Purification and characterization of cycloisomaltotetraose-forming glucanotransferases from Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006.

Glucanotransferases that can synthesize cyclo-{→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→} (CI4) from dextran were purified to homogeneity from the culture supernatant of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006. The molecular mass of both enzymes was estimated to be 86 kDa by SDS-PAGE. The glucanotransferase, named CI4-forming enzyme, from Agreia sp. exhibited the highest activity at pH 6.0 and 40 °C. The enzyme was stable on the pH range of 4.6-9.9 and up to 40 °C. On the other hand, the enzyme from M. trichothecenolyticum exhibited the highest activity at pH 5.7 and 40 °C. The enzyme was stable on the pH range of 5.0-6.9 and up to 35 °C. Both enzymes catalyzed 4 reactions, namely, intramolecular α-1,6-transglycosylation (cyclization), intermolecular α-1,6-transglycosylation, hydrolysis of CI4, and coupling reaction. Furthermore, the CI4-forming enzyme produced CI4 from α-1,6-linked glucan synthesized from starch by 6-α-glucosyltransferase. These findings will enable the production of CI4 from starch.

Gene cloning, expression enhancement in Escherichia coli and biochemical characterization of a highly thermostable amylomaltase from Pyrobaculum calidifontis.

Pcal_0768 gene encoding an amylomaltase, a 4-α-glucanatransferase belonging to family 77 of glycosyl hydrolases, from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. The recombinant protein was produced in E. coli in soluble and active form. However, the expression level was not very high. Analysis of the mRNA of initial seven codons at the 5'-end of the gene revealed the presence of a hair pin like secondary structure. This secondary structure was removed by site directed mutagenesis, without altering the amino acids, which resulted in enhanced expression of the cloned gene. Recombinant Pcal_0768 exhibited optimal amylomaltase activity at 80 °C and pH 6.9. Under these conditions, the specific activity was 690 U/ mg. Recombinant Pcal_0768 was highly thermostable with a half-life of 6 h at 100 °C. It exhibited the highest kcat value among the characterized glucanotransferases. No metal ions were required for activity or stability of the enzyme. Recombinant Pcal_0768 was successfully employed in the synthesis of modified starch for producing thermoreversible gel. To the best of our knowledge, till now this is the most thermostable enzyme among the characterized amylomaltases. High thermostability and starch modification potential make it a novel and distinct amylomaltase.

Expression of a Functional Recombinant Human Glycogen Debranching Enzyme (hGDE) in N. benthamiana Plants and in Hairy Root Cultures.

BACKGROUND: Glycogen storage disease type III (GSDIII, Cori/Forbes disease) is a metabolic disorder due to the deficiency of the Glycogen Debranching Enzyme (GDE), a large monomeric protein (about 176 kDa) with two distinct enzymatic activities: 4-α-glucantransferase and amylo-α-1,6-glucosidase. Several mutations along the amylo-alpha-1,6-glucosidase,4-alphaglucanotransferase (Agl) gene are associated with loss of enzymatic activity. The unique treatment for GSDIII, at the moment, is based on diet. The potential of plants to manufacture exogenous engineered compounds for pharmaceutical purposes, from small to complex protein molecules such as vaccines, antibodies and other therapeutic/prophylactic entities, was shown by modern biotechnology through "Plant Molecular Farming". OBJECTIVE AND METHODS: In an attempt to develop novel protein-based therapeutics for GSDIII, the Agl gene, encoding for the human GDE (hGDE) was engineered for expression as a histidinetagged GDE protein both in Nicotiana benthamiana plants by a transient expression approach, and in axenic hairy root in vitro cultures (HR) from Lycopersicum esculentum and Beta vulgaris. RESULTS: In both plant-based expression formats, the hGDE protein accumulated in the soluble fraction of extracts. The plant-derived protein was purified by affinity chromatography in native conditions showing glycogen debranching activity. CONCLUSION: These investigations will be useful for the design of a new generation of biopharmaceuticals based on recombinant GDE protein that might represent, in the future, a possible therapeutic option for GSDIII.

An ultra-stable glucanotransferase-cum-exoamylase from the hyperthermophile archaeon Thermococcus onnurineus.

