Biosynthesis of nicotinic acid from 3-cyanopyridine by a newly isolated Fusarium proliferatum ZJB-09150

In this study, nitriles were used as sole sources of nitrogen in the enrichments to isolate nitrile-converting microorganisms. A novel fungus named ZJB-09150 possessing nitrile-converting enzymes was obtained with 3-cyanopyridine as sole source of nitrogen, which was identified by morphology, biology and 18S rDNA gene sequence as Fusarium proliferatum. It was found that F. proliferatum had ability to convert nitriles to corresponding acids or amides and showed wide substrate specificity to aliphatic nitriles, aromatic nitriles and ortho-substituted heterocyclic nitriles. The nitrile converting enzymes including nitrilase and nitrile hydratase in ZJB-09150 were induced by ε-caprolactam. Nitrilase obtained in this study showed high activity toward 3-cyanopyridine. It was active within pH 3.0–12.0 and temperature ranging from 25 to 65 °C with optimal at pH 9.0 and temperature 50–55 °C. The enzyme was thermostable and its half-life was 12.5 and 6 h at 45 and 55 °C, respectively. Under optimized reaction conditions, 60 mM 3-cyanopyridine was converted to nicotinic acid in 15 min, which indicated ZJB-09150 has potentials of application in large scale production of nicotinic acid.     

Deep desulfurization of diesel oil and crude oils by a newly isolated Rhodococcus erythropolis strain

The soil-isolated strain XP was identified as Rhodococcus erythropolis. R. erythropolis XP could efficiently desulfurize benzonaphthothiophene, a complicated model sulfur compound that exists in crude oil. The desulfurization product of benzonaphthothiophene was identified as alpha-hydroxy-beta-phenyl-naphthalene. Resting cells could desulfurize diesel oil (total organic sulfur, 259 ppm) after hydrodesulfurization. The sulfur content of diesel oil was reduced by 94.5% by using the resting cell biocatalyst for 24 h at 30 degrees C. Biodesulfurization of crude oils was also investigated. After 72 h of treatment at 30 degrees C, 62.3% of the total sulfur content in Fushun crude oil (initial total sulfur content, 3,210 ppm) and 47.2% of that in Sudanese crude oil (initial total sulfur, 1,237 ppm) were removed. Gas chromatography with pulsed-flame photometric detector analysis was used to evaluate the effect of R. erythropolis XP treatment on the sulfur content in Fushun crude oil, and it was shown that most organic sulfur compounds were eliminated after biodesulfurization.     

Biodesulfurization of benzothiophene and dibenzothiophene by a newly isolated Rhodococcus strain

Rhodococcus sp. KT462, which can grow on either benzothiophene (BT) or dibenzothiophene (DBT) as the sole source of sulfur, was newly isolated and characterized. GC and GC-MS analyses revealed that strain KT462 has the same BT desulfurization pathway as that reported for Paenibacillus sp. A11-2 and Sinorhizobium sp. KT55. The desulfurized product of DBT produced by this strain, as well as other DBT-desulfurizing bacteria such as R. erythropolis KA2-5-1 and R. erythropolis IGTS8, was 2-hydroxybiphenyl. A resting cells study indicated that this strain was also able to degrade various alkyl derivatives of BT and DBT.     

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.     

R-enantioselective hydrolysis of 2,2-dimethylcyclopropanecarboxamide by amidase from a newly isolated strain Brevibacterium epidermidis ZJB-07021

Aims: To isolate new micro-organisms with R-stereospecific amidase activity and to examine their potential as biocatalysts in enantioselective hydrolysis of 2,2-dimethylcyclopropanecarboxamide ( 1 ). Methods and Results: A novel R-stereospecific amidase-producing strain ZJB-07021 was isolated through a sophisticated colorimetric screening method. Based on morphology, physiological tests, Biolog system (GP2) and 16S rRNA sequence, the new isolate was identified as Brevibacterium epidermidis. After 70 min of bioconversion at 35°C, kinetic resolution of (R,S)- 1 by the amidase afforded (S)- 1 in 41·1% yield (>99% ee) and (R)- 2 in 49·9% yield (69·7% ee) with an average E-value of 23. The enantioselectivity was found to be temperature dependent and enhanced from 12·6 at 45°C to 65·9 at 14°C. Conclusions: A novel bacterial strain of B. epidermidis ZJB-07021 producing R-stereospecific amidase was isolated and characterized. The isolate exhibited high E values for kinetic resolution of racemic- 1 to (S)- 1 . Significance and Impact of the Study: To our knowledge, this was the first report on the species B. epidermidis that harboured R-stereospecific amidase. Strain ZJB-07021 could be further improved as a suitable biocatalyst for the stereoselective bioconversion of racemic- 1 after optimization of culture and biotransformation process.     

