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     

Isolation and characterization of a rolling-circle-type plasmid from Rhodococcus erythropolis and application of the plasmid to multiple-recombinant-protein expression

We isolated, sequenced, and characterized the cryptic plasmid pRE8424 from Rhodococcus erythropolis DSM8424. Plasmid pRE8424 is a 5,987-bp circular plasmid; it carries six open reading frames and also contains cis-acting elements, specifically a single-stranded origin and a double-stranded origin, which are characteristic of rolling-circle-replication plasmids. Experiments with pRE8424 derivatives carrying a mutated single-stranded origin sequence showed that single-stranded DNA intermediates accumulated in the cells because of inefficient conversion from single-stranded DNA to double-stranded DNA. This result indicates that pRE8424 belongs to the pIJ101/pJV1 family of rolling-circle-replication plasmids. Expression vectors that are functional in several Rhodococcus species were constructed by use of the replication origin from pRE8424. We previously reported a cryptic plasmid, pRE2895, from R. erythropolis, which may replicate by a theta-type mechanism, like ColE2 plasmids. The new expression vectors originating from pRE8424 were compatible with those derived from pRE2895. Coexpression experiments with these compatible expression vectors indicated that the plasmids are suitable for the simultaneous expression of multiple recombinant proteins.     

Biochemical identification and biophysical characterization of a channel-forming protein from Rhodococcus erythropolis

Organic solvent extracts of whole cells of the gram-positive bacterium Rhodococcus erythropolis contain a channel-forming protein. It was identified by lipid bilayer experiments and purified to homogeneity by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The pure protein had a rather low molecular mass of about 8.4 kDa, as judged by SDS-PAGE. SDS-resistant oligomers with a molecular mass of 67 kDa were also observed, suggesting that the channel is formed by a protein oligomer. The monomer was subjected to partial protein sequencing, and 45 amino acids were resolved. According to the partial sequence, the sequence has no significant homology to known protein sequences. To check whether the channel was indeed localized in the cell wall, the cell wall fraction was separated from the cytoplasmic membrane by sucrose step gradient centrifugation. The highest channel-forming activity was found in the cell wall fraction. The purified protein formed large ion-permeable channels in lipid bilayer membranes with a single-channel conductance of 6.0 nS in 1 M KCl. Zero-current membrane potential measurements with different salts suggested that the channel of R. erythropolis was highly cation selective because of negative charges localized at the channel mouth. The correction of single-channel conductance data for negatively charged point charges and the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.0 nm. The channel-forming properties of the cell wall channel of R. erythropolis were compared with those of other members of the mycolata. These channels have common features because they form large, water-filled channels that contain net point charges.     

Isolation and Characterization of Carbendazim-degrading Rhodococcus erythropolis djl-11

Carbendazim (methyl 1H-benzimidazol-2-yl carbamate) is one of the most widely used fungicides in agriculture worldwide, but has been reported to have adverse effects on animal health and ecosystem function. A highly efficient carbendazim-degrading bacterium (strain dj1-11) was isolated from carbendazim-contaminated soil samples via enrichment culture. Strain dj1-11 was identified as Rhodococcus erythropolis based on morphological, physiological and biochemical characters, including sequence analysis of the 16S rRNA gene. In vitro degradation of carbendazim (1000 mg·L⁻¹) by dj1-11 in minimal salts medium (MSM) was highly efficient, and with an average degradation rate of 333.33 mg·L⁻¹·d⁻¹ at 28°C. The optimal temperature range for carbendazim degradation by dj1-11 in MSM was 25–30°C. Whilst strain dj1-11 was capable of metabolizing cabendazim as the sole source of carbon and nitrogen, degradation was significantly (P<0.05) increased by addition of 12.5 mM NH₄NO₃. Changes in MSM pH (4–9), substitution of NH₄NO₃ with organic substrates as N and C sources or replacing Mg²⁺ with Mn²⁺, Zn²⁺ or Fe²⁺ did not significantly affect carbendazim degradation by dj1-11. During the degradation process, liquid chromatography-mass spectrometry (LC-MS) detected the metabolites 2-aminobenzimidazole and 2-hydroxybenzimidazole. A putative carbendazim-hydrolyzing esterase gene was cloned from chromosomal DNA of djl-11 and showed 99% sequence homology to the mheI carbendazim-hydrolyzing esterase gene from Nocardioides sp. SG-4G.     

