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1.
Crit Rev Food Sci Nutr ; : 1-13, 2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34797201

RESUMO

The controlled release of guest molecules from cyclodextrin (CD) inclusion complexes is very important for specific industrial applications in foods, medicine, cosmetics, textiles, agriculture, environmental protection, and chemical materials. The term "controlled release" encompasses several related methods, including those referred to as immediate release, sustained release and targeted release. Many different CD-based controlled release systems are currently used in practical applications. CD inclusion complexes, CD coupling, supramolecular hydrogels, and supramolecular micelles are among the most common. This review systematically introduces the principles and applications of CD-based controlled release systems, providing a theoretical basis for improving the bioavailability of effective substances and broadening their range of application.

2.
Front Microbiol ; 12: 708480, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335544

RESUMO

Marine extremophiles produce cold-adapted and/or salt-tolerant enzymes to survive in harsh conditions. These enzymes are naturally evolved with unique structural features that confer a high level of flexibility, solubility and substrate-binding ability compared to mesophilic and thermostable homologs. Here, we identified and characterized an amylase, SdG5A, from the marine bacterium Saccharophagus degradans 2-40 T . We expressed the protein in Bacillus subtilis and found that the purified SdG5A enabled highly specific production of maltopentaose, an important health-promoting food and nutrition component. Notably, SdG5A exhibited outstanding cold adaptation and salt tolerance, retaining approximately 30 and 70% of its maximum activity at 4°C and in 3 M NaCl, respectively. It converted 68 and 83% of starch into maltooligosaccharides at 4 and 25°C, respectively, within 24 h, with 79% of the yield being the maltopentaose. By analyzing the structure of SdG5A, we found that the C-terminal carbohydrate-binding module (CBM) coupled with an extended linker, displayed a relatively high negative charge density and superior conformational flexibility compared to the whole protein and the catalytic domain. Consistent with our bioinformatics analysis, truncation of the linker-CBM region resulted in a significant loss in activities at low temperature and high salt concentration. This highlights the linker-CBM acting as the critical component for the protein to carry out its activity in biologically unfavorable condition. Together, our study indicated that these unique properties of SdG5A have great potential for both basic research and industrial applications in food, biology, and medical and pharmaceutical fields.

3.
Food Chem ; 362: 130240, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34119950

RESUMO

The alleviating role of starch derivatives on the quality deterioration of frozen steamed bread dough was investigated in terms of derivative structure, the bread characteristics and dough properties including freezable water contents, yeast activity as well as dough viscoelasticity. The addition of starch derivatives including short-clustered maltodextrin (SCMD), DE2 maltodextrin (MD) and pregelatinized starch (PGS) significantly increased the specific volume and decreased the hardness of steamed bread compared with Control bread after 8-week frozen storage. Lower freezable water content was found in PGS dough than SCMD dough, which was consistent with the results of water absorption index of starch derivatives. The analysis of dough gassing rate and yeast survival ratio demonstrated SCMD could provide more cryoprotection for yeast cells. Meanwhile, a higher elastic module and a more continuous gluten-network structure of SCMD dough were found after 8-week frozen storage. These results indicated starch derivatives especially SCMD were promising to be used as the alternative improvers in frozen dough production.


Assuntos
Pão/análise , Pão/normas , Qualidade dos Alimentos , Congelamento , Amido/farmacologia , Tecnologia de Alimentos , Glutens/química , Saccharomyces cerevisiae/efeitos dos fármacos , Amido/química , Vapor , Viscosidade/efeitos dos fármacos , Água/química , Fermento Seco/análise
4.
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
5.
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
6.
Carbohydr Polym ; 262: 117926, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33838805

RESUMO

Double emulsions are promising carrier systems for foods, pharmaceuticals, and cosmetics. However, their limited stability hinders their practical applications. We used gelatinized starch to develop stable double emulsions as carrier materials. The oil/water/water (O/W/W) double emulsions were formed by 5 wt% native corn starch, while oil/water/oil (O/W/O) double emulsions were formed by 7 wt% native corn starch and high-amylose starch with 60 % and 75 % amylose contents investigated by optical microscopy. Furthermore, the storage stability of double emulsions was revealed by droplet size distribution, microstructure, backscattering, rheological profiles, and low-field nuclear magnetic resonance (LF-NMR) imaging. Results confirmed that the O/W/O double emulsions stabilized by 7 wt% native corn starch had a smaller mean droplet size (11.400 ± 0.424 µm) and excellent storage stability (14 days) than O/W/W and O/W/O double emulsions prepared with high-amylose starch. Such unique double emulsions prepared with gelatinized native corn starch are good candidates of carrier materials.


