Tailor-Made α-Glucans by Engineering the Processivity of α-Glucanotransferases via Tunnel-Cleft Active Center Interconversions.

The function of polysaccharides is intimately associated with their size, which is largely determined by the processivity of transferases responsible for their synthesis. A tunnel active center architecture has been recognized as a key factor that governs processivity of several glycoside hydrolases (GHs), e.g., cellulases and chitinases. Similar tunnel architecture is also observed in the Limosilactobacillus reuteri 121 GtfB (Lr121 GtfB) α-glucanotransferase from the GH70 family. The molecular element underpinning processivity of these transglucosylases remains underexplored. Here, we report the synthesis of the smallest (α1 → 4)-α-glucan interspersed with linear and branched (α1 → 6) linkages by a novel 4,6-α-glucanotransferase from L. reuteri N1 (LrN1 GtfB) with an open-clefted active center instead of the tunnel structure. Notably, the loop swapping engineering of LrN1 GtfB and Lr121 GtfB based on their crystal structures clarified the impact of the loop-mediated tunnel/cleft structure at the donor subsites -2 to -3 on processivity of these α-glucanotransferases, enabling the tailoring of both product sizes and substrate preferences. This study provides unprecedented insights into the processivity determinants and evolutionary diversification of GH70 α-glucanotransferases and offers a simple route for engineering starch-converting α-glucanotransferases to generate diverse α-glucans for different biotechnological applications.

N1019D Mutant of Limosilactobacillus reuteri 121 4,6-α-Glucanotransferase GtfB Significantly Improved Catalytic Activity.

Limosilactobacillus reuteri 121 4,6-α-glucanotransferase GtfB (Lr 121 GtfB), belonging to glycoside hydrolase family 70 (GH70), synthesizes linear isomalto/malto polysaccharides having (α1→6) linkages attached to the nonreducing ends of (α1→4) linked maltose oligosaccharide segments using starch or maltodextrin as a substrate. Since Lr 121 GtfB has low catalytic activity and efficiency, it leads to substrate regeneration and reduced substrate utilization. In this study, we superimposed the crystal structure of Lr 121 GtfB-ΔNΔV with that of L. reuteri NCC 2613 GtfB-ΔNΔV (Lr 2613 GtfB-ΔNΔV) to identify the acceptor binding subsites +1 to +3 and constructed five single-residue mutants and a random mutagenesis of N1019. Compared with the wild-type, N1019D Lr 121 GtfB-ΔN did not alter the product specificity, increased the catalytic activity and efficiency by 420 and 590%, respectively, and maintained >80% relative activity in the pH 3.5-6.5 interval. The findings will contribute to the industrial application of Lr 121 GtfB and provide new solutions for starch synthesis of higher value derivatives.

Bioinformatics and functional selection of GH77 4-α-glucanotransferases for potato starch modification.

4-α-glucanotransferases (4αGTs, EC 2.4.1.25) from glycoside hydrolase family 77 (GH77) catalyze chain elongation of starch amylopectin chains and can be utilized to structurally modify starch to tailor its gelation properties. The potential relationship between the structural design of 4αGTs and functional starch modification is unknown. Here, family GH77 was mined in silico for enzyme candidates based on sub-grouping guided by Conserved Unique Peptide Patterns (CUPP) bioinformatics categorization. From + 12,000 protein sequences a representative set of 27 4αGTs, representing four different domain architectures, different bacterial origins and diverse CUPP groups, was selected for heterologous expression and further study. Most of the enzymes catalyzed starch modification, but their efficacies varied substantially. Five of the 4αGTs were characterized in detail, and their action was compared to that of the industrial benchmark enzyme, Tt4αGT (CUPP 77_1.2), from Thermus thermophilus. Reaction optima of the five 4αGTs ranged from ∼40-60 °C and pH 7.3-9.0. Several were stable for a minimum 4 h at 70 °C. Domain architecture type A proteins, consisting only of a catalytic domain, had high thermal stability and high starch modification ability. All five novel 4αGTs (and Tt4αGT) induced enhanced gelling of potato starch. One, At4αGT from Azospirillum thermophilum (CUPP 77_2.4), displayed distinct starch modifying abilities, whereas T24αGT from Thermus sp. 2.9 (CUPP 77_1.2) modified the starch similarly to Tt4αGT, but slightly more effectively. T24αGT and At4αGT are thus interesting candidates for industrial starch modification. A model is proposed to explain the link between the 4αGT induced molecular modifications and macroscopic starch gelation.