Biotransformation of dl-lactate to pyruvate by a newly isolated Serratia marcescens ZJB-07166

The production of pyruvate using biotransformation from dl-lactate has been recently drawn more and more attentions due to the wide applications of pyruvate in chemicals, drugs, and agrochemicals industries. In the current study, a strain ZJB-07166, which was capable of converting dl-lactate to pyruvate, was newly isolated and characterized and later identified as Serratia marcescens based on the morphology, physiological tests, ATB system and its 16S rDNA sequence. The strain S. marcescens ZJB-07166 was applied in biotransformation of dl-lactate to pyruvate and the detailed time courses for cultivation and biotransformation were investigated. The optimum nitrogen source and carbon source in the microorganism culture for production of lactate dehydrogenase were NH₄Cl and dl-lactate, respectively. The optimum substrate concentration for biotransformation was around 40 mM and EDTA had an obvious stabilizing effect on pyruvate in biotransformation process. The pyruvate production concentration of 210 mM was achieved under the optimum conditions. These results demonstrated that the newly isolated S. marcescens ZJB-07166 was a promising strain for pyruvate production in industrial scale.     

Pseudomonas resinovorans SPR1, a newly isolated strain with potential of transforming eugenol to vanillin and vanillic acid

In this study a novel strain was isolated with the capability to grow on eugenol as a source of carbon and energy. This strain was identified as Pseudomonas resinovorans (GenBank accession no. HQ198585) based on phenotypic characterization and phylogenetic analysis of 16S rDNA gene. The intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and vanillic acid were detected in the culture supernatant during eugenol biotransformation with this strain. The products were confirmed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and spectral data achieved from UV-vis, FTIR and mass spectroscopy. Using eugenol as substrate and resting cells of P. resinovorans SPR1, which were harvested at the end of the exponential growth phase, without further optimization 0.24 g/L vanillin (molar yield of 10%) and 1.1g/L vanillic acid (molar yield of 44%) were produced after 30 h and 60 h biotransformation, respectively. The current work gives the first evidence for the eugenol biotransformation by P. resinovorans.     

Characterization of cellobiohydrolase from a newly isolated strain of Agaricus arvencis

A highly efficient cellobiohydrolase (CBH)-secreting basidiomycetous fungus, Agaricus arvensis KMJ623, was isolated and identified based on its morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular CBH was purified to homogeneity from A. arvencis culture supernatant using sequential chromatography. The relative molecular mass of A. arvencis CBH was determined to be 65 kDa by SDSPAGE and 130 kDa by size-exclusion chromatography, indicating that the enzyme is a dimer. A. arvencis CBH showed a catalytic efficiency (kcat/Km) of 31.8 mM?1 s?1 for p-nitrophenyl-beta-D-cellobioside, the highest level seen for CBH-producing microorganisms. Its internal amino acid sequences showed significant homology with CBHs from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, A. arvencis CBH is distinguished from other CBHs by its high catalytic efficiency.     

Assimilation of Propane and Characterization of Propane Monooxygenase from Rhodococcus erythropolis3/89

The ability of propane-assimilating microorganisms of the genus Rhodococcusto utilize metabolites of the terminal and subterminal pathways of propane oxidation was studied. Propane monooxygenase of Rhodococcus erythropolis3/89 was shown to be an inducible enzyme catalyzing epoxidation and hydroxylation of organic compounds. The optimum conditions for the epoxidation of gaseous and liquid alkenes and the hydroxylation of aromatic carbohydrates were found.     

