Transformation of rifamycin B with immobilized rifamycin oxidase of Curvularia lunata

Rifamycin oxidase of Curvularia lunata was immobilized on polyacrylamide gel. The optimum pH and temperature for immobilized enzyme reaction were 6.5 and 50 °C, respectively. Enzyme stability increased on immobilization and the half lives of immobilized enzyme preparations at 30 and 40 °C were 30 and 11.5 d, respectively. With 2.5 mm beads diffusional resistances were observed. Reusability studies showed that 1 mm size beads gave a higher rate of transformation in comparison with 2 or 2.5 mm beads.     

Optimization of culture conditions for the production of rifamycin oxidase by Curvularia lunata

Maximum activity (8.9 IU/ml) of rifamycin oxidase in Curvularia lunata, grown in shake-flask culture at 28°C and pH 6.5, was after 96 h. Nearly all the glucose was used in 72 h. An initial culture pH of 6.5 and 28°C were optimum for the growth and enzyme production. Among various carbon and organic nitrogen sources, carboxymethylcellulose and peptone were the most effective for enzyme yield. The rate of enzyme production was enhanced when yeast extract was also added to the medium. The optimum medium for the production of rifamycin oxidase contained 10 g each of yeast extract, peptone and carboxymethylcellulose/l and 0.04% (NH₄)₂SO₄.The author is with the Biochemical Engineering Research and Process Development Centre, Institute of Microbial Technology, Post Box 1304, Sector 39-A, Chandigarh 160 014, India     

Effects of different factors on the production of cellulase byCurvularia lunata

A purified culture of the fungal pathogenCurvularia lunata causing leaf blight of pearl millet was used for studies on the production of cell-wall-degrading enzymes. Czapek-Dox medium was found to be the best medium of the five different nutrient media used for the production of cellulase and growth of the fungus. Pectolytic enzymes could not be detected under different cultural conditions. Two-week incubation period, pH 6.0 and temperature 25°C were found to be the most favorable conditions for good growth and maximum production of cellulase by the fungus in present studies. The results are presented and discussed in the light of the earlier findings onC. lunata.     

Characterization of soluble rifamycin oxidase from Curvularia lunata var. aeria

Curvularia lunata var. aeria was grown on yeast extract, peptone and carboxymethylcellulose (YPC) medium for the production of extracellular rifamycin oxidase. The enzyme was partially purified through a Sephadex G-75 column. The half lives of rifamycin oxidase at 30° and 40°C were 9 d and 100 min, respectively. The activation and deactivation energies of the partially purified enzyme, calculated from Arrhenius plots, were 5.80 and 35.10 kcal mol^-1 respectively. The enzyme exhibited a K _m (rifamycin B) value of 0.67 mmol l^-1 and a V _max of 11 μmol h^-1 ml. Three metal ions, Fe²⁺, Ag⁺ and Hg²⁺, inhibited the enzyme in the 10–20 mmol l^-1 metal ion concentration range. Catalytic activity was not affected by the chelating agent, EDTA.     

Microbial transformation of rifamycin B: A new extracellular oxidase fromCurvularia lunata

Summary A highly active extracellular rifamycin oxidase was isolated fromCurvularia lunata var.aeri. The enzyme has a pH optimum of 6.5 and temperature optimum of 50°C.     

Stabilization of rifamycin-B oxidase fromCurvularia lunata

Summary The present investigation deals with the stabilization of rifamycin-B oxidase activity in the culture filtrate ofCurvularia lunata using various methods. It was found that rifamycin-B oxidase activity in the culture filtrate was stable up to 5 days at 5°C, degrading thereafter due to microbial contamination. The stabilization of enzyme activity was carried out by (i) concentration of culture filtrate and (ii) lyophilization, the activity remaining intact for 24 and 75 days, respectively. In another method, the enzyme activity was preserved by addition of 25–30% glycerol to the culture filtrate and the storability of enzyme activity then increased up to 90 days at 5°C. The conversion of rifamycin-B to rifamycin-S using the stabilized rifamycin-B oxidase and fresh culture filtrate were comparable when run under similar conditions. The recovery of rifamycin-S powder from these experiments was not affected in any way in the presence of glycerol. Therefore, the present method of preservation of rifamycin-B oxidase may find industrial application for commerical production of rifamycin-S, which is an important intermediate for the synthesis of an antituberculosis drug, rifamycin.     

