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1.
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.

2.
Food Chem ; 305: 125441, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494499

RESUMO

Samples of granular corn starch were treated with 1,4-α-glucan branching enzyme (GBE) for 20 h using three different methods. These GBE modification methods all increased glycosidic linkage ratio, cyclic glucan content, and proportion of short chains while reducing weight mean molecular weight. The in vitro digestion rates of the modified starches were suppressed. Among these methods, a novel two-stage modification method comprising a 10-h GBE treatment, gelatinization, and a second 10-h GBE treatment, produced samples with the lowest in vitro digestibility. The rapidly digestible starch content was 34.2% lower than that of the control and 18.0% lower than that of the product of one-stage modification with the same duration. Fine structure characterization showed that more cluster structures were proved during the two-stage modification. This two-stage method suppressed the digestibility of corn starch and increased the substrate concentration, showing great potential for the industrial processing of slowly-digestible starchy foods.

3.
Biotechnol Lett ; 2019 Dec 02.
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.

4.
Int J Biol Macromol ; 2019 Nov 18.
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.

5.
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.

6.
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.

7.
Org Lett ; 21(20): 8429-8433, 2019 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-31592676

RESUMO

α-Aminonitrile was an important building block in natural products and key intermedia in organic chemistry. Herein, the direct amination of cyanohydrins with the partner of ammonia to synthesis N-unprotected α-aminonitriles is developed. The reaction proceeds via titanium-catalyzed cyano-borrowing reaction, which features high atom economy and simple operation. A broad range of ketone or aldehyde cyanohydrins was tolerated with ammonia, and the N-unprotected α-aminonitriles were synthesis with moderate to high yields under mild reaction conditions.

8.
J Control Release ; 313: 1-13, 2019 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-31622690

RESUMO

Non-invasive means of insulin administration circumvent some of the inconveniences of injections. Oral administration in particular is convenient, pain-free, and allows favorable glucose homeostasis, but is subject to chemical instability, enzymatic degradation, and poor gastrointestinal absorption. Natural polymeric nanoparticles have emerged as a promising oral delivery system for peptide therapeutics due their safety, biocompatibility, and stability. In this study, self-assembled nanocomposites from chitosan (CS) and insulin-loaded, zein-carboxymethylated short-chain amylose (IN-Z-CSA) nanocomposites were synthesized to improve oral bioavailability of insulin. The optimized IN-Z-CSA/CS0.2% nanocomposites exhibited an average size of 311.32±6.98 nm, a low polydispersity index (0.227±0.01), a negative zeta potential (43.77±1.36 mV), an encapsulation efficiency of 89.6±0.9%, and a loading capacity of 6.8±0.4%. The IN-Z-CSA/CS0.2% nanocomposites were stable in storage conditions. The transepithelial permeability of the N-Z-CSA/CS0.2% nanocomposites was 12-fold higher than that of insulin. Cellular uptake studies revealed that the IN-Z-CSA/CS0.2% nanocomposites were internalized into Caco-2 cells by both endocytosis and a paracellular route. Additionally, in pharmacological studies, orally administered IN-Z-CSA/CS0.2% nanocomposites had a stronger hypoglycemic effect with a relative bioavailability of 15.19% compared with that of IN-Z-CSA1.0% nanocomposites. Furthermore, cell toxicity and in vivo tests revealed that the IN-Z-CSA/CS0.2% nanocomposites were biocompatible. Overall, these results indicate that the IN-Z-CSA/CS0.2% nanocomposites can improve oral bioavailability of insulin and are a promising delivery system for insulin or other peptide/protein drugs.

9.
Int J Biol Macromol ; 2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614157

RESUMO

Naturally fermented and sundried tapioca starch is reportedly the traditional material for polvilho azedo and the primary ingredients of gluten-free food items in Brazil. This study aimed to investigate starch structure and expansion rate, high rate is acclaimed in baking application, changes of tapioca starch and potato starch during combinatorial fermentation and drying. The rate of expansion changed from 3.37 mL/g in native tapioca starch to 3.71 mL/g in fermented oven-dried tapioca starch and 6.97 mL/g in fermented sun-dried tapioca starch, while potato starch sample displayed lesser expansion on all treatments. Rapid viscosity analysis, size-exclusion chromatography, X-ray diffraction (XRD), and attenuated total reflectance Fourier transform infrared (ATR-FTIR) and electron paramagnetic resonance spectrometry (EPR) were performed to determine the structure and investigate its relationship with the expansion rate. Fermentation attacked amorphous area and cleaved glycosidic bonds. Sunlight exposure facilitated complex interactions, and crosslinking increased the molecular weight distribution (MWD) in fermented sun-dried potato starch and led to depolymerization in tapioca starch. EPR revealed an initial free radical distribution in both starches, and our results show that intensity changes in tapioca starch are essential for a high expansion capacity.