Genetic and biochemical characterization of a novel monoterpene epsilon-lactone hydrolase from Rhodococcus erythropolis DCL14

A monoterpene epsilon-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30 degrees C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.     

Cloning and characterisation of a new 2-deoxy-D-ribose-5-phosphate aldolase from Rhodococcus erythropolis

A new 2-deoxy-D-ribose-5-phoshate aldolase (DERA) gene was cloned from Rhodococcus erythropolis strain DSM 311, recombinantly expressed in Escherichia coli, and purified via affinity chromatography which yielded a homo-dimeric enzyme of 44.3 kDa as apparent by size exclusion chromatography. To characterise the enzyme, investigations about pH and temperature tolerance, stability, as well as analyses on resistance to organic solvents and acetaldehyde were performed. In addition, kinetic constants of the new DERA(RE) were compared to respective values of the DERA from E. coli (DERA(EC)). Stability of DERA(RE) turned out to be a crucial factor: The pH for optimal DERA(RE) activity was determined to be 7.0, whereas the highest stability was achieved at pH 9.0 with a half-life of approximately 20 days. The optimal temperature for DERA(RE) activity was 65 °C, but coupled with a rather low stability (half-life of 2 min). The highest stability was achieved at 25 °C. The new enzyme exhibits high resistance to organic solvents and acetaldehyde with a half-life being 2.5× higher compared to DERA(EC) under the exposure of 300 mM acetaldehyde. Hence it has the potential as a new promising biocatalyst with applications in organic synthesis.     

Identification and characterization of a transmissible linear plasmid from Rhodococcus erythropolis BD2 that encodes isopropylbenzene and trichloroethene catabolism.

Rhodococcus erythropolis BD2, which is able to utilize isopropylbenzene as a sole carbon and energy source, was shown to contain a conjugative linear plasmid, pBD2. The estimated size of pBD2 is 208 to 212 kb. Linear plasmid-deficient strains had lost both the isopropylbenzene degradation and trichloroethene degradation characteristics, as well as the arsenite resistance and mercury resistance phenotypes. Reintroduction of pBD2 restored all four characteristics. Conjugational transfer of pBD2 to a plasmidless mutant of strain BD2 and other R. erythropolis strains occurred at frequencies between 3.5 x 10(-5) and 2.6 x 10(-3) transconjugants per recipient. R. erythropolis BD2 degrades isopropylbenzene via 3-isopropylcatechol and 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate. Both isopropylbenzene-oxidizing and meta-cleavage activities were shown to correspond with the presence of pBD2. Southern hybridizations with DNA encoding the toluene dioxygenase structural genes (todC1C2BA) from Pseudomonas putida F1 revealed homology to linear plasmid DNA. These results indicate that the isopropylbenzene degradation pathway encoded by linear plasmid pBD2 is initiated by an isopropylbenzene dioxygenase analogous to toluene dioxygenase.     

Aryl acylamidase from Rhodococcus erythropolis NCIB 12273

Summary A Rhodococcus erythropolis strain was isolated from soil on the basis of its ability to use acetaminophen as the sole source of both carbon and energy for growth. When grown in a complex medium containing an anilide inducer compound, the bacterium exhibited aryl acylamidase (EC 3.5.1.13) activity. This activity was not subject to carbon or nitrogen repression by the growth medium constituents as the enzyme was present throughout the exponential growth phase. The anilide was converted to the corresponding aniline, which was not further degraded. The enzyme was partially purified by a variety of methods including a batch ion exchange procedure, column ion exchange chromatography and hydrophobic interaction chromatography. The enzyme had a maximum activity at around pH 8.0 and had a K_m for acetaminophen of 0.11 mM. Electrochemical assays of aryl acylamidase activity are described. The enzyme is suitable for use as a reagent in the clinical diagnostic measurement of acetaminophen. Offprint requests to: P. A. Vaughan     

