12 research outputs found
Mannan biotechnology: from biofuels to health
<div><p></p><p>Mannans of different structure and composition are renewable bioresources that can be widely found as components of lignocellulosic biomass in softwood and agricultural wastes, as non-starch reserve polysaccharides in endosperms and vacuoles of a wide variety of plants, as well as a major component of yeast cell walls. Enzymatic hydrolysis of mannans using mannanases is essential in the pre-treatment step during the production of second-generation biofuels and for the production of potentially health-promoting manno-oligosaccharides (MOS). In addition, mannan-degrading enzymes can be employed in various biotechnological applications, such as cleansing and food industries. In this review, fundamental knowledge of mannan structures, sources and functions will be summarized. An update on various aspects of mannan-degrading enzymes as well as the current status of their production, and a critical analysis of the potential application of MOS in food and feed industries will be given. Finally, emerging areas of research on mannan biotechnology will be highlighted.</p></div
Antibody characterization via dilution series.
<p>(<b>A</b>) A checkerboard titration experiment of phage-displayed scFv clone bDOA9rb8. Various amount of phage particles were prepared by 10-fold dilution series starting from 10<sup>13</sup>, 10<sup>12</sup>, 10<sup>11</sup>, 10<sup>10</sup>, and 10<sup>9</sup> pfu/well. Each dilution of phage antibody was used to detect different amount of boiled DOA9 antigen at the protein concentration of 10, 5.0, 2.5, 1.3, 0.6 and 0.3 μg/well of total protein, determined by Bradford assay. Each reaction was done in triplicate. The phages were purified by PEG precipitation and re-suspended in PBS buffer at a concentration of 10<sup>11</sup> pfu/μl. Bound phage was detected by anti-M13 HRP, using ABTS as color reagent. The Y-axis indicated the OD value and the SD measured from triplicate wells. (<b>B</b>) Detection limit of phage displayed scFv clone bDOA9rb8 against boiled DOA9. The 5-fold dilution series of boiled DOA9 protein antigen were prepared in sodium carbonate buffer starting from the protein concentration of 10, 2, 0.4, 0.08, 0.02, 0.003 and 0.00 μg/well. The ELISA was performed by using the optimum concentration of phage antibody (10<sup>12</sup> pfu/well), determined from panel A. The average OD<sub>405</sub> nm values and standard errors from triplicate wells are shown.</p
SDS-PAGE and Westernblot analysis of purified soluble scFv antibody.
<p>(<b>A</b>) The soluble scFv antibody against <i>Bradyrhizobium</i> sp. strain DOA9 clone bDOA9rb8 was purified from culture supernatant by IMAC. Lane M: protein molecular weight marker; lane S: culture supernatant input; lane FT: flow-through fraction; lanes W1, W2, and W3 indicate the three wash fractions; lanes E1, E2, and E3 are the three elution fractions. The soluble scFv antibody of approx. 30 kDa can be found in elution fractions 1 and 2. This scFv antibody was used to study the binding specificity in the next step. (<b>B</b>) SDS-PAGE and Western blot analysis of free scFv clone bDOA9rb8 obtained from periplasmic extracts.</p
Selective enrichment of scFv antibodies during the biopanning process.
<p>Selective enrichment of scFv antibodies during the biopanning process.</p
Amino acid sequence and 3D structure of isolated anti-DOA9 rabbit scFv antibody.
<p>The primary amino acid sequence of the clone bDOA9rb8 is shown at the bottom. The flexible linker (G<sub>4</sub>S)<sub>3</sub> that joins V<sub>H</sub> and V<sub>L</sub> segments is indicated. The three complementarity-determining regions (CDRs) are underlined. The 3D structure was done by Phyre<sup>2</sup> software, using the structure of an scFv antibody in complex with an analogue of the main immunogenic region of the acetylcholine receptor (PDB code: 1F3R) as templates. The 3D structures in both space-filling and ribbon models with CDRs of V<sub>H</sub> and V<sub>L</sub> domains are indicated.</p
Specific binding of selected phage-displayed scFv clones.
<p>Phage ELISA results of the binding of scFv antibodies against DOA9 and other antigens in pure culture (<b>A</b>) and plant nodules (<b>B</b>) are shown. The two clones of phage-displayed rabbit scFv, i.e., RB9 and RG8, could bind specifically to only strain DOA9 but not to other <i>Bradyrhizobium</i> strains and <i>Bacillus subtilis</i> 168. Phage-displayed human 3C1 scFv antibody was used as a negative control in this assay. The average OD<sub>405</sub> nm values and standard errors from triplicate wells are shown. Note that in panel B, there was no bacillus nodule because it can’t form nodule in plant roots.</p
Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of <i>Bradyrhizobium</i> sp. DOA9 in both free-living and bacteroid forms
<div><p>A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. <i>Bradyrhizobium</i> sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of <i>Aeschynomene americana</i> in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant–microbe interactions in the future.</p></div
Oligonucleotide primers used for the construction of the immunized rabbit phage library.
<p>Oligonucleotide primers used for the construction of the immunized rabbit phage library.</p
Binding property of free scFv against strain DOA9.
<p>ELISA results of soluble scFv antibodies against bacterial targets in both free-living (<b>A</b>) and bacteroid form (<b>B</b>) in plant nodule are illustrated. Rabbit polyclonal antibody and Phage-display scFv were also used in the assay for comparison. Values are the mean of triplicate wells. Error bars show the standard deviation for each set of data. Panel <b>C</b> illustrates binding properties of free scFv against DOA9 in a checkerboard titration experiment. Serial dilutions of soluble scFv antibody ranging from 50–0 μg were add into wells of ELISA plated immobilized with various concentration of boiled DOA9, ranging from 10 to 0 μg of total protein. The Y-axis indicated the OD value and the SD measured from triplicate wells.</p
Investigation of the morphology of symbiotic nodule using phage-displayed scFv clone bDOA9rb8.
<p>Bright field and immunofluorescence staining images from confocal microscopy of bacteriod form of <i>Bradyrhizobium</i> sp. DOA9 in plant nodules at different magnifications are shown. Sample in <b>A</b> was detected with negative phage clone 3C1; <b>B</b> rabbit polyclonal antibody, or <b>C</b> the phage-displayed scFv clone bDOA9rb8 from this study. Blue color indicated the plate cell wall that was stained with blue fluorophore. Scale bar is 100 μm at 10X magnification and 50 μm at 40X magnifications.</p