10.
Bull Environ Contam Toxicol ; 103(5): 756-762, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31583424

RESUMO

The current study investigated the efficiency and mechanisms of in situ immobilization of artificially Cd-contaminated soils with ferronickel slag (FNS). The available Cd content of soil was measured and the modified European Community Bureau of Reference (BCR) sequential extraction procedure (SEP) was adopted to quantify the evolutions of Cd chemical speciation after the immobilization by the FNS. The results showed that the addition of FNS (5%‒15%) remarkably reduced the available Cd content and increased the pH and cation exchange capacity of soils. The passivation rate of Cd increased from 58.13% to 73.25% as the spiked Cd content rose from 10 to 120 mg kg‒1. The BCR SEP test revealed that the FNS addition substantially reduced the acid soluble fraction and increased the residual fraction of Cd, indicating the reduction of mobility and bioavailability of Cd in soils. The chemical precipitation, ion exchange and surface complexation might be involved in in situ immobilization of Cd-contaminated soils by the FNS.

11.
Viruses ; 11(9)2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31540474

RESUMO

CXC chemokine receptor 4 (CXCR4) is a co-receptor for HIV-1 entry into target cells. Its natural ligand, the chemokine SDF-1, inhibits viral entry mediated by this receptor. However, the broad expression pattern of CXCR4 and its critical roles in various physiological and pathological processes indicate that the direct application of SDF-1 as an entry inhibitor might have severe consequences. Previously, we constructed an effective SDF-1 mutant, SDF-1/54, by deleting the α-helix of the C-terminal functional region of SDF-1. Of note, SDF-1/54 shows remarkable decreased chemotoxic ability, but maintains a similar binding affinity to CXCR4, suggesting SDF-1/54 might better serve as a CXCR4 inhibitor. Here, we found that SDF-1/54 exhibited potent antiviral activity against various X4 HIV-1 strains, including the infectious clone HIV-1 NL4-3, laboratory-adapted strain HIV-1 IIIB, clinical isolates and even drug-resistant strains. By using time-of-addition assay, non-infectious and infectious cell-cell fusion assay and CXCR4 internalization assay, we demonstrated SDF-1/54 is an HIV-1 entry inhibitor. A combination of SDF-1/54 with several antiretroviral drugs exhibited potent synergistic anti-HIV-1 activity. Moreover, SDF-1/54 was stable and its anti-HIV-1 activity was not significantly affected by the presence of seminal fluid, vaginal fluid simulant and human serum albumin. SDF-1/54 showed limited in vitro cytotoxicity to lymphocytes and vaginal epithelial cells. Based on these findings, SDF-1/54 could have a therapeutic potential as an HIV-1 entry inhibitor.

12.
Protein Expr Purif ; 164: 105478, 2019 Dec.
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.

13.
Stem Cell Res Ther ; 10(1): 207, 2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311593

RESUMO

BACKGROUND: The goals of this study were to explore the expression profiles and functional networks of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in mesenchymal stromal cells (MSCs) involved in regulating the function of monocytes and to clarify the mechanisms by which MSCs exert immunoregulatory effects on monocytes. METHODS: MSCs and CD14+ monocytes were separately isolated. The immunoregulatory effects of MSCs on monocytes were determined by flow cytometry. lncRNAs and mRNAs that were differentially expressed (DE) between the control group (MSCs only) and co-culture group (MSCs co-cultured with monocytes) were identified through high-throughput sequencing and bioinformatic analyses and were confirmed by qRT-PCR. Bioinformatic analyses were performed to identify the critical biological functions and signalling pathways involved in MSC-mediated monocyte regulation and to identify the functional networks formed between DE mRNAs and lncRNAs. RESULTS: MSCs showed a strong ability to induce monocyte migration but inhibited monocyte differentiation into M1 macrophages. A total of 145 DE lncRNAs and 768 DE mRNAs were identified between the control and co-culture groups. Significant fold changes in lncRNAs and mRNAs were confirmed by qRT-PCR. GO analysis demonstrated that DE mRNAs and lncRNAs were highly associated with terms related to binding and biological regulation. KEGG analysis revealed 122 significantly regulated pathways, including the cytokine-cytokine receptor pathway and chemokine signalling pathway. Interaction and co-expression networks were constructed for DE mRNAs and lncRNAs, and several key microRNAs were identified in the competitive endogenous RNA (ceRNA) network. Target genes of the DE lncRNAs were analysed to predict critical mRNA-lncRNA axes involved in the immunoregulatory function of MSCs. CONCLUSIONS: Our research describes the lncRNA and mRNA expression profiles and functional networks involved in MSC-mediated regulation of monocytes. These results provide possible molecular mechanisms for the immunoregulatory function of MSCs and may help to elucidate possible molecular therapeutic targets in MSCs for the treatment of autoimmune diseases.

