Mapping haze-komi on rice koji grains using β-glucuronidase expressing Aspergillus oryzae and mass spectrometry imaging

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Response surface studies that elucidate the role of infiltration conditions on Agrobacterium tumefaciens-mediated transient transgene expression in harvested switchgrass (Panicum virgatum)

Agrobacterium tumefaciens-mediated transient expression (agroinfiltration) experiments were performed in harvested switchgrass (Panicum virgatum) leaves to identify the effects of wounding by bead beating, surfactant concentration and vacuum application on in planta β-glucuronidase expression and leaf decay. Expression was scored based on a consistent pattern of visual observations of histochemical staining over the leaf surface as might be observed in stable gene expression in switchgrass leaves. Assays on extracts from leaves were also performed to measure expression levels; however, these assays showed low expression levels, which may have been due to low recombinant protein recovery and decomposition in the leaf. Bead beating was successful for wounding the plant surface, but did not improve the consistency of expression based on histochemical staining observations. Surfactant was necessary for improving contact between the leaf surface and Agrobacterium suspension and consistently improved expression when vacuum application level was low (25 kPa). Increasing vacuum application from 25 to 5 kPa improved expression only when surfactant concentration was low. When a suspension of A. tumefaciens containing 1000 ppm Break-Thru surfactant was added to harvested leaves and 25 kPa vacuum applied, a fairly uniform expression was visualized across the leaf surface within 2–3 days of incubation, suggesting that agroinfiltration is a rapid tool for examining expression of transgenes in switchgrass leaves.     

Oil bodies of transgenicBrassica napus as a source of immobilized β-glucuronidase

The process of immobilizing enzymes is a major cost factor in the utilization of heterogeneous catalysts on an industrial scale. We have developed a new strategy, based on plant genetic manipulation, for the production of foreign peptides associated with the oil body in plant seeds. Seeds of transgenic rapeseed can be produced on a large scale at relatively low cost. Furthermore, oil bodies are readily isolated from seeds by floatation centrifugation. In this paper, we describe some physical and operational properties of an oil body—fusion protein complex and its suitability as a heterogeneous catalyst. Oil bodies from rapeseed, corn, and flax aggregate at pH 5, which facilitates their recovery by floatation. Oil bodies from transgenic rapeseed, carrying the reporter gene β-glucuronidase or the pharmaceutical peptide, hirudin, also aggregate in the same range. This aggregation is reversible. Oil bodies are resistant to a wide range of pH, with some lysis occurring (<10%) at the extremes. They are resistant to shearing forces, such as stirring. The thermal and pH stabilities, as well as the catalytic activity of β-glucuronidase expressed on the oil body surface, are comparable to those of free β-glucuronidase enzyme.     

Detection of Escherichia coli using CMOS array photo sensor-based enzyme biochip detection system

This work presents optical enzyme detection system based on the CMOS array photo sensor and 1 × 3 polymeric enzyme biochip for detecting Escherichia coli in a one-step procedure. This assay, using 4-methylumbelliferyl-β-d-glucuronide (MUG) as a fluorogenic substrate, had a detection limit of 0.1 U/ml for β-glucuronidase (GUD), which was approximately equal to a cell concentration of 10⁶ CFU/ml of E. coli. MUG was incorporated into lauryl tryptose broth at a final concentration of 100 μg/ml for immediate verification of the presence of E. coli in 1 × 3 polymeric enzyme biochip. The 40 strains of E. coli studied all produced GUD. Of another 36 strains of bacteria tested, one strain (Salmonella choleraesuis subsp. choleraesuis) yielded very small amounts of GUD after 24 h incubation. The optical enzyme detection system was sensitive and rapid.     

Adsorption and binding of the transgenic plant proteins, human serum albumin, β-glucuronidase, and Cry3Bb1, on montmorillonite and kaolinite: Microbial utilization and enzymatic activity of free and clay-bound proteins

Human serum albumin (HSA), β-glucuronidase (GUS), and the Cry3Bb1 protein from Bacillus thuringiensis subsp. kumamotoensis are expressed by genetically-modified plants. Commercial samples of these proteins adsorbed and bound rapidly on the clay minerals, kaolinite (K) and montmorillonite (M). Adsorption increased as the concentration of protein increased and then reached a plateau. The greatest amount of adsorption and binding occurred with the Cry3Bb1 protein, of which there was no desorption: 6.7 ±0.21 μg adsorbed and bound μg^− 1 of M; 2.1 ± 0.39 μg adsorbed and bound μg^− 1 of K. With GUS, 2.2 ± 0.29 μg adsorbed and 1.7 ±0.21 μg bound μg^− 1 of M; 1.5 ± 0.28 μg adsorbed and 1.0 ± 0.03 μg bound μg^− 1 of K. HSA was adsorbed and bound the least: 1.2 ±0.04 μg adsorbed and 0.8 ± 0.05 μg bound μg^− 1 of M; 0.4 ± 0.05 μg adsorbed and 0.4 ± 0.03 μg bound μg^− 1 of K. However, X-ray diffraction analyses indicated that only HSA intercalated M, and none of the proteins intercalated K, a nonswelling clay. When bound, the proteins were not utilized for growth by mixed cultures of soil microorganisms, whereas the cultures readily utilized the free (i.e., not adsorbed or bound) proteins as sources of carbon and energy. The enzymatic activity of GUS was significantly enhanced when bound on the clay minerals. These results indicated that recombinant proteins expressed by transgenic plants could persist and function in soil after release in root exudates and from decaying plant residues as the result of the protection provided against biodegradation by binding on clay minerals.