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1.
BMC Plant Biol ; 21(1): 477, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34670492

RESUMO

BACKGROUND: Both underground rhizomes/buds and above-ground Moso bamboo (Phyllostachys heterocycla) shoots/culms/branches are connected together into a close inter-connecting system in which nutrients are transported and shared among each organ. However, the starch storage and utilization mechanisms during bamboo shoot growth remain unclear. This study aimed to reveal in which organs starch was stored, how carbohydrates were transformed among each organ, and how the expression of key genes was regulated during bamboo shoot growth and developmental stages which should lay a foundation for developing new theoretical techniques for bamboo cultivation. RESULTS: Based on changes of the NSC content, starch metabolism-related enzyme activity and gene expression from S0 to S3, we observed that starch grains were mainly elliptical in shape and proliferated through budding and constriction. Content of both soluble sugar and starch in bamboo shoot peaked at S0, in which the former decreased gradually, and the latter initially decreased and then increased as shoots grew. Starch synthesis-related enzymes (AGPase, GBSS and SBE) and starch hydrolase (α-amylase and ß-amylase) activities exhibited the same dynamic change patterns as those of the starch content. From S0 to S3, the activity of starch synthesis-related enzyme and starch amylase in bamboo rhizome was significantly higher than that in bamboo shoot, while the NSC content in rhizomes was obviously lower than that in bamboo shoots. It was revealed by the comparative transcriptome analysis that the expression of starch synthesis-related enzyme-encoding genes were increased at S0, but reduced thereafter, with almost the same dynamic change tendency as the starch content and metabolism-related enzymes, especially during S0 and S1. It was revealed by the gene interaction analysis that AGPase and SBE were core genes for the starch and sucrose metabolism pathway. CONCLUSIONS: Bamboo shoots were the main organ in which starch was stored, while bamboo rhizome should be mainly functioned as a carbohydrate transportation channel and the second carbohydrate sink. Starch metabolism-related genes were expressed at the transcriptional level during underground growth, but at the post-transcriptional level during above-ground growth. It may be possible to enhance edible bamboo shoot quality for an alternative starch source through genetic engineering.


Assuntos
Metabolismo dos Carboidratos/genética , Proteínas de Plantas/metabolismo , Poaceae/genética , Amido/metabolismo , Transcriptoma , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilases/genética , Amilases/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/fisiologia , Brotos de Planta/ultraestrutura , Poaceae/crescimento & desenvolvimento , Poaceae/fisiologia , Poaceae/ultraestrutura , Rizoma/genética , Rizoma/crescimento & desenvolvimento , Rizoma/fisiologia , Rizoma/ultraestrutura
2.
BMC Plant Biol ; 21(1): 479, 2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34674662

RESUMO

Starch branching enzymes (SBEs) are key determinants of the structure and amount of the starch in plant organs, and as such, they have the capacity to influence plant growth, developmental, and fitness processes, and in addition, the industrial end-use of starch. However, little is known about the role of SBEs in determining starch structure-function relations in economically important horticultural crops such as fruit and leafy greens, many of which accumulate starch transiently. Further, a full understanding of the biological function of these types of starches is lacking. Because of this gap in knowledge, this minireview aims to provide an overview of SBEs in horticultural crops, to investigate the potential role of starch in determining postharvest quality. A systematic examination of SBE sequences in 43 diverse horticultural species, identified SBE1, 2 and 3 isoforms in all species examined except apple, olive, and Brassicaceae, which lacked SBE1, but had a duplicated SBE2. Among our findings after a comprehensive and critical review of published data, was that as apple, banana, and tomato fruits ripens, the ratio of the highly digestible amylopectin component of starch increases relative to the more digestion-resistant amylose fraction, with parallel increases in SBE2 transcription, fruit sugar content, and decreases in starch. It is tempting to speculate that during the ripening of these fruit when starch degradation occurs, there are rearrangements made to the structure of starch possibly via branching enzymes to increase starch digestibility to sugars. We propose that based on the known action of SBEs, and these observations, SBEs may affect produce quality, and shelf-life directly through starch accumulation, and indirectly, by altering sugar availability. Further studies where SBE activity is fine-tuned in these crops, can enrich our understanding of the role of starch across species and may improve horticulture postharvest quality.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Produtos Agrícolas/enzimologia , Isoenzimas , Amido/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Motivos de Aminoácidos , Amilopectina/metabolismo , Amilose/metabolismo , Produtos Agrícolas/genética , Produtos Agrícolas/normas , Grão Comestível , Armazenamento de Alimentos , Frutas , Horticultura , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubérculos , Açúcares/metabolismo , Verduras
3.
Enzyme Microb Technol ; 150: 109882, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489035