Solid state cultivation of Curvularia lunata for transformation of rifamycin B to S

Biotransformation of rifamycin B to rifamycin S using two strains of C. lunata namely NCIM 716 and NMU grown on various solid substrates viz., grass, paper, jowar/wheat straw, bran and bagasse was studied. Almost complete biotransformation efficiency of rifamycin B at 0. 06 mM concentration was observed within 24 hr. Among these two strains, C. lunata NMU showed 90% of biotransformation and higher rate of cellulose utilization on solid substrates vis-à-vis reference strain. Cellulase activity of both strains was also studied for exoglucanase, endoglucanase and beta-glucosidase. Column bioreactor studies with bagasse revealed further improvement in biotransformation efficiency of C. lunata NMU.     

Characterization of rifamycin oxidase immobilized on nylon fibers

Summary Rifamycin oxidase, an enzyme used in the biotransformation of rifamycin B to S was immobilized on nylon fibers using glutaraldehyde as the cross linking agent. An activity of 18 U/g of nylon fiber with a binding efficiency of 37% was achieved. The immobilized enzyme showed an operational stability of 7 days and was also protected against thermal inactivation. It exhibited a K_m(app.) of 2.0mM.     

Emulsifier production by steroid transforming filamentous fungus Curvularia lunata. Growth and product characterization.

Curvularia lunata IM 2901, the filamentous fungus used for hydrocortisone manufacture, was found to be able to produce an extracellular emulsifying agent on water-soluble compounds with a yield of 2.6 g l(-1). Cell-free culture broth containing the examined agent caused the formation of stable emulsions with hydrophobic compounds of natural and xenobiotic origin. Vegetable and mineral oils were the best substrates for emulsification. Kinetics of surfactant biosynthesis was classified as a mixed growth-associated and non-growth-associated type. The crude emulsifier was isolated from culture fluid by acetone precipitation. Preliminary chemical characterization showed that the studied bioemulsifier contained 34.0% C, 5.7% H, 1.8% N, 0.15% S and was a complex of protein (25%) and polysaccharide (48%). Sugar component was identified as a polymer of D-glucose.     

Growth characteristics and glucose oxidase production by mutant Penicillium funiculosum strains

The main parameters of growth and glucose oxidase production by the mutant Penicillium funiculosum strains BIM F-15.3, NMM95.132, and 46.1 were studied. The synthesis of extracellular glucose oxidase by these strains was constitutive and occurred following the phase of exponential growth. The mutant strains also synthesized extracellular invertase and cell-associated catalase and glucose oxidase. The syntheses of invertase, the cell-associated enzymes, and extracellular glucose oxidase were found to be maximum between 14 and 18 h, between 48 and 52 h, and by the 96th h of cultivation, respectively. Among the mutants studied, P. funiculosum 46.1 showed the maximal rates of growth and glucose oxidase synthesis.     

Production of laccase byCurvularia sp.

ACurvularia sp. isolated from soil was found to contain laccase activity toward guaiacol as substrate. The organism produced an extracellular laccase in a medium containing yeast extract, peptone and dextrose. Initial medium pH 4.0 and cultivation temperature 30°C were found to be most suitable for maximum enzyme production. The optimum pH and temperature for laccase activity were found to be 5.2 and 50°C, respectively. Under optimum conditions, the enzyme had aK _m (guaiacol) of 0.75 mmol/L and aV of 1.50 CU min⁻¹ ml⁻¹. Some divalent metal ions inhibited laccase activity at very low concentrations.     

Tertiary acetates controlling the mycotoxin production of Curvularia lunata

Summary The biosynthesis of radicinin, which is a mycotoxin excreted from Curvularia lunata cultures, was activated by a system that maintains a constant level of acetate in the medium. The system consists of tertiary alkylacetates sparingly soluble in water, and esterases secreted into the medium by the fungus.     

Studies on fermentative production of rifamycin using Amycolatopsis mediterranei

The fermentative production of rifamycin by Amycolatopsis mediterranei (MTCC17) has been studied. Both qualitative and quantitative aspects of the fermentation were determined by optimizing cultural conditions and medium design to improve the production of rifamycin. A pH value of 7.0, a temperature of 26°C, an aeration rate of 250rev/min for a 50ml volume, a level of inoculum of 10% grown aeration for 48h and a fermentation period of 11days were found to be optimum. Among the nitrogen sources, and culture conditions, peanut meal and aeration were found to be critical for rifamycin production respectively. The above mentioned exercise increased the yields of rifamycin from 350mg/l to 2000mg/l.     

