Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.484
Filtrar
1.
Food Chem ; 320: 126609, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32222658

RESUMO

Bread crumb firming is largely determined by the properties of gluten and starch, and the transformations they undergo during bread making and storage. Amylose (AM) and amylopectin (AP) functionality in fresh and stored bread was investigated with NMR relaxometry. Bread was prepared from flours containing normal and atypical starches, e.g., flour from wheat line 5-5, with or without the inclusion of Bacillus stearothermophilus α-amylase. Initial crumb firmness increased with higher levels of AM or shorter AM chains. Both less extended AM and gluten networks and too rigid AM networks led to low crumb resilience. AP retrogradation during storage increased when crumb contained more AP or longer AP branch chains. Shorter AP branch chains, which were present at higher levels in 5-5 than in regular bread, were less prone to retrogradation, thereby limiting gluten network dehydration due to gluten to starch moisture migration. Correspondingly, crumb firming in 5-5 bread was restricted.


Assuntos
Amilopectina/química , Amilose/química , Pão/análise , Armazenamento de Alimentos , Amilopectina/metabolismo , Amilose/metabolismo , Proteínas de Bactérias , Farinha/análise , Geobacillus stearothermophilus/enzimologia , Glutens/química , Espectroscopia de Ressonância Magnética , Amido/química , Triticum/química , Água , alfa-Amilases/metabolismo
2.
J Mol Biol ; 432(7): 2099-2120, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32067952

RESUMO

ABC importers are membrane proteins responsible for the transport of nutrients into the cells of prokaryotes. Although the structures of ABC importers vary, all contain four conserved domains: two nucleotide-binding domains (NBDs), which bind and hydrolyze ATP, and two transmembrane domains (TMDs), which help translocate the substrate. ABC importers are also dependent on an additional protein component, a high-affinity substrate-binding protein (SBP) that specifically binds the target ligand for delivery to the appropriate ABC transporter. AbnE is a SBP belonging to the ABC importer for arabino-oligosaccharides in the Gram-positive thermophilic bacterium Geobacillus stearothermophilus. Using isothermal titration calorimetry (ITC), purified AbnE was shown to bind medium-sized arabino-oligosaccharides, in the range of arabino-triose (A3) to arabino-octaose (A8), all with Kd values in the nanomolar range. We describe herein the 3D structure of AbnE in its closed conformation in complex with a wide range of arabino-oligosaccharide substrates (A2-A8). These structures provide the basis for the detailed structural analysis of the AbnE-sugar complexes, and together with complementary quantum chemical calculations, site-specific mutagenesis, and isothermal titration calorimetry (ITC) experiments, provide detailed insights into the AbnE-substrate interactions involved. Small-angle X-ray scattering (SAXS) experiments and normal mode analysis (NMA) are used to study the conformational changes of AbnE, and these data, taken together, suggest clues regarding its binding mode to the full ABC importer.


Assuntos
Arabinose/química , Arabinose/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Geobacillus stearothermophilus/enzimologia , Conformação Proteica , Proteínas de Bactérias/genética , Cristalografia por Raios X , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica
3.
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
4.
J Biochem ; 167(2): 203-215, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31617574

RESUMO

Directed evolution using error-prone polymerase chain reaction was employed in the current study to enhance the catalytic efficiency of a thermostable Geobacillus stearothermophilus xylanase XT6 parent. High-throughput screening identified two variants with enhanced activity. Sequencing analysis revealed the presence of a single-amino acid substitution (P209L or V161L) in each variant. The maximum activity of mutant V161L and P209L was at 85°C and 70°C, respectively. Both mutants exhibited maximum activity at pH 7. The thermal and alkaline tolerance of mutant V161L only were markedly improved. The two mutants were more resistant to ethanol inhibition than the parent. Substrate specificity of the two mutants was shifted from beechwood xylan to birchwood xylan. The potential of the two mutants to hydrolyze rice straw and sugarcane bagasse increased. Both turnover number (kcat) and catalytic efficiency (kcat/kM) increased 12.2- and 5.7-folds for variant P209L and 13- and 6.5-folds for variant V161L, respectively, towards birchwood xylan. Based on the previously published crystal structure of extracellular G. stearothermophilus xylanase XT6, V161L and P209L mutation locate on ßα-loops. Conformational changes of the respective loops could potentiate the loop swinging, product release and consequently result in enhancement of the catalytic performance.