Rhodococcus erythropolis 手性醇脱氢酶的克隆表达及其性质

【目的】探讨红串红球菌中一种醇脱氢酶的性质及其对酮酯类及酮类底物的催化能力。【方法】从红串红球菌(Rhodococcus erythropolis ATCC 4277)中获取一段长度为1047 bp的醇脱氢酶(adh)基因,插入载体pET-22b(+)后,在大肠杆菌中进行重组表达。15℃的低温下用自诱导培养基诱导24 h,以苯乙酮为底物测定醇脱氢酶酶活。【结果】测得该诱导条件下重组菌体细胞破碎上清中醇脱氢酶酶活力为2.6 U/mg。经温度、pH耐受性等分析,发现该酶最适pH在6.0-6.5之间,耐受温度可以达到60℃,并且在该温度下保持5 h后,酶活也能保留80%。对于β酮酯类底物的催化反应,以对乙酰乙酸乙酯的催化能力最高。用4-氯乙酰乙酸乙酯(COBE)为底物进行全细胞水相催化反应,经手性液相色谱分析,发现在催化产物以R型4-氯-3羟基丁酸乙酯(CHBE)为主。【结论】该酶在酮酯类的底物转化方面有良好的开发潜力及应用前景。     

Isolation and Characterization of a Rolling-Circle-Type Plasmid from Rhodococcus erythropolis and Application of the Plasmid to Multiple-Recombinant-Protein Expression

We isolated, sequenced, and characterized the cryptic plasmid pRE8424 from Rhodococcus erythropolis DSM8424. Plasmid pRE8424 is a 5,987-bp circular plasmid; it carries six open reading frames and also contains cis-acting elements, specifically a single-stranded origin and a double-stranded origin, which are characteristic of rolling-circle-replication plasmids. Experiments with pRE8424 derivatives carrying a mutated single-stranded origin sequence showed that single-stranded DNA intermediates accumulated in the cells because of inefficient conversion from single-stranded DNA to double-stranded DNA. This result indicates that pRE8424 belongs to the pIJ101/pJV1 family of rolling-circle-replication plasmids. Expression vectors that are functional in several Rhodococcus species were constructed by use of the replication origin from pRE8424. We previously reported a cryptic plasmid, pRE2895, from R. erythropolis, which may replicate by a θ-type mechanism, like ColE2 plasmids. The new expression vectors originating from pRE8424 were compatible with those derived from pRE2895. Coexpression experiments with these compatible expression vectors indicated that the plasmids are suitable for the simultaneous expression of multiple recombinant proteins.     

Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1

Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His₆PheA1 and His₆PheA2 were purified and its catalytic activity characterized. His₆PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His₆PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His₆PheA1 and His₆PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.     

Expression of bovine lactoferrin C-lobe in Rhodococcus erythropolis and its purification and characterization

A Rhodococcus erythropolis expression system for the bovine lactoferrin C-lobe was constructed. The DNA fragments encoding the BLF C-lobe were amplified and cloned into vector pTip LCH1.2. R. erythropolis carrying the pTip-C-lobe was cultured at 30 degrees C with shaking, and expression of the rBLF C-lobe was induced by adding 1 microg/ml (final concentration) thiostrepton. The rBLF C-lobe was isolated in native and denatured (8 M urea) form by Ni-NTA affinity chromatography. To obtain a bioactive rBLF C-lobe, the protein isolated in the denatured form was refolded by stepwise dialysis against refolding buffers. The antibacterial activity of the rBLF C-lobe was tested by the filter-disc plate assay method. The refolded rBLF C-lobe demonstrated antibacterial activity against selected strains of Escherichia coli.     

