Isolation of a Novel Carotenoid,OH-chlorobactene Glucoside Hexadecanoate,and Related Rare Carotenoids from Rhodococcus sp. CIP and Their Antioxidative Activities

In the course of screening for antioxidative carotenoids from bacteria, we isolated and identified a novel carotenoid, OH-chlorobactene glucoside hexadecanoate (4), and rare carotenoids, OH-chlorobactene glucoside (1), OH-γ-carotene glucoside (2) and OH-4-keto-γ-carotene glucoside hexadecanoate (3) from Rhodococcus sp. CIP. The singlet oxygen (¹O₂) quenching model of these carotenoids showed potent antioxidative activities IC₅₀ 14.6 μM for OH-chlorobactene glucoside hexadecanoate (4), 6.5 μM for OH-chlorobactene glucoside (1), 9.9 μM for OH-γ-carotene glucoside (2) and 7.3 μM for OH-4-keto-γ-carotene glucoside hexadecanoate (3).     

Characterization of a secreted cholesterol oxidase from Rhodococcus sp.GKI (CIP 105335)

Extracellular cholesterol oxidase (COX) (EC 1.1.3.6) was produced by Rhodococcus sp. GK1 cells grown in a defined mineral salt medium containing a mixture of phytosterols (sitosterol, campesterol, stigmasterol) as the sole source of carbon and energy. In the same time, the sterols acted as enzyme inducers. The medium was enriched with yeast extract in order to stimulate enzyme secretion. COX was purified from the culture supernatants by affinity-like chromatography on a column packed with kieselguhr and cholesterol. Enzyme bound onto the column was eluted with 0.05 M phosphate buffer pH 7.0 containing Triton X-100 at 0.1% (w/v). Some properties of the purified COX were determined. Its specific activity at pH 7.0 and 30 °C, was around 5.5 units mg^–1. The molecular mass of the enzyme, as estimated by SDS-PAGE, was 59 kDa. Its isoelectrofocusing point was around pH 8.9. The C-5 double bond and the alkyl chain moiety in sterol molecules were necessary for an adequate oxidation of the sterol 3-ol. Enzyme inhibition by the ions (0.1 mM): AsO₂ ^–, Ba²⁺, Co²⁺, Cd²⁺, Cu²⁺, N₃ ^–, Ni²⁺, and Pb²⁺ was negligible (around 10%). However, COX inhibition by 0.1 mM of either Zn²⁺, 2-[(ethylmercurio)-thio]benzoic acid, or Hg²⁺ was 18%, 22% and 93% respectively. Inhibition of activity by Hg²⁺ was significant, even at 1 M. The purified COX (0.1–0.15 mg ml^–1 in 0.05 M phosphate pH 7.0) was relatively heat-stable at temperatures up to 50 °C. At this temperature, the half-life of its activity was around 70 min. However, 90% of the enzyme initial activity was lost by 20 min incubation at 60 °C. The aminoacid sequence of the COX N-terminal segment was: H2N–Ala–Pro–Pro–Val–Ala–Ser–X–Arg–Tyr–X–(Phe)– (X might be 2 Cys residues).     

Isolation of a highly enantioselective epoxide hydrolase from Rhodococcus sp. NCIMB 11216

Summary Whole cells of Rhodococcus sp. NCIMB 11216 catalyze the asymmetric hydrolysis of racemic epoxides giving access to chiral epoxides and diols, which are important chiral building blocks for the synthesis of bioactive compounds. Employing a four-step purification procedure, the epoxide hydrolase responsible for the reaction was isolated and characterized to be a cofactor-independent, soluble monomeric protein of ~35kDa, exhibiting an isoelectric point of 4.7.     