Assuntos
Substituição de Aminoácidos , Biocatálise , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Geobacillus stearothermophilus/enzimologia , Temperatura , Endo-1,4-beta-Xilanases/química , Estabilidade Enzimática , Geobacillus stearothermophilus/genética , Ensaios de Triagem em Larga Escala , Especificidade por Substrato
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.


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
6.
Anal Chim Acta ; 1081: 193-199, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31446958

RESUMO

Isothermal DNA amplification only using a Bacillus stearothermophilus (Bst) DNA polymerase such as loop-mediated isothermal amplification typically entails multiple target sites for primer design and is thereby not suited for the amplification of short gene sequences, for example, the sequences with size below 200 nucleotides (nt). Here we present SLIMP, a novel single enzyme-based isothermal amplification of short gene sequence mediated by both stem-loop and linear primers. In SLIMP, a pair of stem-loop primers and a pair of linear primers are specifically designed to recognize only two target sites. Linear primers in SLIMP are similar as conventional PCR primers, but stem-loop primers are the modified linear primers through attaching a stem-loop structure at their 5'-ends. Attributed to this unique primer design, three basic reaction modes including linear-priming, single stem-loop-priming, and double stem-loop-priming amplifications co-mediate the SLIMP process under the function of Bst DNA polymerase. As a proof-of-concept assay, a synthetic 80 nt sequence from hepatitis B virus S gene was used as the template to develop SLIMP. On performance, SLIMP detection possesses high sensitivity and specificity, good selectivity, and the potential for analysing real sample. Therefore, SLIMP is expected as a novel alternative to amplify short gene sequences using a single enzyme.


Assuntos
Primers do DNA/genética , DNA Viral/análise , DNA Polimerase Dirigida por DNA/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Primers do DNA/química , DNA Viral/genética , Desoxirribonuclease HindIII/química , Geobacillus stearothermophilus/enzimologia , Vírus da Hepatite B/genética , Sequências Repetidas Invertidas , Hibridização de Ácido Nucleico , Estudo de Prova de Conceito , Sensibilidade e Especificidade
7.
Anal Chim Acta ; 1080: 162-169, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31409466

RESUMO

Driven by a bright prospect for rapid, portable and cost-effective point-of-care testing, an assembled Pasteur pipette device to integrate nucleic acid extraction, amplification and detection was developed to detect B. cereus in a sample-to-answer format. Denaturation Bubble-mediated Strand Exchange Amplification (SEA) was chosen to perform isothermal amplification because it requires only a pair of primers and one Bst DNA polymerase. The established SEA can detect as low as 1.0 × 10-13 M genomic DNA of B. cereus, which was comparable with the previously reported method for B. cereus detection. The assembled Pasteur pipette allows sample-to-answer diagnostic in a simple, low-cost, portable, and disposable format. The inherent function of Pasteur pipette enables direct liquid handling without the need of extra pipettes, syringes or pumps. Visual readout was achieved by using a pH sensitive dye, further simplifying result judgment process. The detection limit for B. cereus is 1.0 × 104 CFU/mL in pure cultures, while the detection limit in artificially contaminated milk is 1.0 × 105 CFU/mL without enrichment and 1.0 × 100 CFU/mL following 12 h enrichment. Considering that typical cell counts in food samples associated to food poisoning are 1.0 × 105 to 1.0 × 108 CFU per gram/milliliter B. cereus, our Pasteur pipette is enough sensitive for answer-to-sample diagnosis of B. cereus even directly from foods without enrichment. The whole diagnostic procedure could be completed within 50 min, dramatically decreasing the detection time. In a word, the assembled Pasteur pipette device, combined with a homemade metal bath, possesses great potential for sample-to-answer application in resource-limited settings.


