Production of Squalene in Bacillus subtilis by Squalene Synthase Screening and Metabolic Engineering

Squalene synthase (SQS) catalyzes the conversion of two farnesyl pyrophosphates to squalene, an important intermediate in between isoprene and valuable triterpenoids. In this study, we have constructed a novel biosynthesis pathway for squalene in Bacillus subtilis and performed metabolic engineering aiming at facilitating further exploitation and production of squalene-derived triterpenoids. Therefore, systematic studies and analysis were performed including selection of multiple SQS candidates from various organisms, comparison of expression vectors, optimization of cultivation temperatures, and examination of rate-limiting factors within the synthetic pathway. We were, for the first time, able to obtain squalene synthesis in B. subtilis. Furthermore, we achieved a 29-fold increase of squalene yield (0.26-7.5 mg/L) by expressing SQS from Bacillus megaterium and eliminating bottlenecks within the upstream methylerythritol-phosphate pathway. Moreover, our findings showed that also ispA could positively affect the production of squalene

Further investigation of a peptide extraction method with mesoporous silica using high-performance liquid chromatography coupled with tandem mass spectrometry

Mobil Composition of Matter No. 41 (MCM-41) was the most frequently used mesoporous silica material to extract peptides from complex biological samples. However, there were confusing extraction conditions and large extraction efficiency variance among related reports, which resulted from unclear understanding about the interaction between the material and peptides. In this study, the extraction mechanism was investigated with one set of tryptic peptides by using high-performance liquid chromatography coupled with triple quadrupole mass spectrometry. Generally, hydrophobic interaction and electrostatic attraction were two major driving forces for extraction of peptides, while electrostatic repulsion greatly weakened the interaction between the material and peptides with isoelectric points below the pH. With most peptides positively charged and MCM-41 slightly negatively charged, most efficient extraction was obtained at pH 3, and it was proved that electrostatic and hydrophobic interaction acted in synergy for extraction of all the peptides. A mixed solution of acetonitrile with buffers of high pH or ion strength was demonstrated to be favorable for elution, which performed much better than the commonly used eluate (mixture of acetonitrile with 0.1% trifluoroacetic acid). Finally, under optimum conditions, it was found that extraction efficiency of MCM-41 for protein digest and human serum was greatly improved

Fast Equilibrium Micro-Extraction from Biological Fluids with Biocompatible Core-Sheath Electrospun Nanofibers

Sample preparation methods with high temporal resolution and matrix resistance will benefit fast direct analysis of analytes in a complex matrix, such as drug monitoring in biofluids. In this work, the core-sheath biocompatible electrospun nanofiber was fabricated as a micro-solid phase extraction material. With the poly(N-isopropylacrylamide) (PNIPAAm) as sheath polymer and polystyrene (PS) as core polymer, the fiber membrane was highly hydrophilic and exhibited good antifouling ability to proteins and cells. Its complete expansion in aqueous solution and its nanoscale fiber (100-200 nm) structure offered high mass transfer rate of analytes between liquid and solid phases. The equilibration time of microextraction with this membrane was all shorter than 2 min for eight drugs tested, and the linear ranges covered more than 3 orders of magnitude for most of them. This membrane could be applied to monitor free drugs in plasma and their protein binding kinetics by equilibrium-microextraction with a 2 min temporal resolution. The results showed that the core-sheath electrospun nanofiber membrane would be a better alternative of solid phase material for microextraction with good matrix-resistance ability and high temporal resolution

Rapid solid-phase microextraction of polycyclic aromatic hydrocarbons in water samples by a coated through-pore sintered titanium disk