Isolation and primary characterization of an amidase from Rhodococcus rhodochrous

Amidase (EC 3.5.1.4) was purified to homogeneity from Rhodococcus rhodochrous M8 using isopropanol fractionation and exchange chromatography on Mono Q. The isolated amidase consists of four identical subunits with molecular weight 42+/-2 kD. The activity of the enzyme is maximal at 55-60 degrees C and within the pH range 5-8. The amidase from R. rhodochrous M8 is highly sensitive to such sulfhydryl reagents as Hg2+ and Cu2+. Chelators (EDTA and o-phenanthroline) and serine proteinase inhibitors (PMSF and DIFP) did not inhibit the activity of the enzyme. The enzyme exhibits hydrolytic and acyl transferase activity and does not possess urease activity. Aliphatic amides (acetamide and propionamide) were the best substrates for the amidase from R. rhodochrous M8, whereas bulky aromatic amides were poor substrates of this enzyme. The properties of the isolated enzyme are similar to those found in the corresponding amidase from Arthrobacter sp. J-1 and an amidase with wide substrate specificity from Brevibacterium sp. R312.     

Enhancement of solubilization and biodegradation of petroleum by biosurfactant from Rhodococcus erythropolis HX-2

Abstract

The demand to repair areas contaminated with hydrocarbon products has led to the development of new technologies for the treatment of contaminants in an unconventional method, that is, no physical or chemical methods are used. Biosurfactants are amphiphilic biomolecules produced by microorganisms that can be used in environments contaminated by petroleum products due to their unexceptionable tensile properties. Petroleum degrading strain Rhodococcus erythropolis HX-2 was found to be an effective producer of biosurfactants. The resulting biosurfactant (named NK) exhibits high physicochemical properties in terms of surface activity. It is capable of reducing surface tension from 54.99 to 28.89?mN/m and critical micelle concentration (CMC) is 100?mg/L. NK was found to be a substitute for chemically synthesized surfactants because of its higher solubilization efficiency for petroleum and polycyclic aromatic hydrocarbons, superior to SDS, Tween 80, Triton X-100 and Rhamnolipid (a wide used biosurfactant). In addition, it exhibits favorable emulsion stability over a wide range of pH (3–10), temperature (20–100?°C) and salinity ranges (5–20?g/L). It was found that the addition of biosurfactant can improve the efficiency of petroleum degradation, therefore it has potential applications in bioremediation.

• Highlights

• Rhodococcus erythropolis HX-2 is an effective petroleum degrading strain.

• HX-2 is a potential source of biosurfactant production.

• The biosurfactant NK reduces surface tension and exhibits high emulsification activity.

• The biosurfactant NK is effective over a wide range of temperatures, pH and salinity.

• The biosurfactant NK shows high solubilization efficiency for petroleum as well as polycyclic aromatic hydrocarbons.

     

Characterization of IS1676 from Rhodococcus erythropolis SQ1

To develop a transposable element-based system for mutagenesis in Rhodococcus, we used the sacB gene from Bacillus subtilis to isolate a novel transposable element, IS1676, from R. erythropolis SQ1. This 1693 bp insertion sequence is bounded by imperfect (10 out of 13 bp) inverted repeats and it creates 4 bp direct repeats upon insertion. Comparison of multiple insertion sites reveals a preference for the sequence 5′-(C/T)TA(A/G)-3′ in the target site. IS1676 contains a single, large (1446 bp) open reading frame with coding potential for a protein of 482 amino acids. IS1676 may be similar to an ancestral transposable element that gave rise to repetitive sequences identified in clinical isolates of Mycobacteriumkansasii. Derivatives of IS1676 should be useful for analysis of Rhodococcus strains, for which few other genetic tools are currently available. Received: 1 April 1999 / Received revision: 6 July 1999 / Accepted: 1 August 1999     

Molecular Genetic Markers for Identification of Rhodococcus erythropolis and Rhodococcus qingshengii

Microbiology - The application of restriction analysis of amplification products of the genes rpoC and alkB, encoding the synthesis of the DNA-dependent RNA polymerase β'-subunit and...     