The properties of immobilized whole cell ofHumicola spp. with rifamycin oxidase activity

Summary The whole cell ofHumicola spp. ATCC 20620 with rifamycin oxidase activity was immobilized by copolymerization with acrylamide. The whole cell was defatted by treatment with acetone to reduce the diffusional resistance through the cell membrane. The recovery of enzyme activity after the immobilization step was about 50%. The acetone-defatted cell showed the maximum activity at pH 7.5 for both free and the immobilized forms. No appreciable activity loss could be detected when stored at 4 °C and pH 7.8 for one month, while the half life at 40 °C and pH 8 was decreased to about 8 days. The apparent Km values of rifamycin oxidase for the free and immobilized acetonedefatted cells were 0.3mM and 0.6mM, respectively. The enzyme demonstrated substrate inhibition, but the degree of substrate inhibition was different between two forms of the enzyme preparation. A complete substrate inhibition was observed for the immobilized cell, whereas the enzyme activity was partially inhibited at high substrate concentration in the acetone-defatted cells.     

Improvement of rifamycin B production using mutant strains of Amycolatopsis mediterranei

Two mutant strains of Amycolatopsis mediterranei VA17 and VA18 were isolated using physical (UV) and chemical (NTG) mutagens gave high rifamycin B than the parent type when grown in the same fermentation medium with a pH of 7.2, temperature 32v°C for a period of 12 days. The cultural conditions of both mutant strains are similar to the parent strain except temperature which was higher by 4v°C. By this mutation and selection study, rifamycin B production was improved from 1400 mg/l to 2450 mg/l.     

Inhibition of infectious Rous sarcoma virus production by rifamycin derivative.

A new rifamycin derivative, rifazone-82 (R-82), an inhibitor of viral RNA-dependent DNA polymerase, is selectively toxic to transformed chicken cells in culture. R-82 has now been shown to possess antiviral activity as well. The relatively nontoxic properties of R-82 to nontransformed cells have permitted the execution of experiments examining the effect of a rifamycin derivative on virus reproduction. Addition of low concentrations of R-82 (15 mug/ml) to cultures soon after Rous sarcoma virus infection prevents the spread of infection thoroughout the culture. This inhibition is not dependent on concomitant cellular transformation as identical results were obtained with cells infected with a transformation-defective Rous sarcoma virus. Addition of R-82 to cultures in which all the cells are infected does not substantially affect the yield of physical particles as measured by RNA-dependent DNA polymerase activity and by (3H) uridine incorporation into viral RNA. However, the infectivity of the progeny virus, as measured by focus-forming ability, is decrreased 95 to 99% by R-82 treatment.     

Pharmacokinetics of rifamycin

Rifamycin pharmacokinetics was studied on experimental animals after the antibiotic administration by various routes. Parenteral use of the antibiotic resulted in its high levels in rats and rabbits. Irrespective of the administration route, i. e. intravenous, intramuscular or oral rifamycin satisfactorily penetrated into the rat tissues. The highest antibiotic levels were found in the animal liver. In small amounts the antibiotic was excreted with the urine (about 6 per cent for 4 hours). The extrarenal clearance of rifamycin was lower than the plasmic clearance only by 3 per cent and higher than the kidney clearance almost by 40 times. Rifamycin was bound in close amounts by the blood serum of humans, oxen and rabbits, i. e. by 68, 64 and 56 per cent respectively. The rat organ homogenates bound the antibiotic by 34--72 per cent.     

Effect of uracil on rifamycin SV production by Amycolatopsis mediterranei MV35R

The effect of different organic nitrogen compounds on the production of rifamycin SV by Amycolatopsis mediterranei MV35R and their optimum concentrations have been described. Results obtained indicate that rifamycin SV production increased from 4020 mg l-1 to 4575 mg l-1 when organic nitrogen compound uracil was added at 0.2% (w/v) concentration to the fermentation medium by A. mediterranei MV35R. The rifamycin SV yield was enhanced by 505 mg l-1 using uracil (2 g l-1) when compared with barbital.