Isolation and characterization of a haloalkane halidohydrolase from Rhodococcus erythropolis Y2

Rhodococcus erythropolis strain Y2, isolated from soil by enrichment culture using 1-chlorobutane, was able to utilize a range of halogenated aliphatic compounds as sole sources of carbon and energy. The ability to utilize 1-chlorobutane was conferred by a single halidohydrolase-type haloalkane dehalogenase. The presence of the single enzyme in cell-free extracts was demonstrated by activity strain polyacrylamide gel electrophoresis. The purified enzyme was a monomeric protein with a relative molecular mass of 34 kDa and demonstrated activity against a broad range of haloalkanes, haloalcohols and haloethers. The highest activity was found towards alpha, omega disubstituted chloro- and bromo- C2-C6 alkanes and 4-chlorobutanol. The Km value of the enzyme for 1-chlorobutane was 0.26 mM. A comparison of the R. erythropolis Y2 haloalkane halidohydrolase with other haloalkane dehalogenases is discussed on the basis of biochemical properties and N-terminal amino acid sequence data.     

Production of cholesterol oxidase by a newly isolated Rhodococcus sp.

Fifteen strains of microorganisms with ability to degrade cholesterol were isolated. Among them a Gram-positive, non-motile, non-sporing bacterium with meso-DAP in the cell wall and with a rod-coccus cycle showed the highest ability for cholesterol degradation. It was identified as Rhodococcus sp. strain 2C and was deposited by code 1633 in Persian type culture collection (PTCC). This strain was able to produce high levels of both extracellular and cell-bound cholesterol oxidases in media containing cholesterol as a sole carbon source. The effects of medium composition and physical parameters on cholesterol oxidase production were studied. The optimized medium was found to contain cholesterol 0.15% (w/v), yeast extract 0.3% (w/v), diammonium hydrogen phosphate 0.1% (w/v), Tween 80 (0.05%). The optimum pH and temperature for cholesterol oxidase production in optimized medium were found to be 8–30 °C respectively. Triton X-100 showed the greatest effect in releasing the cell-bound enzyme. The first and most probably the main metabolite of cholesterol degradation was purified and identified as 4-cholestene-3-one.     

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.     

Initial hydrogenation during catabolism of picric acid by Rhodococcus erythropolis HL 24-2.

Rhodococcus erythropolis HL 24-2, which was originally isolated as a 2,4-dinitrophenol-degrading bacterium, could also utilize picric acid as a nitrogen source after spontaneous mutation. During growth, the mutant HL PM-1 transiently accumulated an orange-red metabolite, which was identified as a hydride-Meisenheimer complex of picric acid. This complex was formed as the initial metabolite and further converted with concomitant liberation of nitrite. 2,4,6-Trinitrocyclohexanone was identified as a dead-end metabolite of the degradation of picric acid, indicating the addition of two hydride ions to picric acid.     

Initial hydrogenation during catabolism of picric acid by Rhodococcus erythropolis HL 24-2.

Rhodococcus erythropolis HL 24-2, which was originally isolated as a 2,4-dinitrophenol-degrading bacterium, could also utilize picric acid as a nitrogen source after spontaneous mutation. During growth, the mutant HL PM-1 transiently accumulated an orange-red metabolite, which was identified as a hydride-Meisenheimer complex of picric acid. This complex was formed as the initial metabolite and further converted with concomitant liberation of nitrite. 2,4,6-Trinitrocyclohexanone was identified as a dead-end metabolite of the degradation of picric acid, indicating the addition of two hydride ions to picric acid.     

Decolorization of 1-amino-4-bromoanthraquinone-2-sulfonic acid by a newly isolated strain of Sphingomonas herbicidovorans

A novel isolate of Sphingomonas herbicidovorans could decolorize 1-amino-4-bromoanthraquinone-2-sulfonic acid (bromoamine acid, BAA), an intermediate of anthraquinone dyes, and grow with it as the sole source of carbon. The strain was identified by 16S rRNA gene sequencing and physiological-biochemical test. The optimal condition for both decolorization and cell growth was found at temperature of 30 °C and pH 7.0, respectively. Furthermore, the decolorization efficiency could be enhanced with higher shaking speed. The percentage of BAA decolorization could be over 98% within 24 h even for the initial concentration greater than 1000 mg l^?1. The decolorization kinetics could be reasonably described by the Monod equation. Additional carbon sources such as glucose could enhance the decolorization rate. During the decolorization process, the molecular of BAA cleaved, releasing phthalic acid and an end product which might be benzene derivative substituted by amino, bromo, hydroxyl and sulfonate groups according to the infrared spectral analysis.     

