الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
A previous research, conducted in our laboratory, resulted in a successful recovery of a local promising bacterial isolate (Actinomyces hyovaginalis isolate A11-2) capable of converting vitamin D3 into calcitriol either by using its free cells or cell lysate.
In spite of the rising number of researches on vitamin D3 bioconversion into calcitriol, further deliberate studies on this topic can significantly contribute to improvement of such bioconversion process for application in several fields. Consequently, the goal of this work was the application of different approaches for improvement of calcitriol production from vitamin D3, using Actinomyces hyovaginalis study isolate.
In this research work, the respective isolate produced about 140 µg/L calcitriol under the following conditions: seed culture using nutrient broth, at 200 rpm and 30 °C for two days; main culture (under the same growth conditions) in a medium consisting of 15 g fructose, 15 g defatted soybean,5 g sodium chloride, 2 g calcium carbonate, 1 g dipotassium hydrogen phosphate, 0.2 g sodium fluoride per liter (initial pH of 7.8); substrate (vitamin D3) concentration of 0.2 g/L, added two days after beginning of main culture and remained for four more days bioconversion time under the same growth conditions but with the incubation temperature lowered to 28 °C.
Various optimization experiments were carried out where viable count technique was used to measure the bacterial growth and HPLC assay was used to quantify the production of calcitriol.
Protoplast fusion was attempted using protoplasts prepared from the study isolate and Bacillus species. Three regenerated protoplast hybrids produced higher amounts of calcitriol, from vitamin D3, than that produced by the parent study isolate. The increment in calcitriol production by the three regenerated species (V2B, V3B and V8A), as compared to that of the parent study isolate, was 3.5, 1.4 and 2.8 fold, respectively, under the same culture and reaction conditions. These three regenerated species, V2B, V3B and V8A, were formed by fusion of protoplasts prepared from B. thuringenesis isolate B16 with those of the study isolate. Stability studies revealed that, out of the two hybrids exhibiting the highest vitamin D3 bioconversion abilities, only the hybrid V8A remained stable after twenty repeated subcultures.
An alternative promising approach for vitamin D3 bioconversion into calcitriol, by the study isolate, could be applied depending on the use of cell lysate where about 16 µg calcitriol were produced using the lysate of cells obtained from 1 L culture, in a time not exceeding 6 h.
Consequently, vitamin D3 bioconversion using different protein fractions of the crude cell lysate as well as characterizing the enzyme(s) involved in the bioconversion process, were carried out. The fractionation of the crude cell lysate was accomplished via ion exchange chromatography which revealed that the bioconversion active protein could be totally eluted using a total volume of 80 ml (in the form of 10 ml aliquots) of 50 mM NaCl in Tris-HCl (20 mM, pH 7.4). The eluted protein (chromatographic active fraction) could produce about 30 µg/10 ml calcitriol from the substrate. The optimal bioconversion conditions were pH of 7.8, reaction temperature of 28 °C and reaction time of 6 h.
Furthermore, immobilization of the crude cell lysate was carried out using 2% w/v alginate, which resulted in 140 % increase in calcitriol production from vitamin D3, as compared to that obtained using free cell lysate. The prepared alginate beads, stored in Tris-HCl buffer, were able to be used up to four times over a period of nine days, where only 25% decline in the bioconversion activity was noticed, after the 4th use.
Another experiment was undertaken to immobilize the protein(s), recovered from the chromatographic active fraction, which showed a bioconversion activity about 1.15 fold as that shown by its non-immobilized counterpart. The stored beads could be used up to four times over a period of nine days with only 22% decline in the bioconversion activity, after the 4th use. In addition, it was found out that the storage of the beads, containing protein(s) recovered from the chromatographic active fraction, in Tris-HCL buffer is better for activity than their storage in a dried form. Moreover, the optimal reaction time for the bioconversion process using the immobilized protein(s), was found to be 6 h.
Also, improvement of the bioconversion process, using the study isolate, in a 14 L laboratory fermentor was carried out. The amount of calcitriol produced from vitamin D3, using 4 L working volume of fermentation medium in a batch mode, was found to be about 328 µg/L (about 2 fold as that produced in shake flask). The optimal bioconversion conditions were found to be: inoculum size of 2% v/v; agitation rate of 200 rpm; aeration rate of 1 vvm; initial pH of 7.8 (uncontrolled); addition of vitamin D3 two days after beginning of main culture; using fructose as the medium carbon source and defatted soybean as the nitrogen source.
Finally, an attempt for cloning and expression of the gene, putatively involved in vitamin D3 bioconversion, was carried out. PCR amplification was performed using appropriate conditions and two designed primers as well as the chromosomal DNA of the respective isolate as a PCR template. The obtained PCR product was analyzed using agarose gel electrophoresis. The PCR product of the expected product size (1.2 kb) was successfully cloned into pUCPU21 cloning and pET16b expression vectors. For protein expression, the recombinant plasmid (pET16b-vdh) was transformed into E.coli BL21 (DE3), and the protein expression was undertaken under the control of T7 promoter. SDS-PAGE analysis profile of expressed proteins showed successful gene expression in a soluble form (of about 45 KDa) after IPTG induction. The obtained expressed protein was tested for vitamin D3 bioconversion and results should that the expressed protein was active based on the results obtained, using HPLC and LC-MS analytical methods.