Assuntos
Bacillus cereus/isolamento & purificação , Carga Bacteriana/métodos , Colorimetria/métodos , DNA Bacteriano/análise , Animais , Carga Bacteriana/instrumentação , Proteínas de Bactérias/química , Colorimetria/instrumentação , Corantes/química , DNA Polimerase Dirigida por DNA/química , Contaminação de Alimentos/análise , Geobacillus stearothermophilus/enzimologia , Limite de Detecção , Leite/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Papel
8.
Nat Commun ; 10(1): 3717, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420547

RESUMO

Amine dehydrogenases (AmDHs) catalyse the conversion of ketones into enantiomerically pure amines at the sole expense of ammonia and hydride source. Guided by structural information from computational models, we create AmDHs that can convert pharmaceutically relevant aromatic ketones with conversions up to quantitative and perfect chemical and optical purities. These AmDHs are created from an unconventional enzyme scaffold that apparently does not operate any asymmetric transformation in its natural reaction. Additionally, the best variant (LE-AmDH-v1) displays a unique substrate-dependent switch of enantioselectivity, affording S- or R-configured amine products with up to >99.9% enantiomeric excess. These findings are explained by in silico studies. LE-AmDH-v1 is highly thermostable (Tm of 69 °C), retains almost entirely its catalytic activity upon incubation up to 50 °C for several days, and operates preferentially at 50 °C and pH 9.0. This study also demonstrates that product inhibition can be a critical factor in AmDH-catalysed reductive amination.


Assuntos
Aminoácido Oxirredutases/síntese química , Geobacillus stearothermophilus/enzimologia , Cetonas/metabolismo , Aminação , Aminas , Amônia/metabolismo , Biocatálise , Desaminação , Estereoisomerismo
9.
Talanta ; 204: 248-254, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357289

RESUMO

An ultrasensitive strategy based on sandwich immunoassay coupled with isothermal exponential amplification reaction (IMEXPAR) is proposed for the determination of tumor protein Mucin 1 (MUC1). An immuno-PCR plate was prepared from modification of the primary MUC1-antibody (Ab1) onto the inner-well of the PCR plate. A biotinylated secondary MUC1-antibody tagged with the biotinylated EXPAR primer (P-Ab2) was prepared through biotin-streptavidin reaction. In the presence of target MUC1, sandwich-type combinations were specifically formed in the immuno-PCR plate. With further addition of amplification template, polymerase and nicking enzyme, EXPAR was specifically triggered, producing numerous primer replica in minutes, and greatly enhanced fluorescence of SYBR Green I. The proposed strategy has a good linear relationship with the logarithm of the MUC1 concentration ranging from 3 pM to 3 nM with a limit of detection of 1.63 pM (S/N = 3), which is two orders of magnitude lower than those of other methods. Owing to the specificity of immuno-reaction and EXPAR, the selectivity of the strategy is favorable, even if for the homologous protein. The proposed strategy was further applied for the MUC1 determination in human serum, and a satisfactory recovery range of 98.7%-105.3% was obtained. The strategy can be facilely extended to the ultrasensitive determination of various proteins.


Assuntos
Biomarcadores Tumorais/sangue , Imunoensaio/métodos , Mucina-1/sangue , Técnicas de Amplificação de Ácido Nucleico/métodos , Anticorpos Monoclonais Murinos/imunologia , Sequência de Bases , Biomarcadores Tumorais/imunologia , DNA/química , DNA Polimerase Dirigida por DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Corantes Fluorescentes/química , Geobacillus stearothermophilus/enzimologia , Humanos , Limite de Detecção , Mucina-1/imunologia , Compostos Orgânicos/química , Espectrometria de Fluorescência/métodos , Thermococcus/enzimologia
10.
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.


Assuntos
Amilases/química , Proteínas de Bactérias/química , Geobacillus stearothermophilus/enzimologia , Modelos Moleculares , Conformação Proteica , Sequência de Aminoácidos , Amilases/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Ligação de Hidrogênio , Hidrólise , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Oligossacarídeos/biossíntese , Oligossacarídeos/química , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
11.
Nucleic Acids Res ; 47(13): 6973-6983, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31170294

RESUMO

Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2'-deoxy-2'-fluoro-ß-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.


