| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1999: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Microorganisms were screened by flow cytometry for specific characteristics (hyperproduction, in this study) and such organisms were flow cytometrically sorted. Model systems used were :
(1) a fluorescent carotenoid astaxanthin (ASX) and a microalga Heamatococcus pluvialis, which produces ASX when encysting,
(2) vitamin B1 (B1 ; or thiamin), which is not fluorescent but is chemically transformed to a fluorescent derivative thiochrome, and a sake yeast (Saccharomyces cerevisiae), which produces large amounts of B1, and
(3) a coenzyme pyrroloquinoline quinone (PQQ) and two lactic-acid bacteria Acetobacter acetiand Gluconobacter oxidans, which produce PQQ, mainly as holoenzymes (quinoproteins).
Hyperproduction of these compounds was induced by treatment with mutagenic compounds and hyperproducers were sorted.
Cysts of H.pluvialis were analysed by flow cytometry. The stronger the fluorescence signal was, the larger the size of the cysts was (as microscopic observation), demonstrating sorting of
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H.pluvialis for ASX-hyperproduction. Such cysts were encysted and analysed again to show similar cytographic pattems as those before mutagenesis, suggesting the unstable hyperproducing characteristic obtained.
The sake yeast was cultured under various conditions and cells were encapsulated in gel microdroplets (GMDs), typically and successfully with porous glass filter, to produce microcolonies. After thiochrome reaction under certain conditions, epecially with shorter reaction periods and by quick neutralisation of the reaction mixtures, GMDs could be measured for B1 contents with good correlation with the B1 content of the whole cultue (assayed by thiochrome method) and some percentages of GMDs gave regeneration of living cells.
The lactic-acid bacteria were cultured under various conditions and cytometrically analysed. Variety of cytogrammes was obtained but without well correlation to the PQQ contents in the whole culture. The UV laser used (625 nm) was not suitable for PQQ because of other compounds which interfered PQQ.Use of arc and deuterium lamps might be considered in further study with PQQ. Less
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