Distribution of Aromatic Amino Acid Residues in Substrate-Binding Regions Modulates Substrate Specificity of Microbial Debranching Enzymes.

Debranching enzymes (DBEs) directly hydrolyze α-1,6-glucosidic linkages in glycogen, starch, and related polysaccharides, making them important in the starch processing industry. However, the ambiguous substrate specificity usually restricts synergistic catalysis with other amylases for improving starch utilization. Herein, a glycogen-debranching enzyme from Saccharolobus solfataricus (SsGDE) and two isoamylases from Pseudomonas amyloderamosa (PaISO) and Chlamydomonas reinhardtii (CrISO) were used to investigate the molecular mechanism of substrate specificity. Along with the structure-based computational analysis, the aromatic residues in the substrate-binding region of DBEs played an important role in binding substrates. The aromatic residues in SsGDE appeared clustered, contributing to a small substrate-binding region. In contrast, the aromatic residues in isoamylase were distributed dispersedly, forming a large active site. The distinct characteristics of substrate-binding regions in SsGDE and isoamylase might explain their substrate preferences for maltodextrin and amylopectin, respectively. By modulating the substrate-binding region of SsGDE, variants Y323F and V375F were obtained with significantly enhanced activities, and the activities of Y323F and V375F increased by 30 and 60% for amylopectin, and 20 and 23% for DE4 maltodextrin, respectively. This study revealed the molecular mechanisms underlying the substrate specificity for SsGDE and isoamylases, providing a route for engineering enzymes to achieve higher catalytic performance.

Structural characterization of potato starch modified by a 4,6-α-glucanotransferase B from Lactobacillus reuteri E81.

The recent reports have revealed that increase in amount of α-1,6 linkages by modification of potato starch with enzyme (glycosyltransferases) treatment gains slowly digestible properties to the starch; however, the formation of new α-1,6-glycosidic linkages diminish the thermal resistance of the starch granules. In this study, a putative GtfB-E81, (a 4,6-α-glucanotransferase-4,6-αGT) from L. reuteri E81 was firstly used to produce a short length of α-1,6 linkages. NMR results revealed that external short chains mostly comprised of 1-6 glucosyl units were newly produced in potato starch, and the α-1,6 linkage ratio was significantly increased from 2.9 % to 36.8 %, suggesting that this novel GtfB-E81 might have potentially an efficient transferase activity. In our study, native and GtfB-E81 modified starches showed fundamental similarities with respect to their molecular properties and treatment of native potato starch with GtfB-E81 did not remarkably change thermal stability of the potato starch, which seems to be very prominent for the food industry given the significantly decreased thermal stability results obtained for the enzyme modified starches reported in the literature. Therefore, the results of this study should open up emerging perspectives for regulating slowly digestible characteristics of potato starch in future studies without a significant change in the molecular, thermal, and crystallographic properties.

Multiple approaches of loop region modification for thermostability improvement of 4,6-α-glucanotransferase from Limosilactobacillus fermentum NCC 3057.