RESUMO

Glycogen branching enzymes (GBEs; 1,4-α-glucan branching enzyme; E.C. 2.4.1.18) have so far been described to be capable of both α-1,6-transglycosylation (branching) and α-1,4-hydrolytic activity. The aim of the present study was to elucidate the mode of action of three distantly related GBEs from the glycoside hydrolase family 13 by in depth analysis of the activity on a well-defined substrate. For this purpose, the GBEs from R. marinus (RmGBE), P. mobilis (PmGBE1), and B. fibrisolvens (BfGBE) were incubated with a highly pure fraction of a linear substrate of 18 anhydroglucose units. A well-known and characterized branching enzyme from E. coli (EcGBE) was also taken along. Analysis of the chain length distribution over time revealed that, next to hydrolytic and branching activity, all three GBEs were capable of generating chains longer than the substrate, clearly showing α-1,4-transglycosylation activity. Furthermore, the GBEs used those elongated chains for further branching. The sequential activity of elongation and branching enabled the GBEs to modify the substrate to a far larger extent than would have been possible with branching activity alone. Overall, the three GBEs acted ambiguous on the defined substrate. RmGBE appeared to have a strong preference towards transferring chains of nine anhydroglucose units, even during elongation, with a comparably low activity. BfGBE generated an array of elongated chains before using the chains for introducing branches while PmGBE1 exhibited a behaviour intermediate of the other two enzymes. On the basis of the mode of action revealed in this research, an updated model of the mechanism of GBEs was proposed now including the α-1,4-transglycosylation activity.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucanos , Glicogênio , Especificidade por Substrato
4.
Int J Biol Macromol ; 184: 551-557, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34171255

RESUMO

Modified potato starch with slower digestion may aid the development of new starch derivatives with improved nutritional values, and strategies to increase nutritional fractions such as resistant starch (RS) are desired. In this study, a correspondence between starch structure and enzymatic resistance was provided based on the efficient branching enzyme AqGBE, and modified starches with different amylose content (Control, 100%; PS1, 90%; PS2, 72%; PS3, 32%; PS4, 18%) were prepared. Through SEM observation, NMR and X-ray diffraction analyses, we identified that an increased proportion of α-1,6-linked branches in potato starch changes its state of granule into large pieces with crystallinity. Molecular weight and chain-length distribution analysis showed a decrease of molecular weight (from 1.1 × 106 to 1.1 × 105 g/mol) without an obvious change of chain-length distribution in PS1, while PS2-4 exhibited an increased proportion of DP 6-12 with a stable molecular weight distribution, indicating a distinct model of structural modification by AqGBE. The enhancement of peak viscosity was related to increased hydrophobic interactions and pieces state of PS1, while the contents of SDS and RS in PS1 increased by 37.7 and 49.4%, respectively. Our result provides an alternative way to increase the RS content of potato starch by branching modification.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Burkholderiales/enzimologia , Solanum tuberosum/química , Amido/química , Amilose/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Estrutura Molecular , Peso Molecular , Viscosidade , Difração de Raios X
5.
J Agric Food Chem ; 69(20): 5755-5763, 2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-33988022

