الفهرس 
coverOnly 14 pages are availabe for public view
 |  | from | 151 |  |  |
| | | from | 151 | | |
Abstract
One hundred and eighty bacterial isolates were recovered from different soil collected samples using paraffin baiting technique. Grown bacterial colonies, appearing as white chalky particles around the solidified paraffin wax globules, were collected and screened for vitamin D3 biotransformation activity. Vitamin D3 dissolved in ethanol was added to 2 days old main culture and incubation conditions were 28°C and 200 rpm. Extraction of vitamin D3 and its metabolites was carried out by a modified Bligh and Dyer method using methanol and methylene chloride.
Preliminary analysis, using TLC, showed that five isolates (A11-2, A13-4, A8-4, A26-7 and A26-8) could transform vitamin D3 into 1α, 25-dihydroxyvitamin D3 (calcitriol) with 25-hydroxyvitamin D3 (calcidiol) as an intermediate. Such results were further confirmed using mass spectrometric analyses. The relative intensities of the produced calcidiol and calcitriol by the five positive isolates (A11-2, A13-4, A8-4, A26-7 and A26-8), as analyzed by MS/MS, were compared and it was found that the isolate A11-2 exhibited the highest product intensity for either calcitriol or calcidiol.
The test isolate A11-2 was identified using microscopical, culture and biochemical characteristics as well as Biolog microbial identification system assay. Microscopical, culture and some biochemical characteristics showed great similarity between the test isolate A11-2 and the standard strain, A. autotrophica NRRL B-11275. However, by conducting the Biolog microbial identification system assay, the resulting metabolic fingerprint indicated 100% similarity with Actinomyces hyovaginalis. Thus, interestingly and to the best of our knowledge, the results obtained in the present study revealed, for the first time, that vitamin D3 can be biotransfomed into calcidiol and calcitriol by Actinomyces species.
The Actinomyces hyovaginalis species A11-2 was, then, subjected to different studies to optimize its biotransformation capability of vitamin D3 into calcitriol. The results of these different experiments revealed that the maximum calcitriol production was achieved using nutrient broth as a medium for preculture. The optimal timing of the addition of vitamin D3 was found to be two days after the beginning of the main culture and at concentration of 20 mg% w/v. Optimum culture conditions were found to be 15 g fructose, 15 g skim milk, 1 g dipotassium hydrogen phosphate, 0.2 g sodium fluoride per liter of main culture medium (initial pH of 7.8) and continuing the main culture for 96 hrs after addition of vitamin D3. Such conditions gave calcirtiol production of 220 µg/L.
A study was conducted to test vitamin D3 biotransformation by the cell lysate of the test isolate in comparison to the intact cells. It was found that 118 µg calcitriol was obtained using the lysate of cells obtained from 1 L culture in a time not exceeding 6 hrs.