14.
J Biol Chem ; 294(37): 13740-13754, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31346035

RESUMO

Seminal amyloid fibrils are made up of naturally occurring peptide fragments and are key targets for the development of combination microbicides or antiviral drugs. Previously, we reported that the polysulfonic compound ADS-J1 is a potential candidate microbicide that not only inhibits HIV-1 entry, but also seminal fibrils. However, the carcinogenic azo moieties in ADS-J1 preclude its clinical application. Here, we screened several ADS-J1-like analogs and found that the antiparasitic drug suramin most potently inhibited seminal amyloid fibrils. Using various biochemical methods, including Congo red staining, CD analysis, transmission EM, viral infection assays, surface plasmon resonance imaging, and molecular dynamics simulations, we investigated suramin's inhibitory effects and its putative mechanism of action. We found that by forming a multivalent interaction, suramin binds to proteolytic peptides and mature fibrils, thereby inhibiting seminal fibril formation and blocking fibril-mediated enhancement of viral infection. Of note, suramin exhibited potent anti-HIV activities, and combining suramin with several antiretroviral drugs produced synergistic effects against HIV-1 in semen. Suramin also displayed a good safety profile for vaginal application. Moreover, suramin inhibited the semen-derived enhancer of viral infection (SEVI)/semen-mediated enhancement of HIV-1 transcytosis through genital epithelial cells and the subsequent infection of target cells. Collectively, suramin has great potential for further development as a combination microbicide to reduce the spread of the AIDS pandemic by targeting both viral and host factors involved in HIV-1 sexual transmission.

15.
Int J Biol Macromol ; 138: 394-402, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325505

RESUMO

To better understand structure-function relationships, an X-ray crystal structure of the maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04 (Bst-MFA) with bound acarbose has been determined at 2.2 Å. The structure revealed a classical three-domain fold stabilized by four calcium ions, in which CaI-CaIII form an unprecedented linear metal triad in the interior of domain B. Catalytic residues are deduced to be two aspartic acids and one glutamic acid (Asp234, Glu264, Asp331), and the acarbose is bound to surrounding amino acid residues, mainly through extensive hydrogen bonds. Furthermore, analysis of the structure indicates the existence of at least 8 subsites in Bst-MFA, six glycone sites (-6, -5, -4, -3, -2, -1) and two aglycone sites (+1, +2). Subsite +3 remains to be further explored. Sugar-binding subsites contribute to further presentation of the oligosaccharide-binding mode, which explains the product specificity of Bst-MFA to some extent. In addition, we propose a mechanism by which maltooligosaccharide-forming amylases produce particular maltooligosaccharide products, a result different from that seen with typical α-amylases. Finally, the three-dimensional structure of Bst-MFA complexed with acarbose provides the basis for further studies, designed to increase product specificity.

16.
Int J Biol Macromol ; 138: 966-974, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31356941

RESUMO

In the present study, we investigated the effects of complexing temperatures (75 °C and 95 °C) and oleic acid (OA) content (2%, 6%, 10% and 14%, w/w, dry basis of starch) on the structure and in vitro digestibility of maize starch-OA complexes. The resistant starch content, complexing index, thermal transition temperatures, enthalpy change and relative crystallinity of the complexes prepared at 75 °C were higher than those of corresponding complexes prepared at 95 °C. Additionally, starch-10% OA complexes prepared at 75 °C had the highest resistant starch content (18.82%), complexing index (78.83%) and relative crystallinity (14.62%). Fourier transform infrared spectroscopy revealed that the intermolecular interactions between starch and OA were affected by the complexing temperatures and OA content. Raman spectroscopy indicated that the starch-10% OA complexes prepared at 75 °Ð¡ had a greater short-range molecular order than other complexes. The V-type complexes were further confirmed as V6III polymorph structures according to the spectra obtained by nuclear magnetic resonance spectroscopy. Therefore, we conclude that complexing temperatures and OA content greatly changed the fine structure of the complexes, which further affected their digestibility.

17.
Int J Biol Macromol ; 136: 460-468, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31207329

RESUMO

Inhibition of cyclodextrin glycosyltransferases (CGTases) by their product cyclodextrins limits the efficiency of cyclodextrin production. In an effort to produce variants with good activity but reduced product inhibition, six mutants were constructed at position 603 of the CGTase from Bacillus circulans STB01, which exhibits mixed-type product inhibition. In a kinetic analysis, N603I showed reduced noncompetitive inhibition while N603K, N603H and N603R showed increased noncompetitive inhibition. Unexpectedly, N603E and N603D exhibited reductions in both competitive and noncompetitive product inhibition. Noncompetitive product inhibition is closely related to the interaction between the cyclodextrin and the enzyme in maltose binding site 2 (MBS2). Structural models led to the suggestion that there is increased interaction between maltose binding sites 1 and 2 in mutants N603E and N603D, which may have led to the unexpected results. N603D exhibited a 23.9% greater cyclodextrin yield per gram of enzyme than the wild-type, suggesting it has potential for industrial use. Further reductions in product inhibition may be gained through studies of maltose binding site interactions.