4,6-α-glucanotransferase (4,6-α-GT), as a member of the glycoside hydrolase 70 (GH70) family, converts starch/maltooligosaccharides into α,1-6 bond-containing α-glucan and possesses potential applications in food, medical and related industries but does not satisfy the high-temperature requirement due to its poor thermostability. In this study, a 4,6-α-GT (ΔGtfB) from Limosilactobacillus fermentum NCC 3057 was used as a model enzyme to improve its thermostability. The loops of ΔGtfB as the target region were optimized using directed evolution, sequence alignment, and computer-aided design. A total of 11 positive mutants were obtained and iteratively combined to obtain a combined mutant CM9, with high resistance to temperature (50 °C). The activity of mutant CM9 was 2.08-fold the activity of the wild type, accompanied by a 5 °C higher optimal temperature, a 5.76 °C higher melting point (Tm, 59.46 °C), and an 11.95-fold longer half-life time (t1/2). The results showed that most of the polar residues in the loop region of ΔGtfB are mutated into rigid proline residues. Molecular dynamics simulation demonstrated that the root mean square fluctuation of CM9 significantly decreased by "Breathing" movement reduction of the loop region. This study provides a new strategy for improving the thermostability of 4,6-α-GT through rational loop region modification.

Extracellular recombinant production of 4,6 and 4,3 α-glucanotransferases in Lactococcus lactis.

4,6 α-Glucanotransferase (4,6-α-GTase) and 4,3 α-glucanotransferases (4,3-α-GTase) produced by Lactic Acid Bacteria (LAB) in the GH70 enzyme family have become important due to their catalytic effect on starch and maltodextrins. Their high level of production is necessary for their application at industrial scale. In this respect, both enzymes were expressed extracellularly using Lactococcus lactis as GRAS host. 4,6-α-GTase and 4,3-α-GTase genes from Limosilactobacillus reuteri E81 and Limosilactobacillus fermentum PFC282 respectively were transformed into the plasmid pLEB124 vector having the signal peptide usp45 under the P45 continuous promoter and successfully expressed in Lactococcus lactis MG1363. Western blot screening showed that the relevant enzymes were able to be successfully secreted extracellularly. The Vmax and Km of 4,6-α-GTase were 2.58 µmol min-1 and 0054 mg min-1 whereas 3369 µmol min-1 and 0032 mg min-1 for 4,3-α-GTase respectively. NMR analysis demonstrated the formation of new bonds within the corresponding enzymes. Also, both enzymes were active on maltose, maltoheptaose, maltohexaose and starch and produced malto-oligosaccarides observed by TLC analysis. In conclusion, this study demonstrated first time the extracellular production of 4,6-α-GTase and 4,3-α-GTase with GRAS status that can be useful for starch retrogradation delay and glycaemic index reduction.

Production of Large-Ring Cyclodextrins by Amylomaltases.

Amylomaltase is a well-known glucan transferase that can produce large ring cyclodextrins (LR-CDs) or so-called cycloamyloses via cyclization reaction. Amylomaltases have been found in several microorganisms and their optimum temperatures are generally around 60-70 °C for thermostable amylomaltases and 30-45 °C for the enzymes from mesophilic bacteria and plants. The optimum pHs for mesophilic amylomaltases are around pH 6.0-7.0, while the thermostable amylomaltases are generally active at more acidic conditions. Size of LR-CDs depends on the source of amylomaltases and the reaction conditions including pH, temperature, incubation time, and substrate. For example, in the case of amylomaltase from Corynebacterium glutamicum, LR-CD productions at alkaline pH or at a long incubation time favored products with a low degree of polymerization. In this review, we explore the synthesis of LR-CDs by amylomaltases, structural information of amylomaltases, as well as current applications of LR-CDs and amylomaltases.

Glycogen debranching pathway deduced from substrate specificity of glycogen debranching enzyme.