RESUMO

The 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) catalyzes the formation of α-1,6 branching points in starch and plays a key role in synthesis. To obtain mechanistic insights into the catalytic action of the enzyme, we first determined the crystal structure of GBE from Rhodothermus obamensis STB05 (RoGBE) to a resolution of 2.39 Å (PDB ID: 6JOY). The structure consists of three domains: domain A, domain C, and the carbohydrate-binding module 48 (CBM48). An engineered truncated mutant lacking the CBM48 domain (ΔCBM48) showed significantly reduced ligand binding affinity and enzyme activity. Comparison of the structures of RoGBE with other GBEs showed that CBM48 of RoGBE had a longer flexible loop. Truncation of the flexible loops resulted in reduced binding affinity and activity, thereby substantiating the importance of the optimum loop structure for catalysis. In essence, our study shows that CBM48, especially the flexible loop, plays an important role in substrate binding and enzymatic activity of RoGBE. Further, based on the structural analysis, kinetics, and activity assays on wild type and mutants, as well as homology modeling, we proposed a mechanistic model (called the "lid model") to illustrate how the flexible loop triggers substrate binding, ultimately leading to catalysis.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Rhodothermus , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Glucanos , Rhodothermus/metabolismo , Especificidade por Substrato
6.
Carbohydr Polym ; 262: 117968, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33838833

RESUMO

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.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/química , Glicosídeos/química , Amido/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilose/química , Amilose/metabolismo , Bacillaceae/enzimologia , Digestão , Glicosídeos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Rhodothermus/enzimologia , Amido/metabolismo
7.
Arch Biochem Biophys ; 702: 108821, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33662318

RESUMO

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.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Cianobactérias/enzimologia , Glucanos/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Sequência de Aminoácidos , Domínio Catalítico , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Alinhamento de Sequência
8.
Int J Biol Macromol ; 174: 110-119, 2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33515569

RESUMO

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.


Assuntos
Adsorção/fisiologia , Amido/química , Triticum/química , Zea mays/química , Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilases/química , Amilose/química , Glucana 1,4-alfa-Glucosidase/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Hidrólise , Porosidade , Temperatura , Água/química , alfa-Amilases/química , alfa-Amilases/metabolismo
9.
Food Chem ; 343: 128485, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33172750

RESUMO

The aim of this study was to prepare a supporting carrier, namely highly branched corn starch (HBCS), and to investigate its encapsulation property with ascorbic acid (AA). High amylose corn starch was converted into HBCS via dual enzymatic modification by successively using α-amylase and glycogen branching enzyme. The results showed that the ratio of α-1, 6 linkage of HBCS increased by 1.93%, and a short-to-medium chain length distribution with a compact branched conformation was formed, which suggested HBCS could be a potential highly branched carrier. The HBCS-AA inclusion complex was formed as confirmed by differential scanning calorimetry. The release of AA conformed to the pseudo-Fickian diffusion mechanism and followed the first-order kinetics. Meanwhile, the photostability and thermostability of the embedded AA were moderately enhanced. These findings suggest that HBCS provides new insights into the preparation of wall materials and can be potentially used to deliver AA into food systems.


Assuntos
Ácido Ascórbico/química , Amido/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Cápsulas , alfa-Amilases/metabolismo
10.
J Agric Food Chem ; 68(47): 13791-13797, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33166453

RESUMO

Disulfide bonds play crucial roles in thermostabilization, recognition, or activation of proteins. They are vital in maintaining the respective conformations of globular structures, thereby enhancing thermostability. Bioinformatic approaches provide practical strategies to build disulfide bonds based on structural information. We constructed nine mutants by rational analysis of the 1,4-α-glucan branching enzyme (EC 2.4.1.18) from Geobacillus thermoglucosidans STB02, which catalyzes the synthesis of α-1,6-glucosidic bonds by acting on α-(1,4) and/or α-(1,6) glucosidic linkages. Four of the mutations enhanced thermostability, and five of them had adverse or negligible effects on stability. Circular dichroism spectra and intrinsic fluorescence analysis showed that introducing disulfide bonds might only affect secondary structures. The results also demonstrated that the distances of Cα carbons and thiol groups, as well as the sequence between the two cysteines, need to be considered when designing disulfide bonds.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Geobacillus , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Bacillaceae , Dissulfetos , Estabilidade Enzimática , Geobacillus/genética , Glucanos
11.
Proc Natl Acad Sci U S A ; 117(42): 26503-26512, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33020297

