GBSS mutations in an SBE mutated background restore the potato starch granule morphology and produce ordered granules despite differences to native molecular structure.

Potato starch with mutations in starch branching enzyme genes (SBEI, SBEII) and granule-bound starch synthase gene (GBSS) was characterized for molecular and thermal properties. Mutations in GBSS were here stacked to a previously developed SBEI and SBEII mutation line. Additionally, mutations in the GBSS gene alone were induced in the wild-type variety for comparison. The parental line with mutations in the SBE genes showed a âˆ¼ 40 % increase in amylose content compared with the wild-type. Mutations in GBSS-SBEI-SBEII produced non-waxy, low-amylose lines compared with the wild-type. An exception was a line with one remaining GBSS wild-type allele, which displayed ∼80 % higher amylose content than wild-type. Stacked mutations in GBSS in the SBEI-SBEII parental line caused alterations in amylopectin chain length distribution and building block size categories of whole starch. Correlations between size categories of building blocks and unit chains of amylopectin were observed. Starch in GBSS-SBEI-SBEII mutational lines had elevated peak temperature of gelatinization, which was positively correlated with large building blocks.

The Structure of Maltooctaose-Bound Escherichia coli Branching Enzyme Suggests a Mechanism for Donor Chain Specificity.

Glycogen is the primary storage polysaccharide in bacteria and animals. It is a glucose polymer linked by α-1,4 glucose linkages and branched via α-1,6-linkages, with the latter reaction catalyzed by branching enzymes. Both the length and dispensation of these branches are critical in defining the structure, density, and relative bioavailability of the storage polysaccharide. Key to this is the specificity of branching enzymes because they define branch length. Herein, we report the crystal structure of the maltooctaose-bound branching enzyme from the enterobacteria E. coli. The structure identifies three new malto-oligosaccharide binding sites and confirms oligosaccharide binding in seven others, bringing the total number of oligosaccharide binding sites to twelve. In addition, the structure shows distinctly different binding in previously identified site I, with a substantially longer glucan chain ordered in the binding site. Using the donor oligosaccharide chain-bound Cyanothece branching enzyme structure as a guide, binding site I was identified as the likely binding surface for the extended donor chains that the E. coli branching enzyme is known to transfer. Furthermore, the structure suggests that analogous loops in branching enzymes from a diversity of organisms are responsible for branch chain length specificity. Together, these results suggest a possible mechanism for transfer chain specificity involving some of these surface binding sites.

Stochastic modelling of a three-dimensional glycogen granule synthesis and impact of the branching enzyme.

In humans, glycogen storage diseases result from metabolic inborn errors, and can lead to severe phenotypes and lethal conditions. Besides these rare diseases, glycogen is also associated to widely spread societal burdens such as diabetes. Glycogen is a branched glucose polymer synthesised and degraded by a complex set of enzymes. Over the past 50 years, the structure of glycogen has been intensively investigated. Yet, the interplay between the detailed three-dimensional glycogen structure and the related enzyme activity is only partially characterised and still to be fully understood. In this article, we develop a stochastic coarse-grained and spatially resolved model of branched polymer biosynthesis following a Gillespie algorithm. Our study largely focusses on the role of the branching enzyme, and first investigates the properties of the model with generic parameter values, before comparing it to in vivo experimental data in mice. It arises that the ratio of glycogen synthase over branching enzyme reaction rates drastically impacts the structure of the granule. We deeply investigate the mechanism of branching and parametrise it using distinct lengths. Not only do we consider various possible sets of values for these lengths, but also distinct rules to apply them. We show how combining various values for these lengths finely tunes glycogen macromolecular structure. Comparing the model with experimental data confirms that we can accurately reproduce glycogen chain length distributions in wild type mice. Additional granule properties obtained for this fit are also in good agreement with typically reported values in the experimental literature. Nonetheless, we find that the mechanism of branching must be more flexible than usually reported. Overall, our model provides a theoretical basis to quantify the effect that single enzymatic parameters, in particular of the branching enzyme, have on the chain length distribution. Our generic model and methods can be applied to any glycogen data set, and could in particular contribute to characterise the mechanisms responsible for glycogen storage disorders.

Biomimetic synthesis of maltodextrin-derived dendritic nanoparticle and its structural characterizations.

