Influences of Various Peptide Linkers on the Thermotoga maritima MSB8 Nitrilase Displayed on the Spore Surface of Bacillus subtilis

2017 ◽  
Vol 27 (1) ◽  
pp. 64-71 ◽  
Author(s):  
Huayou Chen ◽  
Zhi Chen ◽  
Bangguo Wu ◽  
Jawad Ullah ◽  
Tianxi Zhang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Fusion Proteins ◽  
Rational Design ◽  
Thermotoga Maritima ◽  
Sodium Dodecyl ◽  
Ph Stability ◽  
Activity Measurement ◽  
Spore Surface ◽  
Outer Coat ◽  
The Stability

In the present study, fusion genes composed of <i>Thermotoga maritima</i> MSB8 nitrilase and <i>Bacillus subtilis</i> 168 outer coat protein CotG were constructed with various peptide linkers and displayed on <i>B. subtilis</i> DB 403 spores. The successful display of CotG-nit fusion proteins on the spore surface of <i>B. subtilis</i> was verified by Western blot analysis and activity measurement. It was demonstrated that the fusion with linker GGGGSEAAAKGGGGS presented the highest thermal and pH stability, which is 2.67- and 1.9-fold of the fusion without linker. In addition, fusion with flexible linker (GGGGS)<sub>3</sub> demonstrated better thermal and pH stability than fusions with linkers GGGGS and (GGGGS)<sub>2</sub>. Fusion with rigid linker (EAAAK) demonstrated better thermal stability than fusions with linkers (EAAAK)<sub>2</sub> and (EAAAK)<sub>3</sub>. Fusions with linker (EAAAK)<sub>2</sub> demonstrated better pH stability than fusions with linkers (EAAAK) and (EAAAK)<sub>3</sub>. In the presence of 1 m<smlcap>M</smlcap> dithiothreitol, 1% (v/v) sodium dodecyl sulfate, and 20% (v/v) ethanol, the optimal linkers of the fusions were MGSSSN, GGGGSEAAAKGGGGS, and (GGGGS)<sub>3</sub>, respectively. In summary, our results showed that optimizing the peptide linkers with different type, length, and amino acid composition of the fusion proteins would be an efficient way to maintain the stability of fusion proteins and thus improve the nitrilase display efficiency, which could provide an effective method for rational design peptide linkers of displayed nitrilase on <i>B. subtilis</i>.

scholarly journals Display of Recombinant Proteins on Bacillus subtilis Spores, Using a Coat-Associated Enzyme as the Carrier

2010 ◽  
Vol 76 (17) ◽  
pp. 5926-5933 ◽  
Author(s):  
Sébastien Potot ◽  
Cláudia R. Serra ◽  
Adriano O. Henriques ◽  
Ghislain Schyns
Keyword(s):  
Bacillus Subtilis ◽  
Coat Protein ◽  
Recombinant Proteins ◽  
Animal Feed ◽  
Active Form ◽  
Bioactive Molecules ◽  
Full Potential ◽  
Spore Surface ◽  
Outer Coat ◽  
Feed Enzymes

ABSTRACT The display of proteins such as feed enzymes at the surface of bacterial spore systems has a great potential use for animal feed. Feed enzymes increase the digestibility of nutrients, leading to greater efficiency in the manufacturing of animal products and minimizing the environmental impact of increased animal production. To deliver their full potential in the gut, feed enzymes must survive the harsh conditions of the feed preparation and the gastrointestinal tract. The well-documented resistance of spores to harsh environments, together with the ability to use proteins that compose the spore as carriers for the display of passenger proteins, suggests that spores could be used as innovative tools to improve the formulation of bioactive molecules. Although some successful examples have been reported, in which abundant structural proteins of the Bacillus subtilis spore outer-coat layer were used as carriers for the display of recombinant proteins, only one convincing example resulted in the display of functional enzymes. In addition, no examples are available about the use of an inner-coat protein for the display of an active passenger enzyme. In our study, we show that the inner-coat oxalate decarboxylase (OxdD) can expose an endogenous phytase, a commonly used feed enzyme for monogastric animals, in an active form at the spore surface. Importantly, despite the higher abundance of CotG outer-coat protein, an OxdD-Phy fusion was more represented at the spore surface. The potential of OxdD as a carrier protein is further documented through the spore display of a bioactive heterologous passenger, the tetrameric β-glucuronidase enzyme from Escherichia coli.

scholarly journals Dispersion of Carbon Nanotubes with “Green” Detergents

Molecules ◽  
2021 ◽  
Vol 26 (10) ◽  
pp. 2908
Author(s):  
Kazuo Umemura ◽  
Ryo Hamano ◽  
Hiroaki Komatsu ◽  
Takashi Ikuno ◽  
Eko Siswoyo
Keyword(s):  
Carbon Nanotubes ◽  
Sodium Dodecyl ◽  
Detergent Solution ◽  
Absorbance Spectroscopy ◽  
Fundamental Research ◽  
The Stability ◽  
Single Walled Cnts ◽  
Better Than ◽  
Walled Cnts ◽  
Cnt Suspensions