Selective biodegradation of S and N heterocycles by a recombinant Rhodococcus erythropolis strain containing carbazole dioxygenase

The carbazole dioxygenase genes were introduced into a dibenzothiophene degrader. The recombinant Rhodococcus erythropolis SN8 was capable of efficiently degrading dibenzothiophene and carbazole simultaneously. SN8 could also degrade various alkylated derivatives of carbazole and dibenzothiophene in FS4800 crude oil by just a one-step bioprocess.     

Initial reactions in the biodegradation of 1-chloro-4-nitrobenzene by a newly isolated bacterium, strain LW1

Bacterial strain LW1, which belongs to the family Comamonadaceae, utilizes 1-chloro-4-nitrobenzene (1C4NB) as a sole source of carbon, nitrogen, and energy. Suspensions of 1C4NB-grown cells removed 1C4NB from culture fluids, and there was a concomitant release of ammonia and chloride. Under anaerobic conditions LW1 transformed 1C4NB into a product which was identified as 2-amino-5-chlorophenol by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. This transformation indicated that there was partial reduction of the nitro group to the hydroxylamino substituent, followed by Bamberger rearrangement. In the presence of oxygen but in the absence of NAD, fast transformation of 2-amino-5-chlorophenol into a transiently stable yellow product was observed with resting cells and cell extracts. This compound exhibited an absorption maximum at 395 nm and was further converted to a dead-end product with maxima at 226 and 272 nm. The compound formed was subsequently identified by 1H and 13C NMR spectroscopy and mass spectrometry as 5-chloropicolinic acid. In contrast, when NAD was added in the presence of oxygen, only minor amounts of 5-chloropicolinic acid were formed, and a new product, which exhibited an absorption maximum at 306 nm, accumulated.     

Mineralization of 4-fluorocinnamic acid by a Rhodococcus strain

A bacterial strain capable of aerobic degradation of 4-fluorocinnamic acid (4-FCA) as the sole source of carbon and energy was isolated from a biofilm reactor operating for the treatment of 2-fluorophenol. The organism, designated as strain S2, was identified by 16S rRNA gene analysis as a member of the genus Rhodococcus. Strain S2 was able to mineralize 4-FCA as sole carbon and energy source. In the presence of a conventional carbon source (sodium acetate [SA]), growth rate of strain S2 was enhanced from 0.04 to 0.14 h^?1 when the culture medium was fed with 0.5 mM of 4-FCA, and the time for complete removal of 4-FCA decreased from 216 to 50 h. When grown in SA-supplemented medium, 4-FCA concentrations up to 1 mM did not affect the length of the lag phase, and for 4-FCA concentrations up to 3 mM, strain S2 was able to completely remove the target fluorinated compound. 4-Fluorobenzoate (4-FBA) was transiently formed in the culture medium, reaching concentrations up to 1.7 mM when the cultures were supplemented with 3.5 mM of 4-FCA. Trans,trans-muconate was also transiently formed as a metabolic intermediate. Compounds with molecular mass compatible with 3-carboxymuconate and 3-oxoadipate were also detected in the culture medium. Strain S2 was able to mineralize a range of other haloorganic compounds, including 2-fluorophenol, to which the biofilm reactor had been exposed. To our knowledge, this is the first time that mineralization of 4-FCA as the sole carbon source by a single bacterial culture is reported.     

Biodegradation of alkali lignin by a newly isolated Rhodococcus pyridinivorans CCZU-B16

Bioprocess and Biosystems Engineering - Based on the Prussian blue spectrophotometric method, one high-throughput screening strategy for screening lignin-degrading microorganisms was built on...