A novel solid-phase microextraction device based on a through-pore sintered titanium disk (ST-SPME) was prepared and evaluated for the rapid extraction of organic pollutants from aqueous samples. The through-pores embedded in a sintered titanium disk were enlarged by HCl etching, yielding more Ti-O functional groups and a rough surface that should benefit the covalent binding of extraction phase with the disk. The large inner surface area (850 mm(2)) of the disk and narrow through-pores (100 gm) significantly increase the extraction capacity and mass transfer rate. In addition, the resistance to water flow of the ST-SPME disk is rather low, yielding a back pressure of only 100 kPa at a flow rate of 50 mL/min. The extracted target compounds are thermally desorbed by a thermal desorption unit and transferred to a GC or GC-MS system. Taking polydimethylsiloxane as the extraction phase and 16 types of polycyclic aromatic hydrocarbons (PAHs) as model analytes, the extraction and desorption conditions were systematically investigated. The optimized extraction time was only 2 min for 100 mL of water sample. Absolute recoveries were between 5.93% and 23.02%, which are similar to that of stir bar sorptive extraction. The LODs and RSDs were 0.06-3.20 ng/L (S/N=3) and 0.57-7.08%, respectively. The method showed good linearity in the range of 0.01-10 AWL with a squared determination coefficient R-2 >= 0.9939. As our method was suitable for the measurement of organic pollutants in the water phase, the ST-SPME/GC-MS method was assessed by analyzing three filtered real environmental samples. Some PAHs were detected at the ng/L level in river water and sea water. (C) 2016 Published by Elsevier B.V

Nanocoating cellulose paper based microextraction combined with nanospray mass spectrometry for rapid and facile quantitation of ribonucleosides in human urine

A rapid and facile analytical method for quantification of ribonucleosides in human urine was developed by the combination of nanocoating cellulose paper based microextraction and nanoelectrospray ionization-tandem mass spectrometry (nESI-MS/MS). Cellulose paper used for microextraction was modified by nano-precision deposition of uniform ultrathin zirconia gel film using a sol-gel process. Due to the large surface area of the cellulose paper and the strong affinity between zirconia and the cis-diol compounds, the target analytes were selectively extracted from the complex matrix. Thus, the detection sensitivity was greatly improved. Typically, the nanocoating cellulose paper was immersed into the diluted urine for selective extraction of target analytes, then the extracted analytes were subjected to nESI-MS/MS detection. The whole analytical procedure could be completed within 10 min. The method was evaluated by the determination of ribonucleosides (adenosine, cytidine, uridine, guanosine) in urine sample. The signal intensities of the ribonuclesides extracted by the nanocoating cellulose paper were greatly enhanced by 136-459-folds compared with the one of the unmodified cellulose paper based microextraction. The limits of detection (LODs) and the limits of quantification (LOQs) of the four ribonucleosides were in the range of 0.0136-1.258 mu g L-1 and 0.0454-4.194 mu g L-1, respectively. The recoveries of the target nucleosides from spiked human urine were in the range of 75.64-103.49% with the relative standard deviations (RSDs) less than 9.36%. The results demonstrate the potential of the proposed method for rapid and facile determination of endogenous ribonucleosides in urine sample

Sheathless interface to match flow rate of capillary electrophoresis with electrospray mass spectrometry using regular-sized capillary

RATIONALE: The flow rate match has been a great challenge when coupling capillary electrophoresis (CE) with electrospray ionization mass spectrometry (ESI-MS). Conventional CE-ESI-MS interfaces used liquid sheath flow, narrowed capillary or additional pressure to meet this requirement; sacrifice of either capillary inner diameter (i.d.) or separation efficiency is often inevitable. Thus, a regular-sized capillary-based sheathless interface would be attractive for flow rate match in CE-MS

Quantification of Low Copy Number Proteins in Single Cells

We have developed an ultrasensitive and highly selective method to quantify low copy number intracellular proteins in a single cell using a low-cost laser-induced fluorescence (LIF) detector and a BV605 fluorescent probe. Active caspase3 proteins in cells were labeled by corresponding antibody-BV605 fluorescent binding, and a cell was injected into a 20 cm x 50 mu m i.d. capillary column, followed by in situ lysis and capillary electrophoresis (CE)-LIF analysis. About seven active caspase3 protein molecules in a detection volume of 91 pL could be detected. In our method, cross-bounding proteins other than active caspase3 could be separated and distinguished by differences of retention time. By using Si photodiode assembly as a fluorescent detector instead of PMT, the dynamic range of the LIF is over 4 orders of magnitude. In this experiment, we found that the number of active caspase3 molecules in 98 single Jurkat cells were from 629 to 12171, reflecting significant heterogeneity among the cells although they were from the same batch. For extended application, it could also be applied to quantify other types of low copy number proteins in a single cell as long as the corresponding antibodies are provided. This high-sensitive method could also be a promising tool for earlier cancer diagnosis and related disease pathway research which is relevant to low copy number proteins. In addition, this low-cost system could also be easily expanded to an array system for high-throughput quantitation of low copy proteins in single cells