New carotenoids from the thermophilic green sulfur bacterium Chlorobium tepidum: 1′,2′-dihydro-γ-carotene, 1′,2′-dihydrochlorobactene, and OH-chlorobactene glucoside ester, and the carotenoid composition of different strains

The complete carotenoid composition of the thermophilic green sulfur bacterium Chlorobium tepidum strain TNO was determined by spectroscopic methods. Major carotenoids were four kinds of carotenes: γ-carotene, chlorobactene, and their 1′,2′-dihydro derivatives (1′,2′-dihydro-γ-carotene and 1′,2′-dihydrochlorobactene). In lesser amounts, hydroxyl γ-carotene, hydroxyl chlorobactene, and their glucoside fatty acid esters were found. The only esterified fatty acid present was laurate, and OH-chlorobactene glucoside laurate is a novel carotenoid. In other strains of C. tepidum, the same carotenoids were found, but the composition varied from strain to strain. The overall pigment composition in cells of strain TNO was 4 mol carotenoids and 40 mol bacteriochlorophyll c per mol bacteriochlorophyll a. The effects of nicotine on carotenoid biosynthesis in C. tepidum differed from those in the thermophilic green nonsulfur bacterium Chloroflexus aurantiacus. Received: 3 February 1997 / Accepted: 6 June 1997     

Isolation and Characterization of a Carbendazim-Degrading Rhodococcus sp. djl-6

Bacterium djl-6, capable of degrading carbendazim, was isolated by continuous enrichment culture originating from carbendazim-treated soil. The isolate was identified as Rhodococcus sp. according to its phenotypic features, physiologic and biochemical characteristics, and phylogenetic analysis. The strain could use carbendazim as sole carbon or nitrogen source. It showed a high average degradation rate of 55.56 mg · L⁻¹ · d⁻¹ in M9 medium amended with carbendazim. High-pressure liquid chromatography–mass spectrometry (HPLC-MS) analysis showed the presence of 2-aminobenzimidazole, benzimidazole, and an unknown metabolite with molecular ions (M⁺) of m/z 104.8 and 118.5. The degradation in the isolate djl-6 seems to be initiated with the cleavage of the methyl carbemate side chain, resulting in the formation of 2-aminobenzimidazole and benzimidazole. This is the first report of the intermediates benzimidazole and 2-aminobenzimidazole found together in the culture filtrate of pure bacterium.     

暗盘孢属YM421黑色素稳定性及其抗氧化活性

对暗盘菌Plectania sp.YM421菌株所产黑色素的稳定性及其抗氧化活性进行了研究。结果表明:该黑色素易溶于氢氧化钠溶液和二甲亚砜,微溶于蒸馏水,不溶于盐酸、无水乙醇、乙酸乙酯、氯仿、二甲苯、丙酮和乙醚;该黑色素在pH≥6时稳定,pH≤5时产生沉淀;对室光、日光、紫外光、亚硫酸钠、Na+和K+稳定;添加苯甲酸钠、柠檬酸钠、蔗糖、葡萄糖、乳糖、麦芽糖对黑色素有增色或护色作用;而双氧水、柠檬酸、VitC、Mg2+、Cu2+、Zn2+、Ca2+、Fe3+、Al3+降低其稳定性;该黑色素与VitC还原能力的EC50值(吸光度为0.5时的浓度)分别为169.6μg/mL和31.88μg/mL,对羟基清除作用的IC50值(清除率为50%时的浓度)分别为360.4μg/mL和183μg/mL。作为一种新型的天然色素,YM421黑色素有望应用于食品、化妆品以及医药等行业。     

Triterpenoid carotenoids and related lipids. Triterpenoid carotenoid aldehydes from Streptococcus faecium UNH 564P.

1. The identification of two novel triterpenoid xanthophylls from Streptococcus faecium UNH 564P is described. 2. Both are aldehydes and were identified as 4,4'-diaponeurosporen-4-al and 4,4'-diapolycopen-4-al. 3. A pathway is presented for the biosynthesis of these and other triterpenoid carotenoids in S. faecium.     