Assuntos
Proteínas de Bactérias/química , DNA Polimerase I/química , Geobacillus stearothermophilus/enzimologia , DNA Polimerase Dirigida por RNA/química , Arabinonucleotídeos/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , DNA Polimerase I/isolamento & purificação , DNA Polimerase I/metabolismo , DNA Bacteriano/metabolismo , Modelos Moleculares , Hibridização de Ácido Nucleico , Nucleosídeos/metabolismo , Ligação Proteica , Conformação Proteica , DNA Polimerase Dirigida por RNA/isolamento & purificação , DNA Polimerase Dirigida por RNA/metabolismo , Relação Estrutura-Atividade , Moldes Genéticos
12.
Food Chem ; 295: 110-119, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174739

RESUMO

Amylose (AM) and amylopectin (AP) functionality during bread making was unravelled with a temperature-controlled time domain proton nuclear magnetic resonance (TD 1H NMR) toolbox. Fermented doughs from wheat flour containing starches with atypical AP chain length distribution and/or AM to AP ratio, or supplemented with Bacillus stearothermophilus α-amylase (BStA) were analyzed in situ during baking and cooling. The gelatinization temperature of starch logically depended on AP crystal stability. It was lower when starch contained a higher portion of short AP branches and higher when starch had higher AP content. During cooling, the onset temperature and extent of AM crystallization were positively related to starch AM content. BStA use resulted in slightly weakened starch networks and increased the starch polymers' mobility at the end of baking. That proton distributions evolved in a way corresponding to starch characteristics supports the suggested interpretation of NMR profiles during baking and cooling.


Assuntos
Amilopectina/química , Amilose/química , Farinha/análise , Triticum/metabolismo , Amilases/metabolismo , Pão/análise , Culinária/métodos , Geobacillus stearothermophilus/enzimologia , Espectroscopia de Prótons por Ressonância Magnética , Amido/química , Temperatura
13.
ACS Sens ; 4(6): 1641-1647, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31188576

RESUMO

Enzymatically induced silver deposition and subsequent electrochemical oxidation have been widely used in electrochemical biosensors. However, this method is ineffective for producing highly enhanced silver deposition for use in ultrasensitive detection. Herein, we report a fast silver deposition method that simultaneously uses three signal amplification processes: (i) enzymatic amplification, (ii) chemical-chemical (CC) redox cycling, and (iii) chemical-enzymatic (CN) redox cycling. DT-diaphorase (DT-D) is used for enzymatic amplification to convert a nitroso compound, a species incapable of directly reducing Ag+ to an amine compound, which can directly reduce Ag+. NADH acts as a reducing agent for the indirect reduction of Ag+ via the two redox cycling processes. 4-Nitroso-1-naphthol is converted to 4-amino-1-naphthol (NH2-N) in the presence of DT-D. NH2-N initiates two redox cycling processes: NH2-N, along with Ag+ and NADH, are involved in the CC redox cycling, whereas NH2-N, along with Ag+, DT-D, and NADH, are involved in the CN redox cycling. Finally, the deposited silver is electrochemically oxidized to produce a signal. When this triple signal amplification strategy for fast silver deposition is applied to an electrochemical immunosensor for detecting parathyroid hormone (PTH), a detection limit as low as ∼100 fg/mL is obtained. The concentrations of PTH in clinical serum determined using the developed immunosensor are found to agree with those measured using a commercial instrument. Thus, the use of this strategy for fast silver deposition is highly promising for ultrasensitive electrochemical detection and biosensing applications.


Assuntos
Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , Compostos Nitrosos/química , Hormônio Paratireóideo/sangue , Prata/química , 1-Naftilamina/análogos & derivados , Anticorpos Imobilizados/imunologia , Proteínas de Bactérias/química , Geobacillus stearothermophilus/enzimologia , Humanos , Limite de Detecção , NAD(P)H Desidrogenase (Quinona)/química , Oxirredução , Hormônio Paratireóideo/imunologia
14.
J Agric Food Chem ; 67(17): 4868-4875, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30995033