Glycogen debranching enzyme (GDE) is bifunctional in that it exhibits both 4-α-glucanotransferase and amylo-α-1,6-glucosidase activity at two distinct catalytic sites. GDE converts the phosphorylase-limit biantennary branch [G-G-G-G-(G-G-G-G↔)G-G- residue, where G = D-glucose, hyphens represent α-1,4-glycosidic bonds, and the double-headed arrow represents an α-1,6-glycosidic bond] into a linear maltooligosyl residue, which is then subjected to phosphorylase, and glycogen degradation continues. The prevailing hypothesis regarding the glycogen debranching pathway was that 4-α-glucanotransferase converts the phosphorylase-limit biantennary branch into the G-G-G-G-G-G-G-(G↔)G-G- residue and amylo-α-1,6-glucosidase cleaves the remaining α-1,6-linked G residue. In the present study, we analyzed the substrate specificities of 4-α-glucanotransferase and amylo-α-1,6-glucosidase using fluorogenic biantennary dextrins such as G-G-G-G-(G-G-G-G↔)G-G-GPA (F4/4/2; where GPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol), G-(G-G-G-G↔)G-G-GPA (F1/4/2), and G-G-G-G-G-G-G-(G↔)G-G-GPA (F7/1/2). Contrary to the prevailing hypothesis, the main branch of F4/4/2 was an important donor substrate component of 4-α-glucanotransferase and did not serve as an acceptor substrate. However, when G-G-G-G-G-GPA was added to the mixture, it successfully accepted a maltotriosyl (G3-) residue from F4/4/2. In addition, amylo-α-1,6-glucosidase exhibited strong activity towards G-G-G-G-(G↔)G-G-GPA but weak activity towards F7/1/2. Furthermore, the debranching activity of GDE towards phosphorylase-limit glycogen substantially increased when methyl α-maltooligosides with lengths equal to or greater than that of methyl α-maltopentaoside (G5-OCH3) were added to the enzyme reaction mixture. Based on these results, we propose the following macroscopic debranching pathway: Via 4-α-glucanotransferase, the G3- residue of the donor branch is transferred to a long (n ≥ 5) linear Gn- residue linked to a different branching G residue.

Characterization of a cold-adapted debranching enzyme and its role in glycogen metabolism and virulence of Vibrio vulnificus MO6-24/O.

Vibrio vulnificus MO6-24/O has three genes annotated as debranching enzymes or pullulanase genes. Among them, the gene encoded by VVMO6_03032 (vvde1) shares a higher similarity at the amino acid sequence level to the glycogen debranching enzymes, AmyX of Bacillus subtilis (40.5%) and GlgX of Escherichia coli (55.5%), than those encoded by the other two genes. The vvde1 gene encoded a protein with a molecular mass of 75.56 kDa and purified Vvde1 efficiently hydrolyzed glycogen and pullulan to shorter chains of maltodextrin and maltotriose (G3), respectively. However, it hydrolyzed amylopectin and soluble starch far less efficiently, and ß-cyclodextrin (ß-CD) only rarely. The optimal pH and temperature of Vvde1 was 6.5 and 25°C, respectively. Vvde1 was a cold-adapted debranching enzyme with more than 60% residual activity at 5°C. It could maintain stability for 2 days at 25°C and 1 day at 35°C, but it destabilized drastically at 40°C. The Vvde1 activity was inhibited considerably by Cu2+, Hg2+, and Zn2+, while it was slightly enhanced by Co2+, Ca2+, Ni2+, and Fe2+. The vvde1 knock-out mutant accumulated more glycogen than the wild-type in media supplemented with 1.0% maltodextrin; however, the side chain length distribution of glycogen was similar to that of the wild-type except G3, which was much more abundant in the mutant. Therefore, Vvde1 seemed to debranch glycogen with the degree of polymerization 3 (DP3) as the specific target branch length. Virulence of the pathogen against Caenorhabditis elegans was attenuated significantly by the vvde1 mutation. These results suggest that Vvde1 might be a unique glycogen debranching enzyme that is involved in both glycogen utilization and shaping of glycogen molecules, and contributes toward virulence of the pathogen.