The genome of the hyperthermophile archaeon Thermococcus onnurineus (strain NA1) encodes a 652 residues-long putative 4-α-glucanotransferase of the GH 57 family which we have expressed in Escherichia coli. The enzyme (TonAmyGT) appears to remove glucose from the reducing end of a donor glucan and transfers it to the non-reducing end of an acceptor glucan, creating a pool of oligosaccharides through disproportionation of any substrate maltooligosaccharide, with maltose acting substantively as the smallest donor glucan as well as the smallest acceptor glucan. Additionally, glucose is also cleaved from maltooligosaccharides and released into solution without being transferred to an acceptor, causing the enzyme to function as an exo-amylase (which can digest starch) in addition to its activity as a glucanotransferase. TonAmyGT functions over a broad range of temperature (20-100 °C) and pH (4.0-9.0), and shows extreme resistance to chemical and thermal denaturation, displaying a melting temperature of 104 °C, at a pressure of 35 psi, in a differential scanning calorimeter. An interesting characteristic is that the glucanotransferase activity shows feedback inhibition through glucose (which the enzyme itself generates), indicating that the exo-amylase and glucanotransferase activities regulate each other.

Purification and characterization of a novel intracellular α-amylase with a wide variety of substrates hydrolysis and transglycosylation activity from Paenibacillus sp. SSG-1.

Intracellular α-amylase was a special glycoside hydrolase in the cytoplasm. We cloned and expressed an intracellular α-amylase, Amy, from Paenibacillus sp. SSG-1. The recombinant enzyme was purified by metal-affinity chromatography, exhibited a molecular mass of 71.7 kDa. Amy exhibited unexpectedly sequence similarity and evolutionary relationships with alpha-glucanotransferase. The docked results of Amy with maltose showed it had similar catalytic residues with α-amylase and glucanotransferase. The substrate specificity experiment showed that Amy could hydrolyze typical substrates into glucose and maltose. It was noteworthy that Amy showed the catalytic capacity of cyclomaltodextrinase and pullulanase. Meanwhile, Amy could transfer sugar molecules and form maltotetraose upon the hydrolysis of substrates. These results indicated that Amy was a novel intracellular α-amylase with distinct catalytic ability characteristics of hydrolyzing glycogen/cyclodextrin/pullulan and transglycosylation. We deduced that Amy may play an important role in utilizing maltooligosaccharides that released from extracellular α-glucan or storage α-glucan (glycogen) in Paenibacillus sp. SSG-1.

Characterization of the Paenibacillus beijingensis DSM 24997 GtfD and its glucan polymer products representing a new glycoside hydrolase 70 subfamily of 4,6-α-glucanotransferase enzymes.

Previously we have reported that the Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 uses the 4,6-α-glucanotransferase GtfD to convert maltodextrins and starch into a reuteran-like polymer consisting of (α1→4) glucan chains connected by alternating (α1→4)/(α1→6) linkages and (α1→4,6) branching points. This enzyme constituted the single evidence for this reaction and product specificity in the GH70 family, mostly containing glucansucrases encoded by lactic acid bacteria (http://www.CAZy.org). In this work, 4 additional GtfD-like proteins were identified in taxonomically diverse plant-associated bacteria forming a new GH70 subfamily with intermediate characteristics between the evolutionary related GH13 and GH70 families. The GtfD enzyme encoded by Paenibacillus beijingensis DSM 24997 was characterized providing the first example of a reuteran-like polymer synthesizing 4,6-α-glucanotransferase in a Gram-positive bacterium. Whereas the A. chroococcum GtfD activity on amylose resulted in the synthesis of a high molecular polymer, in addition to maltose and other small oligosaccharides, two reuteran-like polymer distributions are produced by P. beijingensis GtfD: a high-molecular mass polymer and a low-molecular mass polymer with an average Mw of 27 MDa and 19 kDa, respectively. Compared to the A. chroooccum GtfD product, both P. beijingensis GtfD polymers contain longer linear (α1→4) sequences in their structure reflecting a preference for transfer of even longer glucan chains by this enzyme. Overall, this study provides new insights into the evolutionary history of GH70 enzymes, and enlarges the diversity of natural enzymes that can be applied for modification of the starch present in food into less and/or more slowly digestible carbohydrate structures.

Characterization of the 4,6-α-glucanotransferase GTFB enzyme of Lactobacillus reuteri 121 isolated from inclusion bodies.