RESUMO

The rare sugar l-ribulose is produced from the abundant sugar l-arabinose by enzymatic conversion. An l-arabinose isomerase (AI) from Geobacillus thermodenitrificans was efficiently expressed and encapsulated in Saccharomyces cerevisiae spores. Deletion of the yeast OSW2 gene, which causes a mild defect in the integrity of the spore wall, substantially improved the activity of encapsulated AI, without damaging its superior enzymatic properties of thermostability, pH tolerance,and resistance toward SDS and proteinase treatments. In a 10 mL reaction, 100 mg of dry AI encapsulated in spores produced 250 mg of l-ribulose from 1 g of l-arabinose, indicating a 25% conversion rate. Notably, the product of l-ribulose was directly purified from the reaction solution with an approximately 91% recovery using a Ca2+ ion exchange column. Our results describe not only a facile approach for the production of l-ribulose but also a useful strategy for the enzymatic conversion of rare sugars in "Izumoring".


Assuntos
Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Geobacillus stearothermophilus/enzimologia , Pentoses/metabolismo , Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos/metabolismo , Aldose-Cetose Isomerases/genética , Arabinose/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Saccharomyces cerevisiae/genética , Esporos Fúngicos/genética
15.
Microb Cell Fact ; 18(1): 69, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971250

RESUMO

BACKGROUND: Our laboratory has constructed a Bacillus stearothermophilus α-amylase (AmyS) derivative with excellent enzymatic properties. Bacillus subtilis is generally regarded as safe and has excellent protein secretory capability, but heterologous extracellular production level of B. stearothermophilus α-amylase in B. subtilis is very low. RESULTS: In this study, the extracellular production level of B. stearothermophilus α-amylase in B. subtilis was enhanced by signal peptide optimization, chaperone overexpression and α-amylase mutant selection. The α-amylase optimal signal peptide (SPYojL) was obtained by screening 173 B. subtilis signal peptides. Although the extracellular α-amylase activity that was produced by the resulting recombinant strain was 3.5-fold greater than that of the control, significant quantities of inclusion bodies were detected. Overexpressing intracellular molecular chaperones significantly reduced inclusion body formation and further increased α-amylase activity. Error-prone PCR produced an amylase mutant K82E/S405R (AmySA) with enzymatic activity superior to that of AmyS. Expression of the amySA gene with the SPYojL while overexpressing molecular chaperones resulted in a 7.1-fold improvement in α-amylase activity. When the final expression strain (WHS11YSA) was cultivated in a 3-L fermenter for 92 h, the α-amylase activity of the culture supernatant was 9201.1 U mL-1, which is the highest level that has been reported to date. CONCLUSIONS: This is the first report that describes an improvement of B. stearothermophilus α-amylase extracellular production levels in B. subtilis using these strategies, and this represents the highest extracellular production level ever reported for α-amylase from B. stearothermophilus in B. subtilis. This high-level production provides a basis for enhanced industrial production of α-amylase. These extracellular production level improvement approaches are also expected to be valuable in the expression of other enzymes in B. subtilis.


Assuntos
Bacillus subtilis/genética , Geobacillus stearothermophilus/enzimologia , Chaperonas Moleculares/genética , Sinais Direcionadores de Proteínas , alfa-Amilases/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Fermentação , Expressão Gênica , Geobacillus stearothermophilus/genética , Microbiologia Industrial , Chaperonas Moleculares/metabolismo , Mutação , Regiões Promotoras Genéticas , alfa-Amilases/metabolismo
16.
Analyst ; 144(9): 3064-3071, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30916676

RESUMO

DNA glycosylase (DG) plays a significant role in repairing DNA lesions, and the dysregulation of DG activity is associated with a variety of human pathologies. Thus, the detection of DG activity is essential for biomedical research and clinical diagnosis. Herein, we develop a facile fluorometric method based on the base excision repair (BER) mediated cascading triple-signal amplification for the sensitive detection of DG. The presence of human alkyladenine DNA glycosylase (hAAG) can initiate the cleavage of the substrate at the mismatched deoxyinosine site by endonuclease IV (Endo IV), resulting in the breaking of the DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate strand displacement amplification (SDA) to release primers. The released primers can further bind to a circular template to induce an exponential primer generation rolling circle amplification (PG-RCA) reaction, producing a large number of primers. The primers that resulted from the SDA and PG-RCA reaction can induce the subsequent recycling cleavage of signal probes, leading to the generation of a fluorescence signal. Taking advantage of the high amplification efficiency of triple-signal amplification and the low background signal resulting from single uracil repair-mediated inhibition of nonspecific amplification, this method exhibits a low detection limit of 0.026 U mL-1 and a large dynamic range of 4 orders of magnitude for hAAG. Moreover, this method has distinct advantages of simplicity and low cost, and it can further quantify the hAAG activity from HeLa cell extracts, holding great potential in clinical diagnosis and biomedical research.