Solubilization of carbon nanotubes (CNTs) is a fundamental technique for the use of CNTs and their conjugates as nanodevices and nanobiodevices. In this work, we demonstrate the preparation of CNT suspensions with “green” detergents made from coconuts and bamboo as fundamental research in CNT nanotechnology. Single-walled CNTs (SWNTs) with a few carboxylic acid groups (3–5%) and pristine multi-walled CNTs (MWNTs) were mixed in each detergent solution and sonicated with a bath-type sonicator. The prepared suspensions were characterized using absorbance spectroscopy, scanning electron microscopy, and Raman spectroscopy. Among the eight combinations of CNTs and detergents (two types of CNTs and four detergents, including sodium dodecyl sulfate (SDS) as the standard), SWNTs/MWNTs were well dispersed in all combinations except the combination of the MWNTs and the bamboo detergent. The stability of the suspensions prepared with coconut detergents was better than that prepared with SDS. Because the efficiency of the bamboo detergents against the MWNTs differed significantly from that against the SWNTs, the natural detergent might be useful for separating CNTs. Our results revealed that the use of the “green” detergents had the advantage of dispersing CNTs as well as SDS.

scholarly journals Analysis of some pharmaceuticals in the presence of their synthetic impurities by applying hybrid micelle liquid chromatography

2020 ◽  
Vol 18 (1) ◽  
pp. 377-390
Author(s):  
Dina El Sherbiny ◽  
Mary E. K. Wahba
Keyword(s):  
Liquid Chromatography ◽  
Sodium Dodecyl Sulfate ◽  
Phosphoric Acid ◽  
Mobile Phase ◽  
Sodium Dodecyl ◽  
Uv Detection ◽  
Stability Indicating ◽  
Photolytic Degradation ◽  
Amino Phenol ◽  
The Stability

AbstractA stability-indicating hybrid micelle liquid chromatography accompanied by UV detection was developed for the simultaneous analysis of either paracetamol (PCA) or pseudoephedrine hydrochloride (PSU) with their synthetic impurities. Mixture I contains PCA with p-amino phenol and p-nitro phenol, while mixture II involves the estimation of PSU with benzaldehyde and benzoic acid. Both mixtures were separated using a C18 column that was thermostatically maintained at 40°C and operating under a flow rate of 1.5 mL/min, applying UV detection at 240 nm for mixture I and 220 nm for mixture II. In both cases, the mobile phase consisted of 0.1 M sodium dodecyl sulfate, acetonitrile, and triethylamine (90:10:0.3, v/v/v) and adjusted to pH 4 (mixture I) or pH 3.7 (mixture II) using 2.0 M O-phosphoric acid. The proposed method was validated and successfully applied to assay different pharmaceuticals containing PCA or PSU. Moreover, the stability-indicating nature of the proposed method was proved through applying photolytic degradation procedures for PCA.

Rational Design of the N-Terminal Coding Sequence for Regulating Enzyme Expression in Bacillus subtilis

2021 ◽  
Vol 10 (2) ◽  
pp. 265-276
Author(s):  
Kuidong Xu ◽  
Yi Tong ◽  
Yi Li ◽  
Jin Tao ◽  
Jianghua Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rational Design ◽  
Enzyme Expression ◽  
Coding Sequence

scholarly journals Application of Lipid Class Ratios for Sample Stability Monitoring—Evaluation of Murine Tissue Homogenates and SDS as a Stabilizer

Metabolites ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 277
Author(s):  
Sabrina Krautbauer ◽  
Raquel Blazquez ◽  
Gerhard Liebisch ◽  
Marcus Hoering ◽  
Philip Neubert ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lipid Class ◽  
High Resolution Mass Spectrometry ◽  
Lipolytic Activity ◽  
Sodium Dodecyl ◽  
Lipid Profiles ◽  
Lipolytic Action ◽  
Sample Stability ◽  
Tissue Homogenates ◽  
The Stability

Lipids are a ubiquitous class of structurally complex molecules involved in various biological processes. In the fast-growing field of lipidomics, preanalytical issues are frequently neglected. Here, we investigated the stability of lipid profiles of murine liver, brain, lung, heart, and spleen homogenates by quantitative flow injection analysis using tandem mass spectrometry and high-resolution mass spectrometry. Storage of tissue homogenates at room temperature showed substantial alterations of the lipid profiles reflecting lipolytic action. Therefore, ratios of ceramide to sphingomyelin, lysophosphatidylethanolamine to phosphatidylethanolamine, lysophosphatidylcholine to phosphatidylcholine, and diglyceride to triglyceride were applied to monitor sample stability and the effect of sodium dodecyl sulfate (SDS) as a potential stabilizing agent. The addition of SDS led to a concentration-dependent stabilization of lipid profiles in liver, brain, and heart homogenates, while in lung and spleen homogenates, in particular, the lysophosphatidylethanolamine to phosphatidylethanolamine ratio increased upon addition of SDS. In conclusion, we demonstrated that lipid class ratios reflecting lipolytic activity could be applied to evaluate both the stability of samples and the influence of stabilizers.

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