Slowly digestible property of highly branched α-limit dextrins produced by 4,6-α-glucanotransferase from Streptococcus thermophilus evaluated in vitro and in vivo.

Starch molecules are first degraded to slowly digestible α-limit dextrins (α-LDx) and rapidly hydrolyzable linear malto-oligosaccharides (LMOs) by salivary and pancreatic α-amylases. In this study, we designed a slowly digestible highly branched α-LDx with maximized α-1,6 linkages using 4,6-α-glucanotransferase (4,6-αGT), which creates a short length of α-1,4 side chains with increasing branching points. The results showed that a short length of external chains mainly composed of 1-8 glucosyl units was newly synthesized in different amylose contents of corn starches, and the α-1,6 linkage ratio of branched α-LDx after the chromatographical purification was significantly increased from 4.6% to 22.1%. Both in vitro and in vivo studies confirmed that enzymatically modified α-LDx had improved slowly digestible properties and extended glycemic responses. Therefore, 4,6-αGT treatment enhanced the slowly digestible properties of highly branched α-LDx and promises usefulness as a functional ingredient to attenuate postprandial glucose homeostasis.

Crystal structures of glycogen-debranching enzyme mutants in complex with oligosaccharides.

Debranching is a critical step in the mobilization of the important energy store glycogen. In eukaryotes, including fungi and animals, the highly conserved glycogen-debranching enzyme (GDE) debranches glycogen by a glucanotransferase (GT) reaction followed by a glucosidase (GC) reaction. Previous work indicated that these reactions are catalyzed by two active sites located more than 50 Šapart and provided insights into their catalytic mechanisms and substrate recognition. Here, five crystal structures of GDE in complex with oligosaccharides with 4-9 glucose residues are presented. The data suggest that the glycogen main chain plays a critical role in binding to the GT and GC active sites of GDE and that a minimum of five main-chain residues are required for optimal binding.

Enhancement of large ring cyclodextrin production using pretreated starch by glycogen debranching enzyme from Corynebacterium glutamicum.

Synthesis of large-ring cyclodextrins (LR-CDs) in any significant amount has been challenging. This study enhanced the LR-CDs production by Thermus filiformis amylomaltase (TfAM) enzyme by starch pretreatment using glycogen debranching enzyme from Corynebacterium glutamicum (CgGDE). CgGDE pretreated tapioca starch gave LR-CD conversion of 31.2 ± 2.2%, compared with LR-CDs produced from non-treated tapioca starch (16.0 ± 2.4%). CgGDE pretreatment enhanced amylose content by approximately 30%. Notably, a shorter incubation time of 1 h is sufficient for CgGDE starch pretreatment to produce high LR-CD yield, compared with 6 h required for the commercial isoamylase. High-Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD) and Gel Permeable Chromatography (GPC) revealed that CgGDE is more efficient than the commercial isoamylase in debranching tapioca starch and gave lower molecular weight products. In addition, lower amount of by-products (linear oligosaccharides) were detected in cyclization reaction when using CgGDE-pretreated starch. In conclusion, CgGDE is a highly effective enzyme to promote LR-CD synthesis from starch with a shorter incubation time than the commercial isoamylase.

Amylomaltases in Extremophilic Microorganisms.

Amylomaltases (4-α-glucanotransferases, E.C. 2.4.1.25) are enzymes which can perform a double-step catalytic process, resulting in a transglycosylation reaction. They hydrolyse glucosidic bonds of α-1,4'-d-glucans and transfer the glucan portion with the newly available anomeric carbon to the 4'-position of an α-1,4'-d-glucan acceptor. The intramolecular reaction produces a cyclic α-1,4'-glucan. Amylomaltases can be found only in prokaryotes, where they are involved in glycogen degradation and maltose metabolism. These enzymes are being studied for possible biotechnological applications, such as the production of (i) sugar substitutes; (ii) cycloamyloses (molecules larger than cyclodextrins), which could potentially be useful as carriers and encapsulating agents for hydrophobic molecules and also as effective protein chaperons; and (iii) thermoreversible starch gels, which could be used as non-animal gelatin substitutes. Extremophilic prokaryotes have been investigated for the identification of amylomaltases to be used in the starch modifying processes, which require high temperatures or extreme conditions. The aim of this article is to present an updated overview of studies on amylomaltases from extremophilic Bacteria and Archaea, including data about their distribution, activity, potential industrial application and structure.