Formation of exopolysaccharides by Rhodococcus erythropolis and partial characterization of a heteropolysaccharide of high molecular weight

Summary Polysaccharide formation by Rhodococcus erythropolis was studied using lower mono-, di-and trihydric alcohols, sugars and n-alkanes as carbon sources. Cultural conditions of the organism were examined with regard to polysaccharide production. It was demonstrated that a glycerol substrate, an 30°C incubation temperature and a pH of 7.5 were optimal cultural conditions for polysaccharide formation. Addition of penicillin G in the decelerating growth phase increased the polysaccharide concentration in the culture filtrate to 3.1 g/l. One of the main extracellular heteropolysaccharides formed by Rhodococcus erythropolis consisted of glucose and mannose in the molar ratio 11, a small portion of protein and a trace of glucosamine. The molecular weight was to be 1·14×10⁶.     

Dehalogenation of haloalkanes byRhodococcus erythropolis Y2

Phodococcus erythropolis Y2 produced two types of dehalogenase: a hydrolytic enzyme, that is an halidohydrolase, which was induced by C₃ to C₆ 1-haloalkane substrates, and at least one oxygenase-type dehalogenase induced by C₇ to C₁₆ 1-haloalkanes andn-alkanes. The oxygenase-type activity dehalogenated C₄ to C₁₈ 1-chloroalkanes with an optimum activity towards 1-chlorotetradecane. The halidohydrolase catalysed the dehalogenation of a wide range of 1- and ,-disubstituted haloalkanes and ,-substituted haloalcohols. In resting cell suspensions of hexadecane-grownR. erythropolis Y2 the oxygenase-type dehalogenase had a specific activity of 12.9 mU (mg protein)^–1 towards 1-chlorotetradecane (3.67 mU mg^–1 towards 1-chlorobutane) whereas the halidohydrolase in 1-chlorobutane-grown batch cultures had a specific activity of 44 mU (mg protein)^–1 towards 1-chlorobutane.The significance of the two dehalogenase systems in a single bacterial strain is discussed in terms of their contribution to the overall catabolic potential of the organism.     

Enhanced desulfurization in a transposon-mutant strain of Rhodococcus erythropolis

The dsz desulfurization gene cluster from Rhodococcus erythropolis strain KA2-5-1 was transferred into R. erythropolis strain MC1109, unable to desulfurize light gas oil (LGO), using a transposon-transposase complex. As a result, two recombinant strains, named MC0203 and MC0122, were isolated. Resting cells of strain MC0203 decreased the sulfur concentration of LGO from 120 mg l^–1 to 70 mg l^–1 in 2 h. The LGO-desulfurization activity of strain MC0203 was about twice that of strain MC0122 and KA2-5-1. The 10-methyl fatty acids of strain MC0203 were about 28%–41% that of strain MC1109. It is likely that strain MC0203 had a mutation involving alkylenation or methylation of 9-unsaturated fatty acids caused by the transposon inserted in the chromosome, which increased the fluidity of cell membranes and enhanced the desulfurization activity.     

Production of 3,4-dihydroxyphthalate from phthalate by a membrane-bound two-enzyme system from Rhodococcus erythropolis

 Gram-positive Rhodococcus erythropolis strain S1 formed enzymes for the degradation of phthalate when grown in a phthalate-containing minimal medium. The membrane fraction prepared from phthalate-grown cells by ultrasonication converted phthalate to protocatechuate as the final product. Using two membrane-bound enzymes, phthalate 3,4-dioxygenase (PO) and 3,4-dihydro-3,4-dihydroxyphthalate 3,4-dehydrogenase (PH), prepared by solubilization of the membrane fraction, 3,4-dihydroxyphthalate was selectively obtained from phthalata. Fe²⁺ and Mn²⁺ stimulated the formation of 3,4-dihydroxyphthalate by the membrane-bound PO and PH system. Received: 27 April 1994/Received last revision: 19 August 1994/Accepted: 12 September 1994