RESUMO

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.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Oryza/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/química , Amilopectina/biossíntese , Amilopectina/química , Amilose/biossíntese , Amilose/química , Metabolismo dos Carboidratos , Grão Comestível/genética , Endosperma/metabolismo , Mutação , Oryza/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Sementes/metabolismo , Amido/biossíntese , Sintase do Amido/química , Sintase do Amido/genética , Sintase do Amido/metabolismo
12.
Placenta ; 99: 131-140, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32798765

RESUMO

INTRODUCTION: The placenta performs a range of functions to support fetal growth. In addition to facilitating nutrient transport, the placenta also stores glucose as glycogen, which is thought to maintain fetal glucose supply during late gestation. However, evidence to support such a role is currently lacking. Similarly, our understanding of the dynamics of placental glycogen metabolism in normal mouse pregnancy is limited. METHODS: We quantified the placental glycogen content of wild type C57BL/6JOlaHsd mouse placentas from mid (E12.5) to late (E18.5) gestation, alongside characterising the temporal expression pattern of genes encoding glycogenesis and glycogenolysis pathway enzymes. To assess the potential of the placenta to produce glucose, we investigated the spatiotemporal expression of glucose 6-phosphatase by qPCR and in situ hybridisation. Separate analyses were undertaken for placentas of male and female conceptuses to account for potential sexual dimorphism. RESULTS: Placental glycogen stores peak at E15.5, having increased over 5-fold from E12.5, before declining by a similar extent by E18.5. Glycogen stores were 17% higher in male placentas than in females at E15.5. Expression of glycogen branching enzyme (Gbe1) was reduced ~40% towards term. Expression of the glucose 6-phosphatase isoform G6pc3 was enriched in glycogen trophoblast cells and increased towards term. DISCUSSION: Reduced expression of Gbe1 suggests a decline in glycogen branching towards term. Expression of G6pc3 by glycogen trophoblasts is consistent with an ability to produce and release glucose from glycogen stores. However, the ultimate destination of the glucose generated from placental glycogen remains to be elucidated.


Assuntos
Glicogênio/metabolismo , Placenta/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Gravidez , Trofoblastos/metabolismo
13.
Carbohydr Polym ; 247: 116681, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32829809

RESUMO

Two high amylose (HAM) inbred lines with apparent amylose contents of 55 % and 62 %, respectively, were selected to explore the relationship between molecular structure and gene expression of starch-synthase involved enzymes. GPC analysis of debranched starches showed that the HAM starches (HAMSs) had shorter amylose chains and longer amylopectin chains than normal maize starch (NMS). FACE analysis showed that these HAMSs had a higher content of amylopectin chains of DP > 21. Quantitative Real-Time PCR analysis showed that the HAM lines had specifically low expression of the starch branching enzyme IIb (SBEIIb), and the starch synthase IIIa (SSIIIa) homologue, and high expression of the isoamylase 2 (ISA2), potentially suppressing the generation of amylopectin molecules through deficient branching and excessive debranching process, thereby increasing the relative amylose content. A high expression of GBSS1 was potentially associated with increased short amylose chain lengths in HAMSs.


Assuntos
Amilose/química , Sintase do Amido/genética , Sintase do Amido/metabolismo , Amido/biossíntese , Amido/química , Zea mays/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilopectina/análise , Amilopectina/química , Metabolismo dos Carboidratos , Cromatografia em Gel , Eletroforese/métodos , Isoamilase/metabolismo , Estrutura Molecular , Amido/análise , Zea mays/metabolismo
14.
Carbohydr Polym ; 247: 116729, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32829851