The maltodextrin-derived dendritic nanoparticle was fabricated using microbial branching enzyme and its structural characterizations were investigated. During biomimetic synthesis, molecular weight distribution of maltodextrin substrate with 6.8 × 104 g/mol shifted to the narrower and uniform distribution region with the larger molecular weight up to 6.3 × 106 g/mol (MD12). The enzyme-catalyzed product had the larger size, higher molecular density as well as higher percentage of α-1,6 linkage, accompanying by more chain accumulations of DP 6-12 and disappearance of DP > 24, suggesting the biosynthesized glucan dendrimer had a compact tighter branched structure. The interaction of molecular rotor CCVJ and local structure of dendrimer was monitored, displaying there was a higher intensity related with the numerous nano-pockets at the branch points of MD12. The maltodextrin-derived dendrimers had the single spherical particulate shape with the size range of 10-90 nm. The mathematical models were also established to reveal the chain structuring during enzymatic reaction. The above results showed that the biomimetic strategy for novel dendritic nanoparticle with controllable structure arising from branching enzyme treated maltodextrin, which would help to enlarge the panel of available dendrimer.

Deciphering the structural and functional characteristics of an innovative small cluster branched α-glucan produced by sequential enzymatic synthesis.

Highly branched α-glucan (HBAG) proved to be a promising material as an osmotic agent in peritoneal dialysis solutions. However, high resistance of HBAG to amylolytic enzymes might be a potential drawback for peritoneal dialysis due to its high degree of branching (20-30 %). To address this issue, we designed a small-clustered α-glucan (SCAG) with a relatively low molecular weight (Mw) and limited branching. Structural characteristics revealed that SCAG was successfully synthesized by modifying waxy rice starch (WRS) using sequential maltogenic α-amylase (MA) and starch branching enzyme (BE). The Mw of SCAG was 1.40 × 105 Da, and its (α1 â†’ 6) bonds ratio was 8.93 %, which was below that of HBAG. A relatively short branch distribution was observed in SCAG (CL = 6.27). Short-range orderliness of WRS was reduced from 0.749 to 0.322 with the MABE incubation. Additionally, SCAG had an extremely low viscosity (~12 cP) and nearly no retrogradation. Although the resistance of SCAG to amylolytic enzymes was enhanced by 15.22 % compared with native WRS, the extent was significantly lower than that of HBAG in previous studies. These new findings demonstrate the potential of SCAG as a novel functional α-glucan in food and pharmaceutical applications.

The role of different Wx and BEIIb allele combinations on fine structures and functional properties of indica rice starches.

This study examined the effects of the combinations of Waxy (Wx) and starch branching enzyme IIb (BEIIb) alleles on starch fine structure and functional properties in indica rice cultivars. The results showed that be2b mutant starches with BEIIb deficiency had higher amylose content, shorter amylose long chains, a higher proportion of amylopectin long chains and molecular order, but a lower proportion of amylopectin short chains and relative crystallinity, resulting in higher gelatinization temperature but lower enthalpy and paste viscosity. Compared with the rice lines carrying Wxb allele, Wxa allele contributed to relatively higher amylose content, longer amylopectin chains, less short-range ordered structure and lower relative crystallinity, leading to a little lower gelatinization enthalpy. This study provides new insight into structure-function relations among rice lines with different allele combinations of starch synthesis related genes, which is a useful strategy for rice quality breeding.

The Candida glabrata glycogen branching enzyme structure reveals unique features of branching enzymes of the Saccharomycetaceae phylum.

Branching enzymes (BE) are responsible for the formation of branching points at the 1,6 position in glycogen and starch, by catalyzing the cleavage of α-1,4-linkages and the subsequent transfer by introducing α-1,6-linked glucose branched points. BEs are found in the large GH13 family, eukaryotic BEs being mainly classified in the GH13_8 subfamily, GH13_9 grouping almost exclusively prokaryotic enzymes. With the aim of contributing to the understanding of the mode of recognition and action of the enzymes belonging to GH13_8, and to the understanding of features distinguishing these enzymes from those belonging to subfamily 13_9, we solved the crystal structure of the glycogen branching enzyme (GBE) from the yeast Candida glabrata, CgGBE, in ligand-free forms and in complex with a maltotriose. The structures revealed the presence of a domain already observed in Homo sapiens and Oryza sativa BEs that we named α-helical N-terminal domain, in addition to the three conserved domains found in BE. We confirmed by phylogenetic analysis that this α-helical N-terminal domain is always present in the GH13_8 enzymes suggesting that it could actually present a signature for this subfamily. We identified two binding sites in the α-helical N-terminal domain and in the carbohydrate binding module 48 (CBM48), respectively, which show a unique structural organization only present in the Saccharomycotina phylum. Our structural and phylogenetic investigation provides new insight into the structural characterization of GH13_8 GBE revealing that unique structural features only present in the Saccharomycotina phylum thereby conferring original properties to this group of enzymes.