Assuntos
Emulsões/química , Gelatina/química , Amido/química , Amilose/química , Portadores de Fármacos/química , Humanos , Espectroscopia de Ressonância Magnética/métodos , Tamanho da Partícula , Reologia , Água/química
7.
Food Chem ; 354: 129475, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-33744660

RESUMO

The α-amylases are the most widely used industrial enzymes, and are particularly useful as liquifying enzymes in industrial processes based upon starch. Since starch liquefication is carried out at evaluated temperatures, typically above 60 °C, there is substantial demand for thermostable α -amylases. Most naturally occurring α -amylases exhibit moderate thermostability, so substantial effort has been invested in attempts to increase their thermostability. One structural feature that has the potential to increase protein thermostability is the introduction of salt bridges. However, not every salt bridge contributes to protein thermostability. The salt bridges in amylases have their characteristics in terms of distribution, configuration and location. The summary of these features helps to introduce new salt bridges based on the characteristics. This review focuses on salt bridges of α-amylases, both naturally present and introduced using mutagenesis. Its aim is to provide a bird's eye view of distribution, configuration, location of desirable salt bridges.


Assuntos
Sais/química , alfa-Amilases/metabolismo , Bacillus licheniformis/enzimologia , Sítios de Ligação , Estabilidade Enzimática , Metais/química , Simulação de Dinâmica Molecular , Temperatura , alfa-Amilases/química
8.
Int J Biol Macromol ; 175: 254-261, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33561459

RESUMO

The efficiency of enzymatic cyclodextrin production using cyclodextrin glycosyltransferases (CGTases) is limited by product inhibition. In this study, maltose binding site 2 (MBS2) of the ß-CGTase from Bacillus circulans STB01 was modified to decrease product inhibition. First, two point mutants were prepared at position 599 (A599V and A599N). Then, two double mutants incorporating alanine at position 633 (A599N/Y633A and A599V/Y633A) were prepared. Finally, the entire MBS2 region was replaced by that of the α-CGTase from Paenibacillus macerans JFB05-01 to form multipoint mutant MBS2 ߠ→ α. All five mutants exhibited mixed-type product inhibition, although both the competitive and uncompetitive components of this inhibition were decreased. The total cyclization activities of A599N, A599V and A599V/Y633A were 15.6%, 76.8% and 70.9% lower than that of the wild-type, respectively, while that of A599N/Y633A was 22.4% higher. Among the mutants, only MBS2 ߠ→ α showed catalytic efficiency (kcat/Km) comparable with that of the wild-type. Moreover, A599N, A599N/Y633A and MBS2 ߠ→ α produced cyclodextrin yields 13.1%, 15.8% and 19.7% greater than that of the wild-type, respectively. These results suggest that A599N, A599N/Y633A and MBS2 ߠ→ α may be more suitable than the wild-type for cyclodextrin production.


Assuntos
Bacillus/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Maltose/metabolismo , Bacillus/genética , Proteínas de Bactérias/química , Sítios de Ligação/genética , Ciclização/genética , Ciclodextrinas/metabolismo , Cinética , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Mutação/genética , Paenibacillus/genética , Especificidade por Substrato/genética , beta-Ciclodextrinas/química
9.
J Sci Food Agric ; 101(9): 3742-3748, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33301206

RESUMO

BACKGROUND: Designing a high-concentration (50%, w/w) maltodextrin saccharification process is a green method to increase the productivity of maltose syrup. RESULTS: In this study, a temperature-mediated two-step process using ß-amylase and pullulanase was investigated as a strategy to improve the efficiency of saccharification. During the saccharification process, both pullulanase addition time and temperature adjustment greatly impacted the final maltose yield. These results indicated that an appropriate ß-amylolysis in the first stage (the first 8 h) was required to facilitate saccharification process, with the maltose yield of 8.46% greater than that of the single step saccharification. Molecular structure analysis further demonstrated that a relatively low temperature (50 °C), as compared with a normal temperature (60 °C), in the first stage resulted in a greater number of chains polymerized by at least seven glucose units and a less heterogeneity system within the residual substrate. The molecular structure of the residual substrate might be beneficial for the subsequent cooperation between ß-amylase and pullulanase in the following 40 h (second stage). CONCLUSION: Over a 48 h saccharification, the temperature-mediated two-step process dramatically increased the conversion rate of maltodextrin and yielded significantly more maltose and less byproduct, as compared with a constant-temperature process. The two-step saccharification process therefore offered an efficient and green strategy for maltose syrup production in industry. © 2020 Society of Chemical Industry.


