Effect of low molecular weight regulators on the biosynthesis of rifamycin B by Amycolatopsis mediterranei strains

Formation of differentiation regulators of the A-factor group in representatives of Nocardia and Nocardia-like actinomyces: N. asteroides, N. brasiliensis, Amycolatopsis mediterranei and "Streptomyces listeri" was observed. The effect of the regulators of different nature (barbital, A-factor and B-factor) on biosynthesis of rifamycin B by A. mediterranei strains was studied. It was shown that the A-factor stimulated rifamycin B production in the adifferentiated low active variant isolated from a natural population of the active strain VNIIA 1713 of the rifamycin B-producing culture. B-Factor insignificantly inhibited biosynthesis of rifamycin B in the studied strains of A. mediterranei.     

Characterization of volatile compounds produced by Rhizopus strains grown on agro-industrial solid wastes

Four edible Rhizopus strains were cultivated on eight combinations of solid agro-industrial wastes (cassava bagasse, apple pomace), soyabean, amaranth grain and soyabean oil. Significant differences in growth were observed among strains on the different media studied. The medium containing cassava bagasse with soyabean (5:5 w/w) gave the highest CO₂ production, while Rhizopus oryzae ATCC 34612 was the best producer of volatiles. The aromas of the cultures were light and rather pleasant. The amaranth medium with mineral salts solution produced the highest amount of volatile compounds (VC), demonstrating that the aroma of fermented solid substrates can be improved. The VC production was very rapid, attaining, in most of the cases, its maximum around the first day of culture. These maxima markedly varied according to the medium used.     

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.     

Radiation and fermentation treatment of cellulosic wastes

The effect of radiation pasteurization of sugar cane bagasse and rice straw and fermentation using various strains of fungi were studied for upgrading of cellulosic wastes. The initial contamination by fungi and aerobic bacteria both in bagasse and straw was high. The doses of 30 kGy for sterilization and 8 kGy for elimination of fungi were required. Irradiation effect showed that rice straw contained comparatively radioresistant microorganisms. It was observed that all the fungi (Hericium erinacium, Pleurotus djamor, Ganoderma lucidum, Auricularia auricula, Lentinus sajor-caju, Coriolus versicolor, Polyporus arcularius, Coprinus cinereus) grow extending over the entire substrates during one month after inoculation in irradiated bagasse and rice straw with 3% rice bran and 65% moisture content incubated at 30°C. Initially, sugar cane bagasse and rice straw substrates contained 39.4% and 25.9% of cellulose, 22.9% and 26.9% of hemicellulose, and 19.6% and 13.9% of lignin + cutin, respectively. Neutral detergent fibre (NDF) values decreased significantly in sugar cane bagasse fermented byG. lucidum, A. auricula andP. arcularius, and in rice straw fermented by all the 8 strains of fungi. Acid detergent fibre (ADF) values also decreased in bagasse and rice straw fermented by all the fungi.P. arcularius, H. erinacium, G. lucidum andC. cinereus were found to be the most effective strains for delignification of sugar cane bagasse.     

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.     

玉米弯孢叶斑病菌的形态学分类与RAPD分析

对16个供试弯孢分离株根据其分生孢子的形态进行分类,其中12个为新月弯孢(Curvularia luna-ta),其余4个是画眉草弯孢(C.eragrostidis)。对这些分离株基因组DNA进行RAPD分析发现,一些分属于两种不同弯孢菌的分离株间比同种弯孢的分离株间具有更近的遗传距离,即具有更近的亲缘关系。因此,根据弯孢菌的形态分类和利用RAPD标记的分类结果存在不一致现象。     

Novel assay for screening rifamycin B-producing mutants.

A simple method that allows easy identification of rifamycin B-producing strains is described. This method involves the use of an enzyme, rifamycin oxidase, which converts inactive rifamycin B to active rifamycin S. In this method, colonies to be tested are grown in pairs. The two colonies are then transferred to two plates seeded with a sensitive strain of Staphylococcus aureus, one plate of which contains the enzyme rifamycin oxidase. All paired colonies which show a larger inhibition zone diameter on the enzyme-containing plate are identified as rifamycin B producers.     

Novel assay for screening rifamycin B-producing mutants.

A simple method that allows easy identification of rifamycin B-producing strains is described. This method involves the use of an enzyme, rifamycin oxidase, which converts inactive rifamycin B to active rifamycin S. In this method, colonies to be tested are grown in pairs. The two colonies are then transferred to two plates seeded with a sensitive strain of Staphylococcus aureus, one plate of which contains the enzyme rifamycin oxidase. All paired colonies which show a larger inhibition zone diameter on the enzyme-containing plate are identified as rifamycin B producers.     

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.     