RESUMO

Twenty-four branched maltodextrins were synthesized from eight starches using three thermostable microbial glycogen branching enzymes. The maltodextrins have a degree of branching (DB) ranging from 5 % to 13 %. This range of products allows us to explore the effect of DB on the digestibility, which was quantified under conditions that mimic the digestion process in the small intestine. The rate and extent of digestibility were analyzed using the logarithm of the slope method, revealing that the branched maltodextrins consist of a rapidly and slowly digestible fraction. The amount of slowly digestible maltodextrin increases with an increasing DB. Surprisingly, above 10 % branching the fraction of slowly digestible maltodextrin remains constant. Nevertheless, the rate of digestion of the slowly digestible fraction was found to decline with increasing DB and shorter average internal chain length. These observations increase the understanding of the structural factors important for the digestion rate of branched maltodextrins.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Bactérias/enzimologia , Gelatina/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Glicogênio/metabolismo , alfa-Amilases Pancreáticas/metabolismo , Amido/química , Digestão , Hidrólise , Cinética
15.
Food Chem ; 324: 126855, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32344341

RESUMO

To develop a 1,4-α-glucan branching enzyme (BE) without homology to known allergens, the glgB gene from Bifidobacterium longum was overexpressed under the control of BLMA promoter in Escherichia coli. B. longum BE (BlBE) had a molecular weight of 86.1 kDa and a specific activity of more than 18.5U/mg protein at 25-35 °C and pH 5.5-7.0, and exhibited 30% of the maximum activity at 10 °C. The cold-active BlBE preferred to transfer maltohexaose and introduced DP 4-36 branches into amylose. BlBE also increased the proportion of DP 2-10 branches in amylopectin and decreased its Mw from 1.39 × 106 to 1.16 × 105 g/mol. As the BlBE concentration increased from 0.0 to 0.5U/mg substrate, the retrogradation enthalpy of BlBE-modified wheat starch decreased from 4.50 to 1.83 J/g (p < 0.05) at day 14 and the slowly digestible starch content increased from 2.10% to 17.39% (p < 0.05).


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Bifidobacterium longum/enzimologia , Amido/metabolismo , Triticum/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/classificação , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Sequência de Aminoácidos , Amilopectina/metabolismo , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Peso Molecular , Filogenia , Alinhamento de Sequência , Temperatura , Termodinâmica
16.
Plant Mol Biol ; 103(3): 355-371, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32193789

RESUMO

KEYMESSAGE: Biphasic starch granules in maize ae mutant underwent the weak to strong SBEIIb-defective effect during endosperm development, leading to no birefringence in their exterior due to extended long branch-chains of amylopectin. Biphasic starch granules are usually detected regionally in cereal endosperm lacking starch branching enzyme (SBE). However, their molecular structure, formation mechanism, and regional distribution are unclear. In this research, biphasic starch granules were observed in the inner region of crown endosperm of maize ae mutant, and had poorly oriented structure with comb-like profiles in their exterior. The inner endosperm (IE) rich in biphasic starch granules and outer endosperm (OE) without biphasic starch granules were investigated. The starch had lower amylose content and higher proportion of long branch-chains of amylopectin in IE than in OE, and the exterior of biphasic starch granules had less amylose and more long branch-chains of amylopectin than the interior. Compared with OE, the expression pattern of starch synthesis related enzymes changed significantly in IE. The granule-bound starch synthase I activity within biphasic starch granules decreased slightly. The IE experienced more severe hypoxic stress than OE, and the up-regulated anaerobic respiration pathway indicated an increase in carbon consumption. The starch in IE underwent the SBEIIb-defective effect from weak to strong due to the lack of sufficient carbon inflow, leading to the formation of biphasic starch granules and their regional distribution in endosperm. The results provided information for understanding the biphasic starch granules.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Amido/metabolismo , Zea mays/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/classificação , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Endosperma/enzimologia , Endosperma/ultraestrutura , Amido/ultraestrutura
17.
Food Funct ; 11(3): 2543-2554, 2020 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-32150182

