I= SOCIO – ECONOMIC CONSEQUENCES OF RETURN MIGRATION AND THE ADJUSTMENT PROBLEMS IN PUNJAB Title of Thesis


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Title of Thesis

STUDIES ON THE BIOSYNTHESIS OF ANTIBIOTIC RIFAMYCIN B BY NOCARDIA MEDITERRNEA

Author(s)

Mukhtiar Hassan

Institute/University/Department Details

Islamia University, Bahawalpur / Chemistry

Session

2000

Subject

Chemistry

Number of Pages

178

 

Keywords (Extracted from title, table of contents and abstract of thesis)

Antibiotic Rifamycin B, Nocardia Mediterrnea A TCC 13685, Nocardia mediterranea M-120, Rifamycins, sodium diethyl barbituric acid

 

Abstract

The bacterial culture of Nocardia mediterranea A TCC 13685 was used for production of antitubercular antibiotic rifamycin by submerged fermentation procedure. The culture was improved by sequential mutagenic treatment with NTG (N-methyl-N -nitro-N-nitroso-guanidine) and U. V radiation. The mutant strain was designed as Nocardia mediterranea M-120. In submerged fermentation, 48 hours old (5% v/v) inoculum gave the maximum yield of rifamycin B when the culture was grown in medium containing (g/l) glucose 94; soybean 10; peanut meal 21.4; calcium carbonate 9.5; potassium dihydrogen phosphate 0.4; magnesium sulphate 1.0; (NH4)2S04 8; CuSO4.5H2O 0.0165; ZnSO4.7H2O 0.004; FeSO4.7H2O 0.002 and MgSO4.7H2O 0.001. The addition of sodium diethyl barbituric acid (0.2% w/v) was found best to obtain maximum yield of rifamycin B suppressing the production of other unwanted rifamycins, whereas citrate, propionate and ammonium molybdate were deleted from the medium as they did not increase the rifamycin yield. Carbohydrates in the form of monosaccharide and disaccharides were employed for the production of rifamycin. Among all sugars glucose (9.5%w/v) gave the maximum yield of antibiotic. To economize the feasibility of the process agricultural by products such as com steep liquor, soybean meal, sunflower meal, cotton seed cake and peanut meal etc. were tried. The results obtained by soybean meal were encouraging. But the production of antibiotic was found to be maximal in the presence of 1 % soybean meal and 2.14 % peanut meal. The optimal medium for the submerged fermentation of the rifamycin contained soybean meal, peanut meal, glucose, ammonium sulphate, mineral salts and barbital produced antibiotic rifamycin B 569 ug/ml at the flask level. The environmental factors such as incubation temperature, initial pH, and aeration and agitation rates were also optimized. The optimum rifamycin yield (1150 ug/ml) was obtained in 1.0 L fermenter with working volume of 0.5 L at 28 °C with initial pH of 7.0. The agitation and aeration were 250 rpm and 1.0 L/L/min respectively. A study was made on the mineral requirements of Nocardia mediterranea M-120 for rifamycin production in synthetic medium. It was observed that 100 ppm of iron as FeSO4 has stimulatory effects on the rifamycin production. Zinc, copper and molybdenum were without any effect. However, iron in combination with zinc and manganese gave rather good yield of antibiotic. Magnesium was required at a concentration of 0.1 % and KH2PO4 (0.05 %) for maximum yield of rifamycin, whereas CaCO3 (0.95%) was required for maintenance of constant pH of medium. Organic and inorganic nitrogen sources were tried to enhance the yields of rifamycin B. Ammonium sulphate at a concentration of 0.8 % (w/v) was required for maximal production of rifamycin. The effect of addition of various amino acids on rifamycin production in synthetic as well as basal medium was studied and it was found that the addition of amino acids in the presence of ammonium sulphate was undesirable. However, the presence of L-proline (0.2%) in synthetic medium gave better results as compared to controls. Isolation of rifamycin from broth was carried out by its exhaustive extraction with ethyl acetate, phosphate buffer and choloform. Bioassay, TLC and Spectrophotometeric analysis identified antibiotic rifamycin as rifamycin B.

 

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    Table of Contents  

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1 1 INTRODUCTION 1

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2 2 REVIEW OF LITERATURE \5

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3 3 MATERIALS AND METHODS 64

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4 4 RESULTS AND DISCUSSION 82

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5 5 CONCLUSIONS

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6 6 TABLES

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7 7 FIGURES

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8 8 LITERATURE CITED

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