Usher syndrome type 2A complicated with glycogen storage disease type 3 due to paternal uniparental isodisomy of chromosome 1 in a sporadic patient.

BACKGROUND: The condition of uniparental disomy (UPD) occurs when an individual inherits two copies of a chromosome, or part of a chromosome, from one parent. Most cases of uniparental heterodisomy (UPhD) do not cause diseases, whereas cases of uniparental isodisomy (UPiD), while rare, may be pathogenic. Theoretically, UPiD may cause rare genetic diseases in a homozygous recessive manner. METHODS: A 4-year-old girl presented with congenital hearing loss, developmental delay, hepatomegaly, and other clinical features. She and her parents were genetically tested using trio whole exome sequencing (Trio-WES) and copy number variation sequencing (CNV-seq). In addition, we built a structural model to further examine the pathogenicity of the UPiD variants. RESULTS: Trio-WES identified a paternal UPiD in chromosome 1, and two homozygous pathogenic variants AGL c.4284T>G/p.Tyr1428* and USH2A c.6528T>A/p.Tyr2176* in the UPiD region. We further analyzed the pathogenicity of these two variations. The patient was diagnosed with Usher syndrome type 2A (USH2A) and glycogen storage disease type III (GSD3). CONCLUSIONS: Our study reports a rare case of a patient carrying two pathogenic variants of different genes caused by paternal UPiD, supporting the potential application of Trio-WES in detecting and facilitating the diagnosis of UPD.

Investigation of starch functionality and digestibility in white wheat bread produced from a recipe containing added maltogenic amylase or amylomaltase.

In the crumb of fresh white wheat bread, starch is fully gelatinized. Its molecular and three-dimensional structure are major factors limiting the rate of its digestion. The aim of this study was to in situ modify starch during bread making with starch-modifying enzymes (maltogenic amylase and amylomaltase) and to investigate the impact thereof on bread characteristics, starch retrogradation and digestibility. Maltogenic amylase treatment increased the relative content of short amylopectin chains (degree of polymerization ≤ 8). This resulted in lower starch retrogradation and crumb firmness upon storage, and reduced extent (up to 18%) of in vitro starch digestion for fresh and stored breads. Amylomaltase only modestly shortened amylose chains and had no measurable impact on amylopectin structure. Modification with this enzyme led to slower bread crumb firming but did not influence starch digestibility.

Cloning and characterization of glycogen branching and debranching enzymes from the parasitic protist Trichomonas vaginalis.

The protist Trichomonas vaginalis is an obligate parasite of humans and the causative agent of trichomoniasis, a common sexually transmitted infection. The organism has long been known to accumulate glycogen, a branched polymer of glucose, and to mobilize this reserve in response to carbohydrate limitation. However, the enzymes required for the synthesis and degradation of glycogen by T. vaginalis have been little studied. Previously, we characterized T. vaginalis glycogen synthase and glycogen phosphorylase, the key enzymes of glycogen synthesis and degradation, respectively. We determined that their regulatory properties differed from those of well-characterized animal and fungal enzymes. Here, we turn our attention to how glycogen attains its branched structure. We first determined that the glycogen from T. vaginalis resembled that from a related organism, T. gallinae. To determine how the branched structure of T. vaginalis glycogen arose, we identified open reading frames encoding putative T. vaginalis branching and debranching enzymes. When the open reading frames TVAG_276310 and TVAG_330630 were expressed recombinantly in bacteria, the resulting proteins exhibited branching and debranching activity, respectively. Specifically, recombinant TVAG_276310 had affinity for polysaccharides with long outer branches and could add branches to both amylose and amylopectin. TVAG_330630 displayed both 4-α-glucanotransferase and α1,6-glucosidase activity and could efficiently debranch phosphorylase limit dextrin. Furthermore, expression of TVAG_276310 and TVAG_330630 in yeast cells lacking endogenous glycogen branching or debranching enzyme activity, restored normal glycogen accumulation and branched structure. We now have access to the suite of enzymes required for glycogen synthesis and degradation in T. vaginalis.

