scholarly journals Effects of the 20-Kilodalton Helper Protein on Cry1Ac Production and Spore Formation in Bacillus thuringiensis

2001 ◽  
Vol 67 (12) ◽  
pp. 5362-5369 ◽  
Author(s):  
Zongze Shao ◽  
Ziduo Liu ◽  
Ziniu Yu
Keyword(s):  
Bacillus Thuringiensis ◽  
Recombinant Strain ◽  
Protein Production ◽  
Heliothis Armigera ◽  
High Yield ◽  
Efficient Production ◽  
Crystal Proteins ◽  
Enhanced Production ◽  
Molecular Chaperon

ABSTRACT Bacillus thuringiensis produces large amounts of various pesticidal proteins during the stationary phase. In order to achieve a high yield and form crystals, some pesticidal proteins require the presence of other proteins. Helper protein P20 is required for efficient production of both the Cyt1A and Cry11A crystal proteins in B. thuringiensis subsp. israelensis. Although full-length Cry1 protoxins are usually independent in terms of expression and crystallization in B. thuringiensis, in this study P20 significantly enhanced production of Cry1Ac protoxin (133 kDa) in an acrystalliferous and plasmid-negative strain. In the presence of P20, the yield of Cry1Ac protoxin increased 2.5-fold, and on average the resulting crystals were 1.85 μm long and 0.85 μm wide, three times the size of the crystals formed in the control lacking P20. Correspondingly, the recombinant strain that coexpressed P20 and Cry1Ac exhibited higher toxicity against Heliothis armigera larvae than the control. Furthermore, serious degradation of Cry1Ac in vivo was observed, which has seldom been reported previously. Actually, most protein was completely degraded during synthesis, and after synthesis about one-third of the expressed protoxins were degraded further before crystallization. In this process, P20 protected only nascent Cry1Ac from degradation, indicating that it acted as a molecular chaperon. In addition, spores were smaller and rounder and had a thinner exosporium layer when they were produced in the presence of P20. In summary, Cry1Ac was severely degraded during synthesis; this degradation was effectively relieved by P20, which resulted in enhanced production. Our results indicated that P20 is an effective tool for optimizing protein production in vivo.

scholarly journals Identification and Characterization of Three Previously Undescribed Crystal Proteins from Bacillus thuringiensis subsp.jegathesan

2013 ◽  
Vol 79 (11) ◽  
pp. 3364-3370 ◽  
Author(s):  
Yunjun Sun ◽  
Qiang Zhao ◽  
Liqiu Xia ◽  
Xuezhi Ding ◽  
Quanfang Hu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Bacillus Thuringiensis ◽  
Recombinant Strain ◽  
Fourth Instar ◽  
Gene Promoters ◽  
Content Type ◽  
Crystal Proteins ◽  
Encoding Genes ◽  
Identification And Characterization

ABSTRACTThe total protoxin complement in the parasporal body of mosquitocidal strain,Bacillus thuringiensissubsp.jegathesan367, was determined by use of a polyacrylamide gel block coupled to mass spectrometry. A total of eight protoxins were identified from this strain, including five reported protoxins (Cry11Ba, Cry19Aa, Cry24Aa, Cry25Aa, and Cyt2Bb), as well as three previously undescribed (Cry30Ca, Cry60Aa, and Cry60Ba) in this isolate. It was interesting that the encoding genes of three new protoxins existed ascry30Ca-gap-orf2andcry60Ba-gap-cry60Aa. Thecry30Caand a downstreamorf2gene were oriented in the same direction and separated by 114 bp, andcry60Bawas located 156 bp upstream from and in the same orientation tocry60Aa. The three new protoxin genes were cloned fromB. thuringiensissubsp.jegathesanand expressed in an acrystalliferous strain under the control ofcyt1Agene promoters and the STAB-SD stabilizer sequence. Recombinant strain containing onlycry30Cadid not produce visible inclusion under microscope observation, while that containing bothcry30Caandorf2could produce large inclusions. Cry60Aa and Cry60Ba synthesized either alone or together in the acrystalliferous host could yield large inclusions. In bioassays using the fourth-instar larvae ofCulex quinquefasciatus, Cry60Aa and Cry60Ba alone or together had estimated 50% lethal concentrations of 2.9 to 7.9 μg/ml; however, Cry30Ca with or without ORF2 was not toxic to this mosquito.

scholarly journals Development of a Bioprocess for the Production of Cyclic Lipopeptides Pseudofactins With Efficient Purification From Collected Foam