BACKGROUND: The GTFB enzyme of the probiotic bacterium Lactobacillus reuteri 121 is a 4,6-α-glucanotransferase of glycoside hydrolase family 70 (GH70; http://www.cazy.org ). Contrary to the glucansucrases in GH70, GTFB is unable to use sucrose as substrate, but instead converts malto-oligosaccharides and starch into isomalto-/malto- polymers that may find application as prebiotics and dietary fibers. The GTFB enzyme expresses well in Escherichia coli BL21 Star (DE3), but mostly accumulates in inclusion bodies (IBs) which generally contain wrongly folded protein and inactive enzyme. METHODS: Denaturation followed by refolding, as well as ncIB preparation were used for isolation of active GTFB protein from inclusion bodies. Soluble, refolded and ncIB GTFB were compared using activity assays, secondary structure analysis by FT-IR, and product analyses by NMR, HPAEC and SEC. RESULTS: Expression of GTFB in E. coli yielded > 100 mg/l relatively pure and active but mostly insoluble GTFB protein in IBs, regardless of the expression conditions used. Following denaturing, refolding of GTFB protein was most efficient in double distilled H2O. Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein. When expressed as units of activity obtained per liter E. coli culture, the total amount of ncIB GTFB expressed possessed around 180 % hydrolysis activity and 100 % transferase activity compared to the amount of soluble GTFB enzyme obtained from one liter culture. The product profiles obtained for the three GTFB enzyme preparations were similar when analyzed by HPAEC and NMR. SEC investigation also showed that these 3 enzyme preparations yielded products with similar size distributions. FT-IR analysis revealed extended ß-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs. The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB. CONCLUSION: In view of their relatively high yield, activity and high thermostability, both refolded and ncIB GTFB derived from IBs in E. coli may find industrial application in the synthesis of modified starches.

Molecular mutagenesis at Tyr-101 of the amylomaltase transcribed from a gene isolated from soil DNA.

The wild-type (WT) amylomaltase gene was directly isolated from soil DNA and cloned into a pET19b vector to express in E. coli BL21(DE3). The ORF of this gene consisted of 1,572 bp, encoding an enzyme of 523 amino acids. Though showing 99% sequence identity to amylomaltse from Thermus thermophilus ATCC 33923, this enzyme is unique in its alkaline optimum pH. In order to alter amylomaltase with less coupling or hydrolytic activity to enhance cycloamylose (CA) formation through cyclization reaction, site-directed mutagenesis of the second glucan binding site involving in CA production was performed at Tyr-101. The result revealed that the mutated Y101S enzyme showed a small increase in cyclization activity while significantly decreased disproportionation, coupling and hydrolytic activities. Mutation also resulted in the change in substrate specificity for disproportionation reaction. The WT enzyme preferred maltotriose, while the activity of mutated enzyme was the highest with maltopentaose substrate. Product analysis by HPAEC-PAD demonstrated that the main CAs of the WT amylomaltase were CA29-CA37. Y101S mutation did not change the product pattern, however, the amount of CAs formed by the mutated enzyme tended to increase especially at long incubation time.

Enzymatic synthesis of Acarviosyl-maltooligosaccharides using disproportionating enzyme 1.

Acarbose is a pseudo-tetrasaccharide and one of the most effective inhibitors of glycoside hydrolases. Its derivatives, acarviosyl-maltooligosaccharides, which have longer maltooligosaccharide parts than the maltose unit of acarbose, were synthesized using a disproportionating enzyme partially purified from adzuki cotyledons. The enzyme was identified as a typical type-1 disproportionating enzyme (DPE1) by primary structure analysis. It produced six compounds from 100 mM acarbose and 7.5% (w/v) of maltotetraose-rich syrup. The masses of the six products were confirmed to accord with acarviosyl-maltooligosaccharides with the degrees of polymerization = 5-10 (AC5-AC10) by electrospray ionization mass spectrometry. (1)H and (13)C NMR spectra indicated that AC5-AC10 were α-acarviosyl-(1→4)-maltooligosaccharide, which have maltotriose-maltooctaose respectively in the maltooligosaccharide part. A predominance of AC7 in the products at the early stage of the reaction indicated that DPE1 catalyzes the transfer of the acarviosyl-glucose moiety from acarbose to the acceptors. ACn can be useful tools as new inhibitors of glycoside hydrolases.

Altered large-ring cyclodextrin product profile due to a mutation at Tyr-172 in the amylomaltase of Corynebacterium glutamicum.