Streptococcus agalactiae amylomaltase offers insight into the transglycosylation mechanism and the molecular basis of thermostability among amylomaltases.

Amylomaltase (AM) catalyzes transglycosylation of starch to form linear or cyclic oligosaccharides with potential applications in biotechnology and industry. In the present work, a novel AM from the mesophilic bacterium Streptococcus agalactiae (SaAM), with 18-49% sequence identity to previously reported AMs, was characterized. Cyclization and disproportionation activities were observed with the optimum temperature of 30 °C and 40 °C, respectively. Structural determination of SaAM, the first crystal structure of small AMs from the mesophiles, revealed a glycosyl-enzyme intermediate derived from acarbose and a second acarbose molecule attacking the intermediate. This pre-transglycosylation conformation has never been before observed in AMs. Structural analysis suggests that thermostability in AMs might be mainly caused by an increase in salt bridges since SaAM has a lower number of salt bridges compared with AMs from the thermophiles. Increase in thermostability by mutation was performed. C446 was substituted with A/S/P. C446A showed higher activities and higher kcat/Km values for starch in comparison to the WT enzyme. C446S exhibited a 5 °C increase in optimum temperature and the threefold increase in half-life time at 45 °C, most likely resulting from H-bonding interactions. For all enzymes, the main large-ring cyclodextrin (LR-CD) products were CD24-CD26 with CD22 as the smallest. C446S produced more CD35-CD42, especially at a longer incubation time.

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.

Comparison of Catalyzing Properties of Bacterial 4-α-Glucanotransferases Focusing on Their Cyclizing Activity.

A newly cloned 4-α-glucanotransferase (αGT) from Deinococcus geothermalis and two typical bacterial αGTs from Thermus scotoductus and Escherichia coli (MalQ) were investigated. Among 4 types of catalysis, the cyclization activity of αGTs that produces cycloamylose (CA), a valuable carbohydrate making inclusion complexes, was intensively studied. The new αGT, DgαGT, showed close protein sequence to the αGT from T. scotoductus (TsαGT). MalQ was clearly separated from the other two αGTs in the phylogenetic and the conserved regions analyses. The reaction velocities of disproportionation, cyclization, coupling, and hydrolysis of three αGTs were determined. Intriguingly, MalQ exhibited more than 100-fold lower cyclization activity than the others. To lesser extent, the disproportionation activity of MalQ was relatively low. DgαGT and TsαGT showed similar kinetics results, but TsαGT had nearly 10-fold lower hydrolysis activity than DgαGT. Due to the very low cyclizing activity of MalQ, DgαGT and TsαGT were selected for further analyses. When amylose was treated with DgαGT or TsαGT, CA with a broad DP range was generated immediately. The DP distribution of CA had a bimodal shape (DP 7 and 27 as peaks) for the both enzymes, but larger DPs of CA quickly decreased in the DgαGT. Cyclomaltopentaose, a rare cyclic sugar, was produced at early reaction stage and accumulated as the reactions went on in the both enzymes, but the increase was more profound in the TsαGT. Taken together, we clearly demonstrated the catalytic differences between αGT groups from thermophilic and pathogenic bacteria that and showed that αGTs play different roles depending on their lifestyle.