2020 ◽  
Vol 8 ◽  
Author(s):  
Piotr Biniarz ◽  
Marius Henkel ◽  
Rudolf Hausmann ◽  
Marcin Łukaszewicz
Keyword(s):  
High Performance ◽  
Single Step ◽  
High Yield ◽  
Efficient Production ◽  
Cyclic Lipopeptides ◽  
Enhanced Production ◽  
Working Volume ◽  
New Process ◽  
Co Precipitation ◽  
Surface Active Compounds

Microbial surfactants (biosurfactants) have gained interest as promising substitutes of synthetic surface-active compounds. However, their production and purification are still challenging, with significant room for efficiency and costs optimization. In this work, we introduce a method for the enhanced production and purification of cyclic lipopeptides pseudofactins (PFs) from Pseudomonas fluorescens BD5 cultures. The method is directly applicable in a technical scale with the possibility of further upscaling. Comparing to the original protocol for production of PFs (cultures in mineral salt medium in shaken flasks followed by solvent-solvent extraction of PFs), our process offers not only ∼24-fold increased productivity, but also easier and more efficient purification. The new process combines high yield of PFs (∼7.2 grams of PFs per 30 L of working volume), with recovery levels of 80–90% and purity of raw PFs up to 60–70%. These were achieved with an innovative, single-step thermal co-precipitation and extraction of PFs directly from collected foam, as a large amount of PF-enriched foam was produced during the bioprocess. Besides we present a protocol for the selective production of PF structural analogs and their separation with high-performance liquid chromatography. Our approach can be potentially utilized in the efficient production and purification of other lipopeptides of Pseudomonas and Bacillus origin.

scholarly journals Receptors and Lethal Effect of Bacillus thuringiensis Insecticidal Crystal Proteins to the Anticarsia gemmatalis (Lepidoptera, Noctuidae)

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Lidia Mariana Fiuza ◽  
Neiva Knaak ◽  
Rogério Fernando Pires da Silva ◽  
João Antônio Pêgas Henriques
Keyword(s):  
Bacillus Thuringiensis ◽  
Lethal Effect ◽  
Anticarsia Gemmatalis ◽  
Binding Studies ◽  
Crystal Proteins ◽  
Binding Data ◽  
Lepidoptera Noctuidae ◽  
Insecticidal Crystal Proteins

Bioassays with insecticidal crystal proteins (ICPs) from Bacillus thuringiensis have demonstrated that Cry1Aa, Cry1Ac, and Cry1Ba are the most active toxins on larvae of the Anticarsia gemmatalis. The toxins Cry1Da and Cry1Ea are less toxic, and toxins Cry2Aa are not active. Binding of these ICPs to midgut sections of the A. gemmatalis larvae was studied using streptavidin-mediated detection. The observed staining patterns showed that Cry1Aa and Cry1Ac bound to the brush border throughout the whole length of the midgut. However, the binding sites of Cry1Ba were not evenly distributed in the midgut microvilli. The in vivo assays against larvae of 2nd instar A. gemmatalis confirmed the results from the in vitro binding studies. These binding data correspond well with the bioassay results, demonstrating a correlation between receptors binding and toxicity of the tested ICPs in this insect.

scholarly journals Production of Cry11A and Cry11Ba Toxins in Bacillus sphaericus Confers Toxicity towards Aedes aegyptiand Resistant Culex Populations

1999 ◽  
Vol 65 (7) ◽  
pp. 3021-3026 ◽  
Author(s):  
Pascale Servant ◽  
Marie-Laure Rosso ◽  
Sylviane Hamon ◽  
Sandrine Poncet ◽  
Armelle Delécluse ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Culex Pipiens ◽  
Recombinant Strain ◽  
Bacillus Sphaericus ◽  
Binary Toxin ◽  
Protease Gene ◽  
Gene Encoding ◽  
In Vivo Recombination ◽  
Target Sites

ABSTRACT Cry11A from Bacillus thuringiensis subsp.israelensis and Cry11Ba from Bacillus thuringiensis subsp. jegathesan were introduced, separately and in combination, into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Two loci on theB. sphaericus chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against Aedes aegyptiand Culex pipiens. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to A. aegyptilarvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible C. pipiens larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of C. pipiens SPHAE andCulex quinquefasciatus GeoR to B. sphaericus strain 2297.