Relative importance of branching enzyme isoforms in determining starch fine structure and physicochemical properties of indica rice.

KEY MESSAGE: Down-regulation of starch branching enzymes alters fine structure and starch properties, especially the B-type crystalline pattern and extremely high amylose content identified in the BEIIb-deficiency mutant in the indica rice. The relative importance of the starch branching enzymes in determining the molecular fine structure and starch functional properties were uncovered in this study. An indica rice, Guangluai 4 with high amylose content (AC) and high gelatinization temperature (GT) was used to generate the clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein-9 (Cas9) knockout lines. Five mutant lines were identified including be1-1, be1-2, be2a-1, be2a-2 and be2b-1, and analysis of western blot showed the CRISPR/Cas9 system was successful in inducing mutations in the targeted genes. AC of be2b-1 (34.1%) was greater than that of wild type (WT) (27.4%) and other mutants. Mutations of either BEI or BEIIa did not alter the starch crystallite pattern (A-type). The BEIIb deficiency caused an opaque endosperm phenotype, changed the crystallite pattern from A- to B-type, and dramatically increased the degree of ordered structure, the relative proportion of amylose chains and intermediate to long amylopectin chains, average chain length of amylopectin molecules as well as GT. The BEIIa deficiency had no effect on the proportion of amylose chains, the length of amylopectin intermediate-long chains, conclusion temperature and enthalpy of gelatinization. Down-regulation of BEI increased the proportion of shortest amylopectin chains (fa) but decreased the proportion of long amylopectin chains (fb2 and fb3), leading to a lower GT. It is concluded that the relative importance in determining starch fine structures and functionality was in the order of BEIIb > BEI > BEIIa. Our results provide new information for utilizations of BE-deficient mutants in rice quality breeding.

A structural explanation for the mechanism and specificity of plant branching enzymes I and IIb.

Branching enzymes (BEs) are essential in the biosynthesis of starch and glycogen and play critical roles in determining the fine structure of these polymers. The substrates of these BEs are long carbohydrate chains that interact with these enzymes via multiple binding sites on the enzyme's surface. By controlling the branched-chain length distribution, BEs can mediate the physiological properties of starch and glycogen moieties; however, the mechanism and structural determinants of this specificity remain mysterious. In this study, we identify a large dodecaose binding surface on rice BE I (BEI) that reaches from the outside of the active site to the active site of the enzyme. Mutagenesis activity assays confirm the importance of this binding site in enzyme catalysis, from which we conclude that it is likely the acceptor chain binding site. Comparison of the structures of BE from Cyanothece and BE1 from rice allowed us to model the location of the donor-binding site. We also identified two loops that likely interact with the donor chain and whose sequences diverge between plant BE1, which tends to transfer longer chains, and BEIIb, which transfers exclusively much shorter chains. When the sequences of these loops were swapped with the BEIIb sequence, rice BE1 also became a short-chain transferring enzyme, demonstrating the key role these loops play in specificity. Taken together, these results provide a more complete picture of the structure, selectivity, and activity of BEs.

Structural elements determining the transglycosylating activity of glycoside hydrolase family 57 glycogen branching enzymes.

Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE) catalyze the formation of an α-1,6 glycosidic bond between α-1,4 linked glucooliogosaccharides. As an atypical family, a limited number of GH57GBEs have been biochemically characterized so far. This study aimed at acquiring a better understanding of the GH57GBE family by a systematic sequence-based bioinformatics analysis of almost 2500 gene sequences and determining the branching activity of several native and mutant GH57GBEs. A correlation was found in a very low or even no branching activity with the absence of a flexible loop, a tyrosine at the loop tip, and two ß-strands.

Expression and characterization of 1,4-α-glucan branching enzyme from Microvirga sp. MC18 and its application in the preparation of slowly digestible starch.