Discovery of a new source of rifamycin antibiotics in marine sponge actinobacteria by phylogenetic prediction

Phylogenetic analysis of the ketosynthase (KS) gene sequences of marine sponge-derived Salinispora strains of actinobacteria indicated that the polyketide synthase (PKS) gene sequence most closely related to that of Salinispora was the rifamycin B synthase of Amycolatopsis mediterranei. This result was not expected from taxonomic species tree phylogenetics using 16S rRNA sequences. From the PKS sequence data generated from our sponge-derived Salinispora strains, we predicted that such strains might synthesize rifamycin-like compounds. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis was applied to one sponge-derived Salinispora strain to test the hypothesis of rifamycin synthesis. The analysis reported here demonstrates that this Salinispora isolate does produce compounds of the rifamycin class, including rifamycin B and rifamycin SV. A rifamycin-specific KS primer set was designed, and that primer set increased the number of rifamycin-positive strains detected by PCR screening relative to the number detectable using a conserved KS-specific set. Thus, the Salinispora group of actinobacteria represents a potential new source of rifamycins outside the genus Amycolatopsis and the first recorded source of rifamycins from marine bacteria.     

Screening of rifamycin producing marine sponge bacteria by LC-MS-MS

HPLC-MS-MS has been used for the identification and characterisation of rifamycin B and rifamycin SV in various strains of the marine sponge-derived bacterium Salinispora. Gradient elution using acetonitrile/water/ammonium acetate was used to separate the rifamycins from the matrix and negative ion-electrospray mass spectrometry was used for detection and confirmation. The presence of rifamycin in bacterial extracts was confirmed by matching retention times, parent ion spectra and the fragmentated parent ion spectra of the standard compounds and the bacterial extracts. All strains of the marine sponge bacterium Salinispora tested were found to contain rifamycin thus an alternate actinobacterial source of rifamycin was established.     

11β-Hydroxylation of norethisterone acetate by Curvularia lunata

Abstract: The biotransformation of norethisterone acetate by Curvularia lunata was studied. The 11β-hydroxy-norethisterone acetate produced was identified by mass spectrometry, and ¹H and ¹³C nuclear magnetic resonance after extractive sample preparation.     

Screening of thermophilic anaerobic bacteria for solid substrate cultivation on lignocellulosic substrates

Interest in solid substrate cultivation (SSC) techniques is gaining for biochemical production from renewable resources; however, heat and mass transfer problems may limit application of this technique. The use of anaerobic thermophiles in SSC offers a unique solution to overcoming these challenges. The production potential of nine thermophilic anaerobic bacteria was examined on corn stover, sugar cane bagasse, paper pulp sludge, and wheat bran in submerged liquid cultivation (SmC) and SSC. Production of acetate, ethanol, and lactate was measured over a 10 day period, and total product concentrations were used to compare the performance of different organism-substrate combinations using the two cultivation methods. Overall microbial activity in SmC and SSC was dependent on the organism and growth substrate. Clostridium thermocellum strains JW20, LQRI, and 27405 performed significantly better in SSC when grown on sugar cane bagasse and paper pulp sludge, producing at least 70 and 170 mM of total products, respectively. Growth of C. thermocellum strains in SSC on paper pulp sludge proved to be most favorable, generating at least twice the concentration of total products produced in SmC (p-value < 0.05). Clostridium thermolacticum TC21 demonstrated growth on all substrates producing 30-80 and 60-116 mM of total product in SmC and SSC, respectively. Bacterial species with optimal growth temperatures of 70 degrees C grew best on wheat bran in SmC, producing total product concentrations of 45-75 mM. For some of the organism-substrate combinations total end product concentrations in SSC exceeded those in SmC, indicating that SSC may be a promising alternative for microbial activity and value-added biochemical production.     

Biotransformation of cortexolone to hydrocortisone by molds using a rapid color development assay

Capabilities of 22 molds were assessed for 11beta-hydroxylation of cortexolone (Reichstein's compound S) to hydrocortisone. The biotransformation capability was compared for solid-state and submerged monocultures of the molds under otherwise identical conditions. A novel rapid color development assay and thin layer chromatography were used to qualitatively establish the ability of the fungi to convert cortexolone to hydrocortisone. These assays were validated and supplemented with data from high performance liquid chromatography to obtain quantitative information on the biotransformation. Nearly all the fungi consumed a significant fraction of the cortexolone fed, but only four (i.e. two isolates of Cunninghamella blakesleeana, C. echinulata and Curvularia lunata) yielded measurable quantities of hydrocortisone. Submerged cultures generally gave significantly greater yield of hydrocortisone compared to equivalent solid-state cultures.     

Biodelignification of Lemon Grass and Citronella Bagasse by White-Rot Fungi

Twelve white-rot fungi were grown in solid-state culture on lemon grass (Cymbopogon citratus) and citronella (Cymbopogon winterianus) bagasse. The two lignocellulosic substrates had 11% permanganate lignin and a holocellulose fraction of 58%. After 5 to 6 weeks at 20°C, nine fungi produced a solid residue from lemon grass with a higher in vitro dry matter enzyme digestibility than the original bagasse; seven did the same for citronella. The best fungus for both substrates was Bondarzewia berkeleyi; it increased the in vitro dry matter enzyme digestibility to 22 and 24% for lemon grass and citronella, respectively. The increases were correlated with weight loss and lignin loss. All fungi decreased lignin contents: 36% of the original value for lemon grass and 28% for citronella. Practically all fungi showed a preference for hemicellulose over cellulose.     