Assuntos
DNA Glicosilases/sangue , Reparo do DNA , DNA/química , Ensaios Enzimáticos/métodos , Fluorometria/métodos , Sequência de Bases , DNA Polimerase Dirigida por DNA/química , Desoxirribonuclease IV (Fago T4-Induzido)/química , Fluorescência , Corantes Fluorescentes/química , Geobacillus stearothermophilus/enzimologia , Células HeLa , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Uracila-DNA Glicosidase/química
17.
Nucleic Acids Res ; 47(8): 4136-4152, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30892613

RESUMO

The UvrA2 dimer finds lesions in DNA and initiates nucleotide excision repair. Each UvrA monomer contains two essential ATPase sites: proximal (P) and distal (D). The manner whereby their activities enable UvrA2 damage sensing and response remains to be clarified. We report three key findings from the first pre-steady state kinetic analysis of each site. Absent DNA, a P2ATP-D2ADP species accumulates when the low-affinity proximal sites bind ATP and enable rapid ATP hydrolysis and phosphate release by the high-affinity distal sites, and ADP release limits catalytic turnover. Native DNA stimulates ATP hydrolysis by all four sites, causing UvrA2 to transition through a different species, P2ADP-D2ADP. Lesion-containing DNA changes the mechanism again, suppressing ATP hydrolysis by the proximal sites while distal sites cycle through hydrolysis and ADP release, to populate proximal ATP-bound species, P2ATP-Dempty and P2ATP-D2ATP. Thus, damaged and native DNA trigger distinct ATPase site activities, which could explain why UvrA2 forms stable complexes with UvrB on damaged DNA compared with weaker, more dynamic complexes on native DNA. Such specific coupling between the DNA substrate and the ATPase mechanism of each site provides new insights into how UvrA2 utilizes ATP for lesion search, recognition and repair.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Proteínas de Bactérias/química , Reparo do DNA , DNA Bacteriano/química , Endodesoxirribonucleases/química , Proteínas de Escherichia coli/química , Geobacillus stearothermophilus/enzimologia , ortoaminobenzoatos/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Dano ao DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/genética , Cinética , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica , Thermotoga maritima/química , Thermotoga maritima/enzimologia , Thermotoga maritima/genética , ortoaminobenzoatos/metabolismo
18.
Biochemistry ; 58(12): 1579-1586, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30793594

RESUMO

The aminoglycoside nucleotidyltransferase(4') is an enzyme with high substrate promiscuity and catalyzes the transfer of the AMP group from ATP to the 4'-OH site of many structurally diverse aminoglycosides, which results in the elimination of their effectiveness as antibiotics. Two thermostable variants carrying single-site mutations are used to determine the molecular properties associated with thermophilicity. The thermodynamics of enzyme-ligand interactions showed that one variant (T130K) has properties identical to those of the mesophilic wild type (WT) while the other (D80Y) behaved differently. Differences between D80Y and the T130K/WT pair include the change in heat capacity (Δ Cp), which is dependent on temperature for D80Y but not for WT or T130K. The change in Δ Cp with temperature (ΔΔ Cp) with D80Y is dependent on aminoglycoside only in H2O and remains the same with all aminoglycosides in D2O. Furthermore, the offset temperature ( Toff), the temperature difference that yields identical enthalpies in H2O and D2O, becomes larger with an increase in temperature for WT and T130K but remains mostly unchanged for D80Y. Studies in H2O and D2O revealed that solvent reorganization becomes the major contributor to ligand binding with an increase in temperature for WT and T130K, but changes in low-frequency vibrational modes are the main contributors with D80Y. Data presented in this paper suggest that global properties associated with the enzyme-ligand interactions, such as the thermodynamics of ligand binding, may yield clues about thermophilicity and permit us to distinguish those variants that are simply a more thermostable version of the mesophilic protein.