Isolation and identification of a novel Rhodococcus sp. ML-0004 producing epoxide hydrolase and optimization of enzyme production

Rhodococcus sp. ML-0004, a novel strain for producing epoxide hydrolase, was isolated from soil in this study. The epoxide hydrolase can catalyze the stereo-specific hydrolysis of cis-epoxysuccinic acid to generate l(+)-tartaric acid. By examining physiological, biochemical characteristics and comparing its 16S rDNA gene sequence, it was identified as Rhodococcus opacus, and named R. opacus ML-0004. The optimal conditions for epoxide hydrolase production from R. opacus ML-0004 were also investigated. Propanediol and (NH₄)₂SO₄ were selected as carbon source and nitrogen source, respectively, for the production of R. opacus ML-0004 epoxide hydrolase. The optimal conditions for epoxide hydrolase production were fermentation temperature = 28 °C, pH 7.0, and cultivation time = 26 h. Under these conditions, the maximum epoxide hydrolase activity reached 10.5 U mL⁻¹.     

Rare carotenoids, (3R)-saproxanthin and (3R,2'S)-myxol, isolated from novel marine bacteria (Flavobacteriaceae) and their antioxidative activities

We isolated three orange or yellow pigment-producing marine bacteria, strains 04OKA-13-27 (MBIC08261), 04OKA-17-12 (MBIC08260), and YM6-073 (MBIC06409), off the coast of Okinawa Prefecture in Japan. These strains were classified as novel species of the family Flavobacteriaceae based on their 16S rRNA gene sequence. They were cultured, and the major carotenoids produced were purified by chromatographic methods. Their structures were determined by spectral data to be (3R)-saproxanthin (strain 04OKA-13-27), (3R,2'S)-myxol (strain YM6-073), and (3R,3'R)-zeaxanthin (strains YM6-073 and 04OKA-17-12). Saproxanthin and myxol, which are monocyclic carotenoids rarely found in nature, demonstrated significant antioxidative activities against lipid peroxidation in the rat brain homogenate model and a neuro-protective effect from L-glutamate toxicity.     

Isolation and characterization of a novel Rhodococcus strain with switchable carbonyl reductase and para-acetylphenol hydroxylase activities

In the search for an effective biocatalyst for the reduction of acetophenones with unprotected hydroxy group on the benzene ring, a microorganism, which reduced para-acetylphenol to S-(?)-1-(para-hydroxyphenyl)ethanol under anaerobic conditions, was isolated from soil samples and the 16S rDNA study showed that it was phylogenetically affiliated with species of the genus Rhodococcus and was most similar to Rhodococcus pyridinivorans. Unexpectedly, this strain also hydroxylated para-acetylphenol to give 4-acetylcatechol in presence of oxygen, possessing para-acetylphenol hydroxylase activity. While the reduction of para-acetylphenol had an optimal reaction pH at 7 and a broad optimal temperature range (35–45 °C), the hydroxylation reached the maximum conversion at the pH range of 7–8 and 35 °C. This study identified for the first time a Rhodococcus strain with para-acetylphenol hydroxylase activity, which also contains highly enantioselective carbonyl reductase activity with potential applications for the asymmetric reduction of these less-explored but important ketones such as α-aminoacetophenone, 3′-hydroxyacetophenone and 4′-hydroxyacetophenone. The para-acetylphenol hydroxylase and carbonyl reductase activity are switchable by the reaction conditions.     

亚肉座菌天然产物的分离鉴定及抗肿瘤活性

亚肉座菌能侵染粉虱和介壳虫,具有悠久的生物防治历史,但关于其天然产物的种类及抗肿瘤活性研究尚不深入。本研究借助硅胶柱、HPLC等色谱技术从亚肉座菌发酵液中分离纯化到6个化合物。基于核磁共振和质谱数据解析,化合物1–6依次鉴定为:5α,8α-表二氧-24(R)-甲基胆甾-6,22-二烯-3β-醇5α,8α-epidioxy-24(R)-methylcholesta-6,22-diene-3β-ol(1),杜斯塔宁dustanin(2),3β-乙酸基-15α,22-二羟基何伯烷3β-acetoxy-15α,22-dihydroxyhopane(3),麦角固醇ergosterol(4),7β,15α,22-三羟基何伯烷7β,15α,22-trihydroxyhopane(5)和5-(羟甲基)呋喃-3-羧酸flufuran(6),化合物1和6为首次从虫生真菌中获得。对化合物进行抗肿瘤活性测试发现,化合物1和5对人肝癌细胞(BEL-7404)展示显著的抗肿瘤活性,IC50值分别为28μmol/L和12.4μmol/L,而它们对人正常肝脏细胞(HL-7702)和肾脏上皮细胞(HEK-293T)均未呈现出明显活性。该研究对阐明亚肉座菌代谢产物及其活性提供了重要数据,为开发抗肿瘤新药提供了理论参考。     

Isolation and characterization of a novel methylhopanoid from a facultative methylotrophicCorynebacterium sp.