Nutraceuticals containing modified starch with increased content of slowly-digestible starch (SDS) may reduce the prevalence of obesity, diabetes and cardiovascular diseases due to its slow digestion rate. Enzymatic methods for the preparation of modified starch have attracted increasing attention because of their low environmental impact, safety and specificity. In this study, the efficient glucan branching enzyme McGBE from Microvirga sp. MC18 was identified, and its relevant properties as well as its potential for industrial starch modification were evaluated. The purified McGBE exhibited the highest specificity for potato starch, with a maximal specific activity of 791.21 U/mg. A time-dependent increase in the content of α-1,6 linkages from 3.0 to 6.0% was observed in McGBE-modified potato starch. The proportion of shorter chains (degree of polymerization, DP < 13) increased from 29.2 to 63.29% after McGBE treatment, accompanied by a reduction of the medium length chains (DP 13-24) from 52.30 to 35.99% and longer chains (DP > 25) from 18.51 to 0.72%. The reduction of the storage modulus (G') and retrogradation enthalpy (ΔHr) of potato starch with increasing treatment time demonstrated that McGBE could inhibit the short- and long-term retrogradation of starch. Under the optimal conditions, the SDS content of McGBE-modified potato starch increased by 65.8% compared to native potato starch. These results suggest that McGBE has great application potential for the preparation of modified starch with higher SDS content that is resistant to retrogradation.

Two 1,4-α-glucan branching enzymes successively rearrange glycosidic bonds: A novel synergistic approach for reducing starch digestibility.

Enzymatically rearranging α-1,4 and α-1,6 glycosidic bonds in starch is a green approach to regulating its digestibility. A two-step modification process successively catalyzed by 1,4-α-glucan branching enzymes (GBEs) from Rhodothermus obamensi STB05 (Ro-GBE) and Geobacillus thermoglucosidans STB02 (Gt-GBE) was investigated as a strategy to reduce the digestibility of corn starch. This dual GBE modification process caused a reduction of 25.8 % in rapidly digestible starch fraction in corn starch, which were more effective than single GBE-catalyzed modification with the same duration. Structural analysis indicated that the dual GBE modified product contained higher branching density, more abundant short branches, and shorter external chains than those in single GBE-modified product. These results demonstrated that a moderate Ro-GBE treatment prior to starch gelatinization caused several suitable alterations in starch molecules, which promoted the transglycosylation efficiency of the following Gt-GBE treatment. This dual GBE-catalyzed modification process offered an efficient strategy for regulating starch digestibility.

Cyanobacterial branching enzymes bind to α-glucan via surface binding sites.

Besides their catalysis, specific interactions between starch/glycogen processing enzymes and their substrates have been reported. Multiple branching enzyme (BE) isoforms, BE1, BE2, and BE3, have been found in a limited number of cyanobacterial species that are characterized by amylopectin accumulation. Seven surface binding sites (SBSs) located away from the active site have been identified in crystal structures of cyanobacterial BE1 from Crocosphaera subtropica (Cyanothece sp.) ATCC 51142 (51142BE1). In the present study, binding affinity toward amylopectin, amylose, and glycogen was investigated for wild-type 51142BE1 and its mutants (residues at SBSs important for sugar-binding were replaced by alanine). These enzymes showed retarded mobility during electrophoresis in non-denaturing polyacrylamide gels in the presence of polysaccharides. This was caused by interactions between the enzymes and the polysaccharides, enabling calculation of the dissociation constants (Kd values) of the enzymes toward the polysaccharides. Mutational analysis indicated that particular domains of the protein (domains A and C) were involved in the polysaccharide binding. Kd values toward the polysaccharides were also measured for 10 BE isoforms (five BE1, three BE2, and two BE3) from 5 cyanobacterial strains. All BEs displayed much lower Kd values (higher affinity) toward amylopectin and amylose than toward glycogen, as described for plant BEs. In addition, one BE2 displayed exceptionally high Kd values (low affinity), while two BE3 exhibited multiple Kd values to all polysaccharides. These results could be ascribed to sequence variations in the SBSs, irrespective of the catalytic specificity.

Comparison of functional properties of porous starches produced with different enzyme combinations.