Assuntos
Aminoglicosídeos/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Nucleotidiltransferases/metabolismo , Cianobactérias/enzimologia , Escherichia coli/genética , Geobacillus stearothermophilus/enzimologia , Ligantes , Ligação Proteica , Isoformas de Proteínas/metabolismo , Staphylococcus aureus/enzimologia , Temperatura , Termodinâmica
19.
Anal Biochem ; 570: 27-31, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30738758

RESUMO

Previously, we developed a kinase cycling method using creatine kinase and pyruvate kinase (RMPK) both from rabbit muscle in the presence of an excess amount of ATP and IDP for the quantitative determination of substrate. To our surprise, the RMPK cycling reaction was 10-fold more efficient using Mn2+ rather than Mg2+. Here, we investigated PK from Geobacillus stearothermophilus (GSPK) as an alternative source of enzyme. Spectrophotometric real-time detection was accomplished by coupling the reaction to ADP-dependent glucokinase (ADP-GK) together with glucose-6-phosphate dehydrogenase (G6PD). The rate of increase in absorbance of NADH at 340 nm was monitored. GSPK displayed an even greater preference than RMPK for Mn2+ over Mg2+ in the cycling reaction with ATP and GDP or ATP and IDP. The much lower Km values for the substrate in the presence of Mn2+ rather than Mg2+ are consistent with the results of the cycling reaction observed in this study.


Assuntos
Proteínas de Bactérias/metabolismo , Geobacillus stearothermophilus/enzimologia , Manganês/metabolismo , Piruvato Quinase/metabolismo , Trifosfato de Adenosina/metabolismo , Íons/química , Cinética , Magnésio/química , Magnésio/metabolismo , Manganês/química , Especificidade por Substrato
20.
FEBS Open Bio ; 9(2): 384-395, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30761262

RESUMO

Cyclodextrin glucanotransferases (CGTases) convert α-1,4-glucans to cyclic oligosaccharides (cyclodextrins, CD), which have found applications in the food and the pharmaceutical industries. In this study, we used two CGTases with different cyclization activities, product specificities, and pH and temperature optima to construct chimeric variants for the synthesis of large-ring CD. We used (a) a synthetic thermostable CGTase mainly forming α- and ß-CD (CD6 and CD7) derived from Geobacillus stearothermophilus ET1/NO2 (GeoT), and (b) a CGTase with lower cyclization activity from the alkaliphilic Bacillus sp. G825-6, which mainly synthesizes γ-CD (CD8). The A1, B, A2, and CDE domains of the G825-6 CGTase were replaced with corresponding GeoT CGTase domains by utilizing a megaprimer cloning approach. A comparison of the optimum temperature and pH, thermal stability, and CD products synthesized by the variants revealed that the B domain had a major impact on the cyclization activity, thermal stability, and product specificity of the constructed chimera. Complete suppression of the synthesis of CD6 was observed with the variants GeoT-A1/B and GeoT-A1/A2/CDE. The variant GeoT-A1/A2/CDE showed the desired enzyme properties for large-ring CD synthesis. Its melting temperature was 9 °C higher compared to the G825-6 CGTase and it synthesized up to 3.3 g·L-1 CD9 to CD12, corresponding to a 1.8- and 2.3-fold increase compared to GeoT and G825-6 CGTase, respectively. In conclusion, GeoT-A1/A2/CDE may be a candidate for the further development of CGTases specifically forming larger CD.


Assuntos
Ciclodextrinas/biossíntese , Glucosiltransferases/metabolismo , Temperatura , Bacillus/enzimologia , Ciclização , Ciclodextrinas/química , Estabilidade Enzimática , Geobacillus stearothermophilus/enzimologia , Concentração de Íons de Hidrogênio , Especificidade da Espécie
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA