Stability comparison of four lipases and catalytic mechanism during the synthesis of 1,3-di-oleic-2-medium chain triacylglycerols in a trace water-in-oil system: Experimental analyses and computational simulations.

In the present study, a kind of structured lipids, namely 1,3-di-oleic-2-medium chain (OMO) triacylglycerols, were synthesized through lipase-catalyzed reactions using coconut oil and rapeseed acid as materials in a trace water-in-oil system. Experimental analysis and computational simulations were undertaken to compare the stability of four lipases including Lipozyme RMIM, Lipozyme TLIM, Novozym 435, and Aspergillus oryzae immobilized lipase (AOIM), and illustrate catalytic mechanism of Novozym 435 during the synthesis of OMO. Fourier transform infrared and molecular dynamics simulation results demonstrated that a decrease in ordered structure (α-helix and ß-sheet) led to a reduction in enzyme activity. Compared with Lipozyme RMIM and Novozym 435, Lipozyme TLIM and AOIM exhibited better stability due to a short-chain lid in TLIM, which covers activity sites, and hydrogen bonds formed between activity center of AOIM and water. Among four lipases, AOIM exhibited best catalytic performance: a OMO yield of 30.7% at 3 hr and a good stability of long term (48 hr). Density functional theory results demonstrated that specifically, during the synthesis of OMO triacylglycerol, the addition of Novozym 435 (derived from Candida antarctica lipase B, CALB) substantially lowered reaction barriers (64.4 KJ/mol with CALB vs. 332.7 KJ/mol with no lipase), aiding in the generation of OMO because of the formations of transitional tetrahedral intermediates. A trace water-in-oil system was a green and efficient alternative for lipase-catalyzed production of OMO, and this study provided crucial insights into the stability/instability and catalytic mechanisms of lipase in the synthesis of structured lipids. PRACTICAL APPLICATIONS: We compared the stability of Lipozyme RMIM, Lipozyme 435, Lipozyme TLIM, and AOIM during the synthesis of the OMO triacylglycerols in a trace water-in-oil system, where exhibited a high catalytic activity of lipase in water-oil interface. AOIM had the highest stability and showed the best catalytic performance due to the formation of hydrogen bonds. Besides, for the first time, the transition tetrahedral structure was revealed in the enzymatic synthesis of medium- and long-chain triacylglycerols. This study provides a rational approach to compare lipase stability and a possible hint to choose appropriate enzyme in a specific catalytic condition.

Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.

Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers.

VHH phage-based competitive real-time immuno-polymerase chain reaction for ultrasensitive detection of ochratoxin A in cereal.

Phage display-mediated immuno-polymerase chain reaction (PD-IPCR) is an ultrasensitive detection technology that combines the advantages of immuno-PCR and phage display. The phage particle, which displayed antibody fragments including single-chain fragment variable (scFv), variable domain of heavy-chain antibodies (VHH), and antigen-binding fragment (Fab) on the surface can be directly used in IPCR, supplying both the detection antibody and deoxyribonucleic acid (DNA) template. In this work, we used ochratoxin A (OTA) as a model system to study the capacity of PD-IPCR in the detection of toxic small molecular weight compounds, especially mycotoxins. An alpaca-derived VHH library was constructed and subjected to four cycles of panning. In total, 16 clones with four unique sequences were selected by competitive binding with OTA. The clone VHH-28 resulted in the lowest 50% inhibitory concentration of 0.31 ng/mL in the phage enzyme-linked immunosorbent assay (ELISA) and was selected to develop the VHH phage-based real-time immuno-PCR (RT-IPCR). The detection limit of the VHH phage-based RT-IPCR was 3.7 pg/L, with a linear range of 0.01-1000 pg/mL. This method was compared with conventional ELISA, and validation results indicated the reliability of VHH phage-based RT-IPCR in the detection of OTA in cereal samples. This study provides a new idea for the ultrasensitive detection of mycotoxins and other toxic small molecular weight compounds.

Isolation and characterization of recombinant variable domain of heavy chain anti-idiotypic antibodies specific to aflatoxin B1.

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F(ab')2 fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic (anti-Id) responses. Antigen-specific elution method was used for panning private anti-Id VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay (ELISA). Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.