Aroma production by spores ofPenicillium roqueforti on a synthetic medium

Summary The synthesis of 2-heptanone from the sodium salt of octanoic acid by spores of five strains ofPenicillium roqueforti was studied. The strains showed a high disparity in kinetic behavior. The one selected, which was originally isolated from blue cheese, had a good resistance to substrate inhibition along with a good apparent biotransformation yield (close to 60%). An activator was needed in the incubation medium. The loss of activity of aging spores was reduced by the activator compounds; ethanol exhibited the highest efficiency. When spores were produced on buckwheat seeds with a solid state fermentation technique, the medium itself was an activator source. When the biotransformation reaction was carried out in a stirred aerated fermentor, the volatile loss by air-stream stripping had to be taken into account. No ketone metabolism occurred with the strain used.     

A Study of the cellulases produced by three mesophilic actinomycetes grown on bagasse as substrate

The cellulases that strains of Streptomyces albogriseolus, S. nitrosporeus, and Micromonospora melanosporea produce when grown on untreated ballmilled bagasse were investigated. Optimum conditions for extracellular cellulase production and activity were determined to be growth at pH 6.7-7.4 and 25-35 degrees C for 4-5 days and assay at pH 5.0-6.0 and 45-55 degrees C, respectively. The endoglucanases were thermally stable at 50 degrees C, but the Avicelases had a half-life of approximately 24 h at this temperature. Nearly half of the endoglucanases and almost all of the Avicelases were absorbed on ballmilled bagasse after 15 min incubation at 50 degrees C. The beta-glucosidases were found to be mainly intracellular or cell wall bound. These mesophilic actinomycetes concomitantly produced xylanases and beta-xylosidases with cellulases that, apart from cellobiose and glucose, also release xylose from bagasse. This feature may be advantageous in the commerical application of the enzymes of mesophilic actinomycetes for the saccharification of natural cellulosic substrates.     

Stability of rifamycin B in aqueous solutions

Stability of rifamycin B in aqueous solutions at various values of pH and temperature was studied. It was shown that inactivation of the antibiotic in neutral and alkaline solutions proceeded according to the first order equation. In acid solutions rifamycin B was oxidized with air oxygen to form rifamycin O.     

Rifamycins: an insight into biological activity based on structural investigations

The structures of two compounds of the antibiotic rifamycin series have been studied by X-ray methods: rifamycin B, C₃₉H₄₉NO₁₄, which is active on bacterial RNA polymerase, and rifamycin Y, C₃₉H₄₇NO₁₅, which is devoid of any antibacterial activity. In both cases their p-iodoanilide derivative has been used and the final crystal structure data are now given.A comparative study of the structures of rifamycin B and rifamycin Y, and of the structures of two other ansa-compounds with similar activity on bacterial RNA polymerase, tolypomycin Y and streptovaricin C, together with the analysis of the activity ratios of a series of semisynthetic rifamycins, makes it possible to suggest that the following constitutional and conformational features are common to all the ansa-compounds with anti-bacterial activity: (i) a naphthoquinone or naphthohydroquinone nucleus with free oxygen functions, O(1) and O(2), on C(1) and C(8) respectively: (ii) an amide nitrogen attached to C(2); (iii) a 17-membered chain bridging the chromophoric group; (iv) two free hydroxyl groups attached to atoms C(21) and C(23) of the ansa-bridge and in the same relative positions with respect to each other and with respect to O(1) and O(2). However, the constitution and the conformation of the remaining part of the ansa-bridge can be rather different in different compounds.     

RNA polymerase-rifamycin. A molecular model of inhibition

By fluorimetric titration of Rifs (E. coli B) and Rifr (E. coli rpoB255) RNA polymerases with rifamycin, the mutant polymerase was demonstrated to bind rifamycin. A comparison of spatial structures of rifamycin and dinucleotide fragment of RNA in the hybrid with DNA revealed their similarity. Taking into account this structural similarity and also the fact that two phosphodiester bonds can be formed by RNA polymerase in the presence of rifamycin, a model for the inhibition mode was proposed. According to this model, rifamycin occupies the place of two terminal nucleotides of synthesized, but not translocated pentanucleotide in the transcribing complex. Asp-516 of the wild type beta-subunit was assumed to form a hydrogen bond with the rifamycin C(23) hydroxyl group. On the base of this model, reduced "cycling" synthesis of tetra-, penta-... up to decanucleotides by the Rifr RNA polymerase, in comparison with Rifs, was predicted.