To obtain porous starch granules with higher absorption capacities, three types of enzyme combinations were adopted to modify wheat and maize starches: (1) sequential α-amylase (AA) â†’ glucoamylase (GA); (2) sequential branching enzyme (BE) â†’ GA; and (3) sequential AA→BE→GA. The results indicated that AA→BE→GA treatment had a most optimal influence on porous starches. Compared to AA→GA and BE→GA, the mesopores in wheat starch granules treated with AA→BE→GA decreased by 52.82 and 48.70%, respectively. Conversely, the macropores increased by 216.68 and 138.18%, respectively. While for maize starch, the percentages of mesopores and macropores hardly changed after three enzyme combinations. Comparing the three enzyme treatments showed that pore volume (0.005 and 0.007 cm3/g) and pore size (36.35 and 26.54 nm) were largest in the AA→BE→GA treated wheat and maize starches, respectively. Compared to the AA→GA and BE→GA, the adsorption capacities for oil, dye and heavy metal ions, wheat starch treated with AA→BE→GA increased by 46.61 and 242.33%, and 44.52 and 134.41%, and 28.83 and 271.72%, respectively. Correspondingly, that of maize starch increased by 29.71 and 133.29%, and 42.92 and 79.93%, and 28.16 and 161.43%, respectively. These results may provide a new and valuable enzyme combination for optimising porous starch granules with higher absorption capacities.

Enzymatic properties of an efficient glucan branching enzyme and its potential application in starch modification.

Glucan branching enzymes (GBEs, EC 2.4.1.18) catalyze the formation of α-1,6-linked branch in starch, which is important for the starch modification with prospective properties. In this study, the aqGBE gene encoding an efficient glucan branching enzyme was cloned from Aquabacterium sp. strain A7-Y and successfully expressed in Escherichia coli BL21 (DE3). The specific activity of the purified recombinant enzyme rAqGBE was 2850 U/mg with potato starch as the optimal substrate, and the Km and Vmax values of rAqGBE were 1.18 mg/mL and 588.2 µmol/min/mg, respectively. Enzymological characterization showed that rAqGBE exhibits its optimal activity under the condition of 40 °C and pH 7.0, respectively, which is independent of calcium ions. Otherwise, rAqGBE-treated potato starch showed different chain length distribution compared with control, the numbers of short chains (degree of polymerization, DP < 7) and long chains (DP > 25) increased from 4.5% to 9.6% and 6.1%-15.7% after enzymatic treatment, respectively. In starch anti-ageing assay, with minimum usage of 0.8 mg rAqGBE per g starch, the rAqGBE-treated potato starch exhibited reduced retrogradation properties. Our results indicate that the branching enzyme AqGBE may therefore be a promising tool for the enzymatic modification of starch.

Inactivation of rice starch branching enzyme IIb triggers broad and unexpected changes in metabolism by transcriptional reprogramming.

Starch properties can be modified by mutating genes responsible for the synthesis of amylose and amylopectin in the endosperm. However, little is known about the effects of such targeted modifications on the overall starch biosynthesis pathway and broader metabolism. Here we investigated the effects of mutating the OsSBEIIb gene encoding starch branching enzyme IIb, which is required for amylopectin synthesis in the endosperm. As anticipated, homozygous mutant plants, in which OsSBEIIb was completely inactivated by abolishing the catalytic center and C-terminal regulatory domain, produced opaque seeds with depleted starch reserves. Amylose content in the mutant increased from 19.6 to 27.4% and resistant starch (RS) content increased from 0.2 to 17.2%. Many genes encoding isoforms of AGPase, soluble starch synthase, and other starch branching enzymes were up-regulated, either in their native tissues or in an ectopic manner, whereas genes encoding granule-bound starch synthase, debranching enzymes, pullulanase, and starch phosphorylases were largely down-regulated. There was a general increase in the accumulation of sugars, fatty acids, amino acids, and phytosterols in the mutant endosperm, suggesting that intermediates in the starch biosynthesis pathway increased flux through spillover pathways causing a profound impact on the accumulation of multiple primary and secondary metabolites. Our results provide insights into the broader implications of perturbing starch metabolism in rice endosperm and its impact on the whole plant, which will make it easier to predict the effect of metabolic engineering in cereals for nutritional improvement or the production of valuable metabolites.

Bacterial 1,4-α-glucan branching enzymes: characteristics, preparation and commercial applications.

The 1,4-α-glucan branching enzymes (GBEs) are ubiquitously distributed in animals, microorganisms and plants. These enzymes modify the structure of both starch and glycogen; changing the frequency and position of branches by forming new α-1,6-glucosidic linkages. In organisms, controlling the number and distribution of branches is an irreplaceable process that maintains the physiological state of starch and glycogen in the cell. The process is also the foundation for the industrial applications of GBEs. So far, a number of GBEs have been identified in eukaryotes and prokaryotes as researchers searched for GBEs with optimal properties. Among them, bacterial GBEs have received particular attention due to the convenience of heterologous expression and industrial applications of GBEs from bacteria than GBEs from other sources. The advantages of bacterial GBEs in potential applications stimulated the investigations of bacterial GBEs in terms of their structure and properties. However, full exploitation of GBEs in commercial applications is still in its infancy because of the disadvantages of currently available enzymes and of limited imagination with respect to future possibilities. Thus, in this review, we present an overview of the bacterial GBEs including their structure, biochemical properties and commercial applications in order to depict the whole picture of bacterial GBEs.

New insights into amylose and amylopectin biosynthesis in rice endosperm.

How various isoforms of rice-starch biosynthesis enzymes interact during amylose and amylopectin synthesis is explored. The chain-length distributions of amylopectin and amylose from 95 varieties with different environmental and genetic backgrounds were obtained using size- exclusion chromatography, and fitted with biosynthesis-derived models based on isoforms of starch synthase (SSI-SSIV), starch branching enzyme (SBE, including SBEI and SBEII) and granule-bound starch synthase (GBSS) that are involved in amylose and amylopectin synthesis. It is usually thought that these are synthesized by separate enzymes. However, the amount of longer amylopectin chains correlated with that of shorter amylose chains, indicating that GBSS, SBE and SS affect both amylose and amylopectin synthesis. Further, the activity of GBSS in amylose correlated with that of SS in amylopectin. This new understanding of which enzymes are suggested by the statistics to be involved in both amylose and amylopectin synthesis could help rice breeders develop cereals with targeted properties.

Improving waxy rice starch functionality through branching enzyme and glucoamylase: Role of amylose as a viable substrate.

The low solubility, gel strength and shear resistance of waxy rice limit utility in large-scale and widespread food applications. Herein, functional properties of waxy rice starch (WRS) have been improved through sequential branching enzyme (BE) and glucoamylase (GA) treatment with wheat amylose (WA), rice amylose (RA) and corn amylose (CA) as the substrate. The results suggest that, compared to the BE treatment, the sequential GA→BE treatment significantly improves the WRS properties. Among the three substrates, WA appears to be suited highly for the WRS through the GA→BE treatment. GABE-WRS/WA had the highest content of α-1,6-glucosidic linkages, increased number of short chains, shortest average chain length, increased crystallinity, enhanced solubility, paste clarity and swelling power, elevated gelatinization temperature and enthalpy, increased viscosity, strongest gels, and highest shear-resistance. Overall, the sequential GA→BE treatment appears to be suitable to modify the functional properties of waxy rice starch toward developing more palatable and large-scale food products.

Expression and characterization of an extremely thermophilic 1,4-α-glucan branching enzyme from Rhodothermus obamensis STB05.

A gene encoding 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) from the extremely thermophilic bacterium Rhodothermus obamensis STB05 was successfully cloned and expressed in Escherichia coli. Extracellular expression of the recombinant enzyme (R.o-GBE) was achieved with a yield of 1080 mg/L. Then it was purified and further characterized biochemically. R.o-GBE was optimally active at pH 7.0 and 65 °C. It remained stable at temperatures up to 80 °C and had a half-life at 85 °C of approximately 31 min. Far-UV circular dichroism and intrinsic fluorescence analyses revealed that high temperatures reduced its activity by changing the secondary and tertiary structure of R.o-GBE. The enzyme had broad pH stability between pH 3.0 and 11.0 at 4 °C, and preferred weakly acidic conditions at high temperatures. None of the metal ions enhanced the activity of R.o-GBE, but Ca2+ may be required for its activity. Its specific activity with amylopectin was 6651 U/mg, which is much higher than that reported for other GBEs. Its excellent thermostability, broad pH stability, and high specific activity make R.o-GBE highly suitable for industrial applications.