Several compounds such as a methylhopanoid and carotenoids have been isolated and characterized from a facultative methylotrophicCorynebacterium sp., a vitamin B¹² producer. A novel pentacyclic triterpene, 2-methyl-22-hydroxyhopane has been identified by IR,₁H-and¹³C-NMK and mass spectrometry. During the purification procedure a red pigment has been characterized as a mixture of several carotenoids by TLC and UV-VIS spectroscopy.     

Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis. Isolation and physiological characterization

Rhodococcus erythropolis cells growing in a cholesterol-free glycerol-containing mineral medium displayed very low levels of a cell-wall-bound cholesterol oxidase activity. Addition of cholesterol induced a marked increase in the synthesis of this enzyme, which reached a maximum within 6 days and was subsequently followed by the appearance of extracellular cholesterol oxidase in the culture broth. Significant levels of induction were only achieved when cholesterol emulsified with Tween 80. The presence of chloramphenicol at the time of induction completely prevented the emergence of both enzymatic forms, suggesting the requirement of de novo protein synthesis. Upon transfer of cholesterol-growing cultures to fresh medium lacking cholesterol, the extracellular cholesterol oxidase was quickly erased, while the activity of the particulate enzyme decreased sharply. The electrophoretic pattern on native Western blotting as well as on sodium dodecyl sulphate/polyacrylamide gels, together with kinetic data, strongly support the idea that the particulate and extracellular cholesterol oxidases are two different forms of the same enzyme with an estimated molecular mass of 55 kDa. Received: 26 September 1996 / Received revision: 30 December 1996 / Accepted: 4 January 1997     

Elucidation of a carotenoid biosynthesis gene cluster encoding a novel enzyme, 2,2'-beta-hydroxylase, from Brevundimonas sp. strain SD212 and combinatorial biosynthesis of new or rare xanthophylls

A carotenoid biosynthesis gene cluster mediating the production of 2-hydroxyastaxanthin was isolated from the marine bacterium Brevundimonas sp. strain SD212 by using a common crtI sequence as the probe DNA. A sequence analysis revealed this cluster to contain 12 open reading frames (ORFs), including the 7 known genes, crtW, crtY, crtI, crtB, crtE, idi, and crtZ. The individual ORFs were functionally analyzed by complementation studies using Escherichia coli that accumulated various carotenoid precursors due to the presence of other bacterial crt genes. In addition to functionally identifying the known crt genes, we found that one (ORF11, named crtG) coded for a novel enzyme, carotenoid 2,2'-beta-hydroxylase, which showed intriguingly partial homology with animal sterol-C5-desaturase. When this crtG gene was introduced into E. coli accumulating zeaxanthin and canthaxanthin, the resulting transformants produced their 2-hydroxylated and 2,2'-dihydroxylated products which were structurally novel or rare xanthophylls, as determined by their nuclear magnetic resonance and high-performance liquid chromatography/photodiode array detector/atmospheric pressure chemical ionization mass spectrometry spectral data. The new carotenoid produced was suggested to have a strong inhibitory effect on lipid peroxidation.     