Assuntos
Glicosídeo Hidrolases/química , Maltose/análise , Polissacarídeos/química , Biocatálise , Glucose , Hidrólise , Temperatura
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.
J Agric Food Chem ; 68(44): 12400-12412, 2020 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-33084325

RESUMO

Molecular structure of starch in daily diet is closely associated with diabetes management. By enzymatically reassembling α-1,4 and α-1,6 glycosidic bonds in starch molecules, we have synthesized an innovative short-clustered maltodextrin (SCMD) which slowly releases glucose during digestion. Here, we investigated the potential benefits of the SCMD-containing diet using diabetic db/db mice. As compared to a diet with normal starch, this dietary style greatly attenuated hyperglycemia and repaired symptoms associated with diabetes. Additionally, in comparison with acarbose (an α-glucosidase inhibitor) administration, the SCMD-containing diet more effectively accelerated brown adipose activation and improved energy metabolism of db/db mice. Furthermore, the SCMD-containing diet was a more suitable approach to improving the intestinal microflora than acarbose administration, especially the proliferation of Mucispirillum, Akkermansia, and Bifidobacterium. These results reveal a novel strategy for diabetes management based on enzymatically rebuilding starch molecules in the daily diet.


Assuntos
Diabetes Mellitus Tipo 2/dietoterapia , Diabetes Mellitus Tipo 2/metabolismo , Carboidratos da Dieta/metabolismo , Microbioma Gastrointestinal , Polissacarídeos/metabolismo , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Diabetes Mellitus Tipo 2/microbiologia , Carboidratos da Dieta/análise , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polissacarídeos/química , Amido/metabolismo
12.
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
13.
J Agric Food Chem ; 68(3): 838-844, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31896254

RESUMO

Maltooligosaccharide-forming amylases (MFAses) are promising tools for a variety of food industry applications because they convert starch into functional maltooligosaccharides. The MFAse from Bacillus stearothermophilus STB04 (BstMFAse) is a thermostable enzyme that preferentially produces maltopentaose and maltohexaose. An X-ray crystal structure of the BstMFAse-acarbose complex suggested that mutation of glycine 109 would increase its maltohexaose specificity. Using site-directed mutagenesis, glycine 109 was replaced with several different amino acids. Mutant-containing asparagine (G109N), aspartic acid (G109D), and phenylalanine (G109F) produced 36.1, 42.4, and 39.0% maltohexaose from starch, respectively, which was greater than that produced by the wild-type (32.9%). These mutants also exhibited substantially altered oligosaccharide hydrolysis patterns in favor of maltohexaose production. Homology models suggested that the mutants form extra interactions with the substrate at subsite -6, which were responsible for the enhanced maltohexaose specificity of BstMFAse. The results of this study support the proposition that binding of the substrate's nonreducing end in the nonreducing end-subsite of the MFAse active center plays a crucial role in its product specificity.


Assuntos
Amilases/genética , Amilases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Geobacillus stearothermophilus/enzimologia , Oligossacarídeos/metabolismo , Amilases/química , Proteínas de Bactérias/química , Sítios de Ligação , Geobacillus stearothermophilus/genética , Mutagênese Sítio-Dirigida , Oligossacarídeos/química , Engenharia de Proteínas , Amido/química , Amido/metabolismo , Especificidade por Substrato
14.
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
15.
Int J Biol Macromol ; 154: 1303-1313, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31751711

RESUMO

The maltooligosaccharide-forming amylases (MFAses) degrade starch into maltooligosaccharides which potentially benefit human diet and grow popular in food processing, but little has been studied about their product specificity and structures. We focused on this topic and provide evidence through an X-ray crystal structure of the maltotetraose (G4)-forming amylase from Pseudomonas saccharophila STB07 (MFAps), as well as co-crystal structures of MFAps with G4 and with pseudo-maltoheptaose (pseudo-G7) determined at up to 1.1 Å resolution. G4 and pseudo-G7 occupy active cleft subsites -4 to -1 and -4 to +3 respectively. Binding induces conformational changes in the active sites except Asp193, working as the base catalyst. Comparison of the MFAps structure with those of other α-amylases revealed obvious differences in the loop structures providing dominant interactions between protein and substrate in the non-reducing side of the active sites cleft. These structures at the non-reducing end may govern the G4 specificity of MFAps and also be relevant to its exo-type action pattern.


Assuntos
Amilases/química , Amilases/metabolismo , Comamonadaceae/enzimologia , Maltose/análogos & derivados , Concentração de Íons de Hidrogênio , Maltose/metabolismo , Modelos Moleculares , Domínios Proteicos , Especificidade por Substrato , Temperatura
16.
Bioresour Technol ; 296: 122323, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31698224

RESUMO

Effects of solid-state fermentation on rapid drying and spoilage prevention of potato pulp were evaluated. Pectin hydrolyzing and antibacterial ability of pectinase-secreting Aspergillus aculeatus and Bacillus subtilis were compared. A. aculeatus grew better in potato pulp, with highest pectinase yield of 342.71 ±â€¯5.09 U/mL and rapid pH reduction to 3.76 ±â€¯0.01. Next generation sequencing showed that the abundance of genera Candida and Enterobacter, which probably caused undesirable fermentation and spoilage, were significantly reduced after inoculation with A. aculeatus. In addition, fermentation with A. aculeatus significantly reduced water holding capacity from 16.63 ±â€¯0.36 g/g to 7.78 ±â€¯0.12 g/g, which resulted in lower viscosity and water binding capacity, and concomitantly significantly decreased moisture content from 76.05 ±â€¯0.24% to 12.95 ±â€¯0.19% after filtration and airflow drying. These results suggested that solid-state fermentation might be a promising technology for efficient processing and utilization of potato pulp.


