Shinji Kamiya

Use of magnetic particles isolated from magnetotactic bacteria for enzyme immobilization

SummaryWe report the novel use of magnetic particles isolated from magnetotactic bacteria. Magnetotactic bacteria were collected from enriched sludge by use of a magnetic harvesting apparatus. Magnetic particles separated from magnetotactic bacteria were shown to be pure magnetite. Glucose oxidase and uricase were immobilized on magnetic particles. The activity of glucose oxidase immobilized on biogenic magnetites was 40 times that immobilized on artificial magnetites or Zn-ferrite particles. Both glucose oxidase and uricase coupled with biogenic magnetic particles retained their activities when they were reused 5 times.

Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

Abstract Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1–103xa0ng/ml.

Design and application of a new cryptic-plasmid-based shuttle vector for Magnetospirillum magneticum.

ABSTRACT A 3.7-kb cryptic plasmid designated pMGT was found in Magnetospirillum magneticum MGT-1. It was characterized and used for the development of an improved expression system in strain AMB-1 through the construction of a shuttle vector, pUMG. An electroporation method for magnetic bacteria that uses the cryptic plasmid was also developed.

Enhancement of magnetic particle production by nitrate and succinate fed-batch culture of Magnetospirillum sp. AMB-1

Magnetospirillum sp. AMB-1 is a magnetic bacterium, which is capable of growing under air atmosphere. This bacterium was employed to make bacterial magnetic particles (BMPs). AMB-1 only makes BMPs during logarithmic growth phase under anaerobic conditions. Since it requires nitrate as a nitrogen source, control of nitrate concentration in the medium was necessary. The fed-batch culture of AMB-1 was carried out by adding nitric acid and succinate as nitrogen and carbon source respectively. One liter of AMB-1 culture produced 0.34 g of dry cells and 4.5 mg of BMPs. BMP production by AMB-1 cultivated in the fed-batch culture was found to be seven times higher than that cultivated in the batch culture.

Electrochemical luminescence-based homogeneous immunosensor

Abstract Luminol capable of generating electrochemical luminescence was employed as a label of immunoassay. A luminol-labeled antibody generated luminescence upon anodic oxidation and the luminescence decreased markedly in the presence of antigen. The decrease was correlated to the antigen concentration as low as 10 −11 g/ml antigen. The proposed method was operated in an optoelectrochemical cell. A miniaturized and sensitive immunosensing can be developed by fabricating a small cell with a very thin layer.

Production of a Protein (Enzyme, Antibody, Protein A)-Magnetite Complex by Genetically Engineered Magnetic Bacteria Magnetospirillum Sp. AMB-1

Magnetospirillum sp. AMB-1 is a magnetic bacterium which synthesizes intracellular particles of magnetite (Fe3O4). A genomic DNA fragment required for the synthesis of magnetic particles was previously isolated from this strain. The complete nucleotide sequence of this fragment has been determined by us. An open reading frame (ORF), designated magA, encodes a polypeptide which represents an iron transport protein. Intracellular localization of the MagA protein was studied using magA—luc fusion proteins. The luc gene was cloned downstream of the magA hydrophilic C-terminal domain. The fusion protein was detected on the surface of the lipid bilayer covering the magnetic particles. The MagA protein on the bacterial magnetic particle (BMP) was detected by immunoassay using an anti-luciferase antibody. The N- and C-termini of MagA protein were found outside the BMP membrane. These results show the utility of magA fusions for detecting multi functional proteins on BMP. Recombinant protein-BMP complex production has been carried out using the fed-batch culture by adding nitric acid and succinate as nitrogen and carbon source.