RESUMO

A novel amylopectin-based cyclic architecture was fabricated, arising from microbial branching enzyme treated waxy rice starch. The recombinant enzyme had a molecular weight of 72.0 kDa, and exhibited optimum activity at pH 7.0 and 75 °C. During the cyclization reaction catalyzed by a branching enzyme, the molecular weight of amylopectin rapidly decreased for the initial 2 h, and then very slowly decreased, tapering off at approximately 1.8 × 105 g mol-1 at 12 h. The number of A-chain fractions greatly increased, whereas the percentage of B-chain fractions decreased after enzymatic modification, accompanied by more α-1, 6 linkage formation. The core ring structure as a glucoamylase-resistant fraction had a number-average degree of polymerization of 21, which was constructed by 19 glucose units linked with, 2 glucosyl stubs at the O-6-position of the cyclic glucan through α-1,4 and α-1,6 linkages. Similar to large-ring cyclodextrin with equal glucose units, this cyclic glucan had a cavity geometry with two-circular loops and short stubs in perpendicular planes. Moreover, this cyclic glucan could complex with iodine for the host-guest formation. These results revealed the potential application of the amylopectin-based cyclic glucan as a good delivery system to encapsulate and protect bioactive ingredients.


Assuntos
Amilopectina/química , Amilopectina/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilopectina/biossíntese , Proteínas de Bactérias/metabolismo , Biotecnologia , Fenômenos Químicos , Ciclização , Geobacillus stearothermophilus/enzimologia , Hidrólise , Peso Molecular , Oryza , Amido
18.
Food Chem ; 316: 126348, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32044699

RESUMO

The 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02 (GtGBE, EC 2.4.1.18) does not possess the thermostability required by modified starch industry. To increase its thermostability, a rational design strategy was used to introduce additional salt bridges into GtGBE. The strategy involved in mutation of individual residues to form "local" two-residue salt bridges. Accordingly, five of local salt bridges (Q231R-D227, Q231K-D227, T339E-K335, T339D-K335, and I571D-R569 mutants) were separately introduced into GtGBE. The half-times of these mutants at 60 °C were 17% to 51% longer than that of wild-type. Subsequently, these two-residue salt bridges were extended to form salt bridge networks (Q231R/K-D227-D131H, T339D/E-K335-I291H, and I571D-R569-R617H mutants). Among these mutants, except I571D-R569-R617H, the half-times of Q231R/K-D227-D131H, T339D/E-K335-I291H mutants at 60 °C were 15%, 17%, 21% and 17% longer than those of the corresponding two-residue salt bridges, respectively. The results showed that design and introduction of salt bridges improves enzyme thermostability in GtGBE.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Geobacillus/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Estabilidade Enzimática/efeitos dos fármacos , Geobacillus/genética , Mutação , Cloreto de Sódio/farmacologia , Temperatura
19.
Crit Rev Biotechnol ; 40(3): 380-396, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31996051

RESUMO

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.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Bactérias/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Animais , Bactérias/genética , Proteínas de Bactérias , Biotecnologia , Glucanos , Humanos , Modelos Moleculares , Amido/biossíntese
20.
Int J Biol Macromol ; 155: 987-994, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31712143

RESUMO

Resistant starch (RS) is helpful in controlling and preventing metabolic syndrome relevant diseases. However, the RS content of natural starch and modified starch produced by enzymatic method is generally low. To solve this problem, we selected the glycogen branching enzyme from Vibrio vulnificus (VvGBE) and investigated its application. Firstly, it was expressed in E. coli with the enzyme activity was 53.33 U/mL, and its optimum temperature and pH was 35 °C and 7.5, respectively. The half-life of VvGBE at 35 °C was 10 h, and the enzyme was most stable at pH 9.5. When we used the recombinant enzyme to treat corn starch, the content of RS increased by 19.41%, which was higher than that achieved with other enzymes. More specially, the conversion of slowly digestible starch to RS, which was only demonstrated in chemical modification, was accomplished. The fine structure of the modified starch was further investigated. Results showed that the number of short chains (DP < 13) increased to 90.58%, and the α-1,6 linkages ratio increased from 7.19% to 15.64%. The increase of short chains and α-1,6 linkages may contribute to high RS content. This study can provide a reference for the development of modified starch with lower digestibility.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Recombinantes/metabolismo , Amido/química , Vibrio vulnificus/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Proteínas de Bactérias/genética , Digestão , Humanos , Proteínas Recombinantes/genética
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