In silico and in vivo study of radio-iodinated nefiracetam as a radiotracer for brain imaging in mice

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
M. H. Sanad ◽  
A. B. Farag ◽  
S. F. A. Rizvi
Keyword(s):  
Brain Imaging ◽  
Radiochemical Purity ◽  
High Yield ◽  
High Uptake ◽  
Oxidizing Agent ◽  
Receptor Ligand ◽  
Nootropic Agent ◽  
Biodistribution Studies

Abstract This study presents development and characterization of a radiotracer, [125I]iodonefiracetam ([125I]iodoNEF). Labeling with high yield and radiochemical purity was achieved through the formation of a [125I]iodoNEF radiotracer after investigating many factors like oxidizing agent content (chloramines-T (Ch-T)), substrate amount (Nefiracetam (NEF)), pH of reaction mixture, reaction time and temperature. Nefiracetam (NEF) is known as nootropic agent, acting as N-methyl-d-aspartic acid receptor ligand (NMDA). The radiolabeled compound was stable, and exhibited the logarithm of the partition coefficient (log p) value of [125I]iodonefiracetam as 1.85 (lipophilic). Biodistribution studies in normal mice confirmed the suitability of the [125I]iodoNEF radiotracer as a novel tracer for brain imaging. High uptake of 8.61 ± 0.14 percent injected dose/g organ was observed in mice

scholarly journals Additive-Enhanced Exfoliation for High-Yield 2D Materials Production

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 601
Author(s):  
Dinh-Tuan Nguyen ◽  
Hsiang-An Ting ◽  
Yen-Hsun Su ◽  
Mario Hofmann ◽  
Ya-Ping Hsieh
Keyword(s):  
Large Scale ◽  
2D Materials ◽  
Transition Metal Dichalcogenides ◽  
Spectroscopic Characterization ◽  
High Yield ◽  
Nanometer Scale ◽  
Efficient Production ◽  
Device Performance ◽  
Metal Dichalcogenides ◽  
Scale Deposition

The success of van-der-Waals electronics, which combine large-scale-deposition capabilities with high device performance, relies on the efficient production of suitable 2D materials. Shear exfoliation of 2D materials’ flakes from bulk sources can generate 2D materials with low amounts of defects, but the production yield has been limited below industry requirements. Here, we introduce additive-assisted exfoliation (AAE) as an approach to significantly increase the efficiency of shear exfoliation and produce an exfoliation yield of 30%. By introducing micrometer-sized particles that do not exfoliate, the gap between rotor and stator was dynamically reduced to increase the achievable shear rate. This enhancement was applied to WS2 and MoS2 production, which represent two of the most promising 2D transition-metal dichalcogenides. Spectroscopic characterization and cascade centrifugation reveal a consistent and significant increase in 2D material concentrations across all thickness ranges. Thus, the produced WS2 films exhibit high thickness uniformity in the nanometer-scale and can open up new routes for 2D materials production towards future applications.

scholarly journals A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Enantiomeric Excess ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

AbstractIn this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg−1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

scholarly journals Anticancer Compounds Derived from Marine Diatoms

Marine Drugs ◽  
2020 ◽  
Vol 18 (7) ◽  
pp. 356 ◽  
Author(s):  
Hanaa Ali Hussein ◽  
Mohd Azmuddin Abdullah
Keyword(s):  
Unsaturated Fatty Acids ◽  
High Surface ◽  
High Surface Area ◽  
Drug Carriers ◽  
Growth Cycle ◽  
In Vivo Studies ◽  
High Yield ◽  
Anti Cancer ◽  
Nano Medicine

Cancer is the main cause of death worldwide, so the discovery of new and effective therapeutic agents must be urgently addressed. Diatoms are rich in minerals and secondary metabolites such as saturated and unsaturated fatty acids, esters, acyl lipids, sterols, proteins, and flavonoids. These bioactive compounds have been reported as potent anti-cancer, anti-oxidant and anti-bacterial agents. Diatoms are unicellular photosynthetic organisms, which are important in the biogeochemical circulation of silica, nitrogen, and carbon, attributable to their short growth-cycle and high yield. The biosilica of diatoms is potentially effective as a carrier for targeted drug delivery in cancer therapy due to its high surface area, nano-porosity, bio-compatibility, and bio-degradability. In vivo studies have shown no significant symptoms of tissue damage in animal models, suggesting the suitability of a diatoms-based system as a safe nanocarrier in nano-medicine applications. This review presents an overview of diatoms’ microalgae possessing anti-cancer activities and the potential role of the diatoms and biosilica in the delivery of anticancer drugs. Diatoms-based antibodies and vitamin B12 as drug carriers are also elaborated.

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