Corynebacterium glutamicum amylomaltase (CgAM) catalyzes the formation of large-ring cyclodextrins (LR-CDs) with a degree of polymerization of 19 and higher. The cloned CgAM gene was ligated into the pET-17b vector and used to transform Escherichia coli BL21(DE3). Site-directed mutagenesis of Tyr-172 in CgAM to alanine (Y172A) was performed to determine its role in the control of LR-CD production. Both the recombinant wild-type (WT) and Y172A enzymes were purified to apparent homogeneity and characterized. The Y172A enzyme exhibited lower disproportionation, cyclization, and hydrolysis activities than the WT. The k(cat)/K(m) of the disproportionation reaction of the Y172A enzyme was 2.8-fold lower than that of the WT enzyme. The LR-CD product profile from enzyme catalysis depended on the incubation time and the enzyme concentration. Interestingly, the Y172A enzyme showed a product pattern different from that of the WT CgAM at a long incubation time. The principal LR-CD products of the Y172A mutated enzyme were a cycloamylose mixture with a degree of polymerization of 28 or 29 (CD28 or CD29), while the principal LR-CD product of the WT enzyme was CD25 at 0.05 U of amylomaltase. These results suggest that Tyr-172 plays an important role in determining the LR-CD product profile of this novel CgAM.

Characterization of 4-alpha-glucanotransferase from Synechocystis sp. PCC 6803 and its application to various corn starches.

A putative 4-alpha-glucanotransferase (alphaGTase) gene from Synechocystis sp. PCC 6803 was identified being composed of 1505 nucleotides, and the overexpressed protein was purified with an affinity chromatography. The recombinant alphaGTase had about 57kDa of molecular mass when judged by SDS-PAGE analysis. The optimum reaction condition of the alphaGTase was shown to be pH 7 at 45 degrees C in 50mm phosphate buffer. This enzyme displayed transglycosylating activity on various maltooligosaccharides, of which the smallest donor and acceptor molecules were determined to be maltose and glucose, respectively. Various corn starches consisting of different proportions of amylopectin and amylose were incubated with the recombinant alphaGTase. The change in molecular weight distribution of alphaGTase-modified starch was analyzed by HPSEC. The reaction pattern of alphaGTase showed substantial decrease in amylopectin and increase in the peak corresponding to cycloamylose (CA). The production yield of CA tended to increase from 5 to 30% along with the increase in the apparent amylose content in corn starch, which suggested that linear amylose chain would be preferred to produce CA in the alphaGTase treatment. The detectable minimum degree of polymerization (DP) of CA was shown to be 22 by MALDI-TOF-MS analysis. As another action mode of alphaGTase, the rearrangement of amylopectin branch-chain distribution occurred without hydrolysis to small oligosaccharides. After isoamylolysis, alphaGTase-treated starch displayed the increase in DP 4-9 and longer than DP 21 when the relative proportion of branch chains in amylopectin was determined by HPAEC.

Characterization of a novel debranching enzyme from Nostoc punctiforme possessing a high specificity for long branched chains.

A novel debranching enzyme from Nostoc punctiforme PCC73102 (NPDE) exhibits hydrolysis activity toward both alpha-(1,6)- and alpha-(1,4)-glucosidic linkages. The action patterns of NPDE revealed that branched chains are released first, and the resulting maltooligosaccharides are then hydrolyzed. Analysis of the reaction with maltooligosaccharide substrates labeled with (14)C-glucose at the reducing end shows that NPDE specifically liberates glucose from the reducing end. Kinetic analyses showed that the hydrolytic activity of NPDE is greatly affected by the length of the substrate. The catalytic efficiency of NPDE increased considerably upon using substrates that can occupy at least eight glycone subsites such as maltononaose and maltooctaosyl-alpha-(1,6)-beta-cyclodextrin. These results imply that NPDE has a unique subsite structure consisting of -8 to +1 subsites. Given its unique subsite structure, side chains shorter than maltooctaose in amylopectin were resistant to hydrolysis by NPDE, and the population of longer side chains was reduced.

Function of second glucan binding site including tyrosines 54 and 101 in Thermus aquaticus amylomaltase.

Amylomaltase from Thermus aquaticus catalyzes three types of transglycosylation reaction, as well as a weak hydrolytic reaction of alpha-1,4 glucan. From our previous study [Fujii et al., Appl. Environ. Microbiol., 71, 5823-5827 (2005)], tyrosine 54 (Y54) was identified as an amino acid controlling the reaction specificity of this enzyme. Since Y54 is not located around the active site but in the proposed second glucan binding site that is 14 A away from catalytic residues, the functions of Y54 and the second glucan binding site are of great interest. In this study, we introduced mutations into another tyrosine (Y101) in the second glucan binding site. The obtained mutated enzymes were subjected to all four types of enzyme assay and the effects of mutations on the reaction specificities of these enzymes were comprehensively investigated. These studies indicated that the amino acid substitution at Y54 or Y101 for removing their aromatic side chain increases cyclization activity (intra-molecular transglycosylation reaction) but decreases disproportionation, coupling and hydrolytic activities (inter-molecular reactions). The superimposition of the reported structures of the enzyme with and without substrate analog revealed the occurrence of a conformational change in which a donor binding site becomes open. From lines of evidence, we conclude that the binding of glucan substrate to the second glucan binding site through an interaction with the aromatic side chains of Y54 and Y101 is a trigger for the enzyme to take a completely active conformation for all four types of activity, but prevents the cyclization reaction to occur since the flexibility of the glucan is restricted by such binding.

Activation of 4-alpha-glucanotransferase activity of porcine liver glycogen debranching enzyme with cyclodextrins.

Glycogen debranching enzyme (GDE) is a single polypeptide chain containing distinct active sites for 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. Debranching of phosphorylase limit dextrin from glycogen is carried out by cooperation of the two activities. We examined the effects of cyclodextrins (CDs) on debranching activity of porcine liver GDE using a fluorogenic branched dextrin, Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5/84), as a substrate. B5/84 was hydrolyzed by the hydrolytic action of 4-alpha-glucanotransferase to B5/81 and maltotriose. The fluorogenic product was further hydrolyzed by the amylo-alpha-1,6-glucosidase activity to the debranched product, Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (G8PA), and glucose. alpha-, beta- and gamma-CDs accelerated the liberation of B5/81 from B5/84, indicating that the 4-alpha-glucanotransferase activity was activated by CDs to remove the maltotriosyl residue from the maltotetraosyl branch. This led to acceleration of B5/84 debranching. The extent of 4-alpha-glucanotransferase activation increased with CD concentration before reaching a constant value. This suggests that there is an activator binding site and that the binding of CDs stimulates 4-alpha-glucanotransferase activity. In the porcine liver, glycogen degradation may be partially stimulated by the binding of a glycogen branch to this activator binding site.

Purification of glycogen debranching enzyme from porcine brain: evidence for glycogen catabolism in the brain.

Amylo-1,6-glucosidase from porcine brain was purified to homogeneity by ammonium sulfate fractionation, followed by sequential steps of liquid chromatography on DEAE-Sephacel, Sephacryl S-300, and Super Q. The purified enzyme had both maltooligosaccharide transferase and amylo-1,6-glucosidase activities within a single polypeptide chain, and the combination of these two activities removed the branches of phosphorylase limit dextrin. Based on these results, the purified enzyme was identified as a glycogen debranching enzyme (GDE). The molecular weight of the brain GDE was 170,000 by gel-filtration and 165,000 by reducing SDS-PAGE. The pH profile of maltooligosaccharide transferase activity coincided with that of the amylo-1,6-glucosidase activity (pH optimum at 6.0). The existence of GDE as well as glycogen phosphorylase in the brain explains brain glycogenolysis fully and supports the hypothesis that glycogen is a significant source of energy in this organ.

Characterisation of disproportionating enzyme from wheat endosperm.

Disproportionating enzyme or D-enzyme (EC 2.4.1.25) is an alpha-1,4 glucanotransferase which catalyses cleavage and transfer reactions involving alpha-1,4 linked glucans altering (disproportionating) the chain length distribution of pools of oligosaccharides. While D-enzyme has been well characterised in some plants, e.g. potato and Arabidopsis, very little is known about its abundance and function in cereals which constitute the major source of starch worldwide. To address this we have investigated D-enzyme in wheat (Triticum aestivum). Two putative D-enzyme cDNA clones have been isolated from tissue-specific cDNA libraries. TaDPE1-e, from an endosperm cDNA library, encodes a putative polypeptide of 575 amino acid residues including a predicted transit peptide of 41 amino acids. The second cDNA clone, TaDPE1-l, from an Aegilops taushii leaf cDNA library, encodes a putative polypeptide of 579 amino acids including a predicted transit peptide of 45 amino acids. The mature polypeptides TaDPE1-e and TaDPE1-l were calculated to be 59 and 60 kDa, respectively, and had 96% identity. The putative polypeptides had significant identity with deduced D-enzyme sequences from corn and rice, and all the expected conserved residues were present. Protein analysis revealed that D-enzyme is present in the amyloplast of developing endosperm and in the germinating seeds. D-enzyme was partially purified from wheat endosperm and shown to exhibit disproportionating activity in vitro by cleaving maltotriose to produce glucose as well as being able to use maltoheptaose as the donor for the addition of glucans to the outer chains of glycogen and amylopectin.

Crystallization and preliminary X-ray crystallographic study of disproportionating enzyme from potato.

Disproportionating enzyme (D-enzyme; EC 2.4.1.25) is a 59 kDa protein that belongs to the alpha-amylase family. D-enzyme catalyses intramolecular and intermolecular transglycosylation reactions of alpha-1,4 glucan. A crystal of the D-enzyme from potato was obtained by the hanging-drop vapour-diffusion method. Preliminary X-ray data showed that the crystal diffracts to 2.0 A resolution and belongs to space group C222(1), with unit-cell parameters a = 69.7, b = 120.3, c = 174.2 A.

Transglycosylation activity of Dictyoglomus thermophilum amylase A.

Amylase A from Dictyoglomus thermophilum is a thermophilic enzyme and has about 40% identity with 4-alpha-glucanotransferase (GTase) from Thermococcus litoralis, and both of these enzymes belong to family 57 glycosyl hydrolase. Since the transglycosylation activity of T. litoralis GTase has been well characterized, the substrate specificity and reaction products of amylase A from D. thermophilum were examined. alpha-1,4 Glucan was produced from maltooligosaccharides, and glucoamylase-resistant molecules (cycloamyloses) were produced from longer chain amylose (average molecular mass 200 kDa). It has been reported that amylase A from D. thermophilum hydrolyzes starch, but in this study it was found that the enzyme was also able to use maltooligosaccharides and long chain amylose as substrate and has transglycosylation activity.

A synergistic reaction mechanism of a cycloalternan-forming enzyme and a D-glucosyltransferase for the production of cycloalternan in Bacillus sp. NRRL B-21195.

Cycloalternan-forming enzyme (CAFE) was first described as the enzyme that produced cycloalternan from alternan. In this study, we found that a partially purified preparation of CAFE containing two proteins catalyzed the synthesis of cycloalternan from maltooligosaccharides, whereas the purified CAFE alone was unable to do so. In addition to the 117 kDa CAFE itself, the mixture also contained a 140 kDa protein. The latter was found to be a disproportionating enzyme (DE) that catalyzes transfer of a D-glucopyranosyl residue from the non-reducing end of one maltooligosaccharide to the non-reducing end of another, forming an isomaltosyl residue at the non-reducing end. CAFE then transfers the isomaltosyl residue to the non-reducing end of another isomaltosyl maltooligosaccharide, to form an alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-(1-->4)-maltooligosaccharide, and subsequently catalyzes a cyclization to produce cycloalternan. Thus, DE and CAFE act synergistically to produce cycloalternan directly from maltodextrin or starch.

Glycogen debranching enzyme in bovine brain.

Glycogen debranching enzyme was partially purified from bovine brain using a substrate for measuring the amylo-1,6-glucosidase activity. Bovine cerebrum was homogenized, followed by cell-fractionation of the resulting homogenate. The enzyme activity was found mainly in the cytosolic fraction. The enzyme was purified 5,000-fold by ammonium sulfate precipitation, anion-exchange chromatography, gel-filtration, anion-exchange HPLC, and gel-permeation HPLC. The enzyme preparation had no alpha-glucosidase or alpha-amylase activities and degraded phosphorylase limit dextrin of glycogen with phosphorylase. The molecular weight of the enzyme was 190,000 and the optimal pH was 6.0. The brain enzyme differed from glycogen debranching enzyme of liver or muscle in its mode of action on dextrins with an alpha-1,6-glucosyl branch, indicating an amino acid sequence different from those of the latter two enzymes. It is likely that the enzyme is involved in the breakdown of brain glycogen in concert with phosphorylase as in the cases of liver and muscle, but that this proceeds in a somewhat different manner. The enzyme activity decreased in the presence of ATP, suggesting that the degradation of brain glycogen is controlled by the modification of the debranching enzyme activity as well as the phosphorylase.