lower than in the wild type, suggesting that the mutation had a pleiotropic effect on the SSI activity. In contrast, the activities of BEI, BEIIa, ADP-Glc pyrophosphorylase, isoamylase, isoamylase, pullulanase, and Suc synthase were not affected by the mutation. Therefore, it is stressed that the function of BEIIb cannot be complemented by BEIIa and BEI. These results strongly suggest that BEIIb plays a specific role in the transfer of short chains, which might then be extended by SS to form the A and B(1) chains of amylopectin cluster in rice endosperm.     

Kinetic expression for maltose production from soluble starch by simultaneous use of beta-amylase and debranching enzymes

The kinetics of the hydrolysis of soluble starch by simultaneous use of beta-amylase and either isoamylase or pullulanse was studied experimentally for a wide range of subtrate and enzyme concentrations. A kinetic expression was constituted for maltose production by beta-armylase, which was stimulated by an increase in linear linkage portions due to the debranching enzyme on amylopectin molecules. As a result, calculations by the kinetic expression agreed with time course data under various conditions.     

Comparative biochemistry of α-glucan-utilization in pseudomonas amyloderamosa and Pseudomonas saccharophila

Growth patterns on and utilization of various α-glucans were investigated in Pseudomonas amyloderamosa and P. saccharophila. Maltose, maltodextrins (average chain length 7 glucosyl units) and glycogen supported excellent growth of both organisms and were extensively metabolized, although glycogen utilization in P. saccharophila was preceded by a prolonged lag phase. P. amyloderamosa produced limited growth on amylopectin and the carbohydrate was only partly degraded. It seemed likely that many of the unit chains liberated from amylopectin had a length exceeding the substrate range accepted by the maltodextrin permease (transport) system. A correlation was established between the pH of the medium and the utilization of glycogen and amylopectin for growth in P. amyloderamosa. The carbohydrates were at least partly utilizable at pH 6.0, whereas they could not support any growth at pH 6.5. Most likely, the lack of growth at the higher pH reflected the low activity of isoamylase at this pH. The enzyme patterns of maltodextrin catabolism in the two bacteria were established. Intracellularly, maltodextrin phosphorylase and 4-α-glucanotransferase occurred in both. Degradation of extracellular α-glucans was mediated by a mainly intracellular isoamylase in P. amyloderamosa, whereas P. saccharophila possessed an extracellular α-amylase and a firmly cell-bound pullulanase.     

A comparative study of edible canna (Canna edulis) starch from different cultivars. Part II. Molecular structure of amylose and amylopectin

Molecular structures of starches isolated from Japanese-green, Thai-green and Thai-purple cultivars of edible canna (Canna edulis Ker) were investigated. The absolute amylose content ranged from 19 to 25%. Degrees of polymerization (DPn) values of amylose determined by fluorescence-labeling method were 1590 for Thai-purple, 1620 for Japanese-green and 1650 for Thai-green cultivars. Mole% of branched fraction of amyloses from edible canna starches examined by a HPLC system after β-amylolysis of labeled amyloses was 13–16%. Branch chain-length distributions of amylopectin analyzed by HPSEC after debranching with isoamylase, followed by fluorescence-labeling of unit chain, showed bimodal distribution with the DPn range of 25–28. The amylopectin of edible canna starches contained high amounts of organic phosphorus (391–420 ppm). The distribution profile of phosphorylated chains, separated from non-phosphorylated chains by DEAE-Sephadex A-50 chromatography, indicated that the phosphate groups were located mostly in long B-chains of amylopectin molecules.     

Mode of Loss of Phosphatidyl-ethanolamine in Autoxidation

This report identifies and describes the chemical structure of granular material that accumulates in the cytoplasm of Selenomonas ruminantium grown in glucose or lactate medium. The granular material was identified to be glycogen. Its molecular weight was about 2 x 10⁷ daltons. Conversion into maltose with α-amylase, β-amylase, and isoamylase was 61%, 37%, and 15%, respectively. The glycogen was digested completely by joint action of β-amylase and isoamylase, and its conversion into maltose was 103%. The average chain length of the glycogen was 23.5. The maximum absorption of the iodine complex of the glycogen was at 520 nm. These results led us to conclude that the chemical structure of this glycogen was similar to that of plant amylopectin, unlike normal Microbiol or animal glycogen so far known. When S. ruminantium was grown in glucose medium, the amount of glycogen in cells reached about 260 µg/mg dry weight of cells during late exponential phase and early stationary phase.     

Three isoforms of isoamylase contribute different catalytic properties for the debranching of potato glucans

Isoamylases are debranching enzymes that hydrolyze alpha-1,6 linkages in alpha-1,4/alpha-1,6-linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism.     

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.