Assuntos
Solanum tuberosum , Aspergillus , Dessecação , Fermentação , Poligalacturonase
17.
Appl Biochem Biotechnol ; 190(3): 1010-1022, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31654380

RESUMO

By sequence comparison, the majority of 1,4-α-glucan-branching enzymes (GBEs) consist of an N-terminal carbohydrate-binding domain, a TIM-barrel catalytic domain, and a C-terminal all-beta domain. Among these structures, the GBE from Geobacillus thermoglucosidans STB02 uniquely has a highly charged 26-amino-acid C-terminal extension, whose functional roles are the least understood. In this research, the functional significance of the C-terminal domain in GBE from G. thermoglucosidans STB02 and its extension were assessed using a C-terminal deletion analysis. Mutants lacking of more than 7 residues of the C-terminal all-beta domain could not be detected in lysates of their Escherichia coli expression strains, suggesting that an intact all-beta domain is required for structural stability. In contrast, truncation of the C-terminal extension resulted in greater stability and solubility than the wild type, as well as a lower sensitivity to the presence of added metal ions. Comparison of this mutant with the wild type suggests that the interaction of metal ions with the C-terminal extension influences performance of this enzyme.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Geobacillus/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/isolamento & purificação , Sequência de Aminoácidos , Estabilidade Enzimática , Escherichia coli/genética , Metais/metabolismo , Mutação , Solubilidade
18.
Biotechnol Lett ; 42(2): 295-303, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31792661

RESUMO

OBJECTIVES: Analyze the thermostability, mode of action, and product specificity of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04 (Bst-MFA) from the biochemical and structural point of view. RESULTS: Using three-dimensional co-crystal structure of Bst-MFA with acarbose as a guide, experiments were performed to analyze the thermostability, mode of action and product specificity of Bst-MFA. The results showed that the Ca2+-Ca2+-Ca2+ metal triad of Bst-MFA is responsible for its high thermostability. Multiple substrate binding modes, rather than one productive binding mode determined by non-reducing end recognition, are in accordance with an endo-type mode of action. Significant interactions between subsites - 5 and - 6 and glucosyl residues at the non-reducing end explain the maltopentaose (G5) and maltohexaose (G6) specificity of Bst-MFA. CONCLUSIONS: Bst-MFA is a thermostable enzyme that preferentially produces G5 and G6, with an endo-type mode. The understanding of structure-function relationships provides the foundation for future efforts to the modification of Bst-MFA.


Assuntos
Amilases/química , Amilases/metabolismo , Geobacillus stearothermophilus/enzimologia , Oligossacarídeos/metabolismo , Amilases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Cristalografia , Estabilidade Enzimática , Geobacillus stearothermophilus/química , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato , Termodinâmica
19.
Appl Microbiol Biotechnol ; 103(23-24): 9433-9442, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31676918

RESUMO

The maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04 (Bst-MFA) randomly cleaves the α-1,4 glycosidic linkages of starch to produce predominantly maltopentaose and maltohexaose. The three-dimensional co-crystal structure of Bst-MFA with acarbose highlighted the stacking interactions between Trp139 and the substrate in subsites - 5 and - 6. Interactions like this are thought to play a critical role in maltopentaose/maltohexaose production. A site-directed mutagenesis approach was used to test this hypothesis. Replacement of Trp139 by alanine, leucine, or tyrosine dramatically increased maltopentaose production and reduced maltohexaose production. Oligosaccharide degradation indicated that these mutants also enhance productive binding of the substrate aglycone, leading to a high maltopentaose yield. Therefore, the aromatic stacking between Trp139 and substrate is suggested to control product specificity and the oligosaccharide cleavage pattern.


Assuntos
Aminoácidos/química , Amilases/química , Amilases/genética , Geobacillus stearothermophilus/enzimologia , Oligossacarídeos/biossíntese , Sítios de Ligação , Geobacillus stearothermophilus/genética , Hidrólise , Modelos Moleculares , Mutagênese Sítio-Dirigida , Especificidade por Substrato
20.
Protein Expr Purif ; 164: 105478, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31421223

RESUMO

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.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Proteínas de Bactérias/genética , Rhodothermus/genética , Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodothermus/química , Rhodothermus/metabolismo
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