Isolation and characterization of an atrazine-degrading Rhodococcus sp. strain MB-P1 from contaminated soil

Aims:  The aim of this study is to isolate and characterize organisms capable of utilizing high concentration atrazine from the contaminated sites.
Methods and Results:  A selective enrichment was used for isolating atrazine-degrading organisms from the contaminated sites resulting in isolation of an efficient atrazine-degrading organism designated as strain MB-P1. On the basis of 16S rRNA gene sequencing, total cellular fatty acid analysis and physiological and biochemical tests, strain MB-P1 was identified as a member of genus Rhodococcus . High performance liquid chromatography was performed to identify the atrazine degradation intermediates demonstrating that the degradation proceeds via formation of 'de-ethylatrazine' and 'de-isopropylatrazine'. Further, plasmid curing by SDS method showed atrazine-degrading gene(s) to be plasmid-encoded.
Conclusions:  We have successfully isolated a Rhodococcus sp. strain MB-P1 which is capable of utilizing atrazine as sole source of carbon and energy at very high concentrations of 1000 ppm. The pathway for degradation of atrazine has also been determined. The metabolic gene(s) responsible for atrazine degradation was found to be plasmid-encoded.
Significance and Impact of the Study:  Rhodococcus sp. strain MB-P1 could be used as an ideal model system for in-situ degradation and restoration of ecological niches which are heavily contaminated with atrazine.     

Microbial transformation of squalene: Formation of a novel ketone from squalene by a Rhodococcus sp.

Summary A Rhodococcus sp., isolated from soil, was able to use squalene as the sole carbon source. The principal metabolic product from squalene was detected by thin layer and high performance liquid chromatography, and identified as 2, 6, 10, 15, 19, 23-hexamethyltetraco-sa-2, 6, 10, 14, 18, 22-hexaen-12-one by nuclear magnetic resonance, infrared, ultra-violet, and mass spectrometry.     

Isolation, characterization and antioxidative activity of C-phycocyanin from Limnothrix sp. strain 37-2-1

C-phycocyanin (C-PC) is a blue colored accessory photosynthetic pigment found in cyanobacteria. Some of the medicinal properties of Spirulina have been attributed to this pigment, which includes anticancer, antioxidant, and anti-inflammatory activity. We have screened cyanobacteria isolated from freshwater habitats in Florida for their high content of C-PC. Of 125 strains tested, one filamentous strain identified as Limnothrix sp. was selected for further research. This strain produced 18% C-PC of total dry biomass. Here we describe a simple method for obtaining C-PC of high purity without the use of ion exchange chromatography. The procedure is based on pigment precipitation from the cell lysate with an appropriate concentration of ammonium sulfate, then purification with activated carbon and chitosan, followed by a sample concentration using tangential flow filtration. We have shown that when the lower concentration of ammonium sulfate was used, C-PC with higher purity index was recovered. Characterization of C-PC from Limnothrix showed that it had an absorbance maximum at 620nm and fluorescence at 639nm. The molecular mass of intact C-PC was estimated to be ~50kDa with α and β subunits forming dimmers. When C-PC content per unit biomass was compared to that of marketed Spirulina powder, we found that Limnothrix was superior. C-phycocyanin from Limnothrix had an antioxidative activity on DPPH free radicals similar to that found in a natural antioxidant - rutin.     

Identification of carotenoid cleavage dioxygenases from Nostoc sp. PCC 7120 with different cleavage activities

Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes that oxidatively cleave carotenoids into apocarotenoids. Dioxygenases have been identified in plants and animals and produce a wide variety of cleavage products. Despite what is known about apocarotenoids in higher organisms, very little is known about apocarotenoids and CCDs in microorganisms. This study surveyed cleavage activities of ten putative carotenoid cleavage dioxygenases from five different cyanobacteria in recombinant Escherichia coli cells producing different carotenoid substrates. Three CCD homologs identified in Nostoc sp. PCC 7120 were purified, and their cleavage activities were investigated. Two of the three enzymes showed cleavage of beta,beta-carotene at the 9,10 and 15,15' positions, respectively. The third enzyme did not cleave full-length carotenoids but cleaved the apocarotenoid beta-apo-8'-carotenal at the 9,10 position. 9,10-Apocarotenoid cleavage specificity has previously not been described. The diversity of carotenoid cleavage activities identified in one cyanobacteria suggests that CCDs not only facilitate the degradation of photosynthetic pigments but generate apocarotenals with yet to be determined biological roles in microorganisms.