18.
Environ Pollut ; 249: 843-850, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30953946

RESUMO

A novel carbonaceous material (NCM), prepared by the pyrolyzation of the oily sludge of tank bottom, was proposed to remove Cr(VI) from a synthetic solution for the first time. The effects of initial Cr(VI) concentration, NCM dosage and initial solution pH on Cr(VI) removal and the adsorption kinetics, the adsorption isothem were investigated. The removal mechanism was studied by comparing the surface properties of NCM before and after the Cr(VI) removal. The results showed that NCM can effectively remove Cr(Ⅵ) from the synthetic solution with the increase of solution pH at equilibrium. At the initial Cr(Ⅵ) concentrations of 40, 100, 150 and 250 mg/L and NCM dosages of 1, 3, 6 and 8 g/L with initial solution pH of 2, the removal efficiency of Cr(VI) was 95.5, 96.8, 95.2 and 81.2%, and the solution pH at equilibrium reached 2.3, 3.5, 5.8 and 7.5, respectively. NCM was suitable for Cr(Ⅵ) removal while the initial Cr(VI) concentration was less than 100 mg/L and initial solution pH was lower than 2.5. Most of Cr(VI) was removed by the reduction of Fe2+ and S2- in NCM to Cr(III) and with the generation of stable FeCr2O4. Some Cr(VI) may be removed by reacting with Fe2+ and Ca2+ to produce CaCrO4 and FeCrO4 on the NCM surface. The dissolution of CaAl2Si2O8 and CaS in the solution increased the solution pH at equilibrium. NCM has been proved to be a material with dual functions both chemical reduction and adsorption.


Assuntos
Aluminossilicato de Cálcio/química , Compostos de Cálcio/química , Cromo/análise , Esgotos/química , Sulfetos/química , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Adsorção , Concentração de Íons de Hidrogênio , Cinética , Modelos Teóricos , Óleos/química , Oxirredução , Soluções , Propriedades de Superfície , Águas Residuárias/química
19.
Int J Biol Macromol ; 134: 247-254, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31026525

RESUMO

A new biodegradable, renewable, and environmentally friendly starch-based adhesive for wood-based panels was synthesized. The synthesis was conducted by grafting polymerization of vinyl acetate (VAC) monomer onto corn starch and crosslinking polymerization with N-methylol acrylamide (NMA). Compared with the traditional starch-based wood adhesive, the water resistance of starch-based adhesive with NMA (SWA-N) was greatly improved to more than 1 MPa; this exceeds the Chinese standard by 40%. The results from various analyses, including particle size, contact angle, thermogravimetric analysis (TGA), Fourier-transform infrared (FTIR), confocal Raman microscopy (CRM) and 13C-CPMAS, indicated that such improved performance is due to increased crosslinking density and formation of complex network structure. Such complex network structure was found to inhibit excessive expansion of the adhesive during high temperature pressing and water absorption. As a result, the internal structure of the adhesive remained intact when subjected to hot pressing and placed in wet conditions.

20.
Int J Biol Macromol ; 132: 759-765, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30953720

RESUMO

1,4-α-Glucan branching enzyme (GBE, EC. 2.4.1.18), which plays a key role in the synthesis of starch and glycogen, has been overexpressed in E. coli as an intracellular enzyme by many researchers. In this study, it was found that the GBEs from Geobacillus thermoglucosidans and Rhodothermus obamensis were secreted into the culture medium when they were expressed separately, in E. coli. This occurred despite the absence of any signal peptide. In fact, although bioinformatics tools predicted that both of these proteins would localize to the cytoplasm, a high level of expression and non-classical secretion was found to achieve without addition of the inducer isopropyl ß-d-thiogalactopyranoside. Further experiments revealed that secretion was a two-step process that occurred via the periplasmic space. Results excluded the involvement of the Sec pathway or the TAT pathway. Instead, the findings indicated a relationship between cell membrane integrity and the secretion of the two GBEs, and suggested that their N-termini play an essential role in their expression and secretion.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Escherichia coli/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/química , Permeabilidade da Membrana Celular , Estabilidade Enzimática , Escherichia coli/citologia , Geobacillus/enzimologia , Rhodothermus/enzimologia
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