Monitoring of an Alkaline 2,4,6-Trichlorophenol-Degrading Enrichment Culture by DNA Fingerprinting Methods and Isolation of the Responsible Organism, Haloalkaliphilic Nocardioides sp. Strain M6.

A site situated near Alkali Lake (Oregon) and highly contaminated by chloroaromatic compounds was chosen for isolation of alkaliphilic chlorophenol-degrading bacteria. Prolonged cultivation of an enrichment culture followed by successive transfers resulted in a strong increase in the 2,4,6-trichlorophenol (2,4,6-TCP) degradation rate. Repetitive extragenic palindromic PCR and amplified ribosomal DNA restriction analysis were applied to distinguish members of the enrichment culture and monitor them during the enrichment procedure.

Bioconversion of acrylonitrile to acrylamide using a thermostable nitrile hydratase.

Although providing an attractive route for production of acrylamide from acrylonitrile, utilization of nitrile hydratase enzymes has been limited by the requirement for low temperature bioconversion conditions. This report summarizes a search for thermostable nitrile hydratases from aerobic moderate thermophiles screened for ability to grow on acrylonitrile at concentrations to 1% at elevated temperatures. A new isolate Bacillus sp.

Degradation of benzyldimethylalkylammonium chloride by Aeromonas hydrophila sp. K.

Aims: The aim of this work was to study the biodegradation of benzyldimethylalkylammonium chloride (BAC) by Aeromonas hydrophila sp. K, an organism isolated from polluted soil and capable of utilizing BAC as sole source of carbon and energy.

Methods And Results: High performance liquid chromatography and gas chromatography-mass spectrometry (GC-MS) analysis was used to study BAC degradation pathway.

Cobalt activation of Bacillus BR449 thermostable nitrile hydratase expressed in Escherichia coli.

Expression of nitrile hydratase enzymes utilized in a new "green" process for acrylamide production has proven difficult in Escherichia coli owing to lack of a cobalt transport system to introduce the required cobalt ion into this host. We describe the expression of a thermostable nitrile hydratase from a moderate thermophile Bacillus sp. BR449 in E.

Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp. BR449 into Escherichia coli.

A moderate thermophile, Bacillus sp. BR449 was previously shown to exhibit a high level of nitrile hydratase (NHase) activity when growing on high levels of acrylonitrile at 55 degrees C. In this report, we describe the cloning of a 6.

Cloning and expression of the limonene hydroxylase of Bacillus stearothermophilus BR388 and utilization in two-phase limonene conversions.

A 3.6-kb fragment of Bacillus stearothermophilus BR388 chromosomal DNA that confers growth on limonene to Escherichia coli has been sequenced, revealing a single open reading frame encoding a single subunit limonene hydroxylase containing 444 amino acid residues. This enzyme proved capable of limonene hydroxylation to a mixture of carveol and perillyl alcohol as well as dehydrogenation of these products to carvone and perillyl aldehyde.

Cloning of a wide-spectrum amidase from Bacillus stearothermophilus BR388 in Escherichia coli and marked enhancement of amidase expression using directed evolution*.

A 1.6-kb DraI-HindIII DNA fragment from Bacillus stearothermophilus BR388 chromosomal DNA encoding a wide-spectrum amidase was cloned into Escherichia coli DH5alpha. With acrylamide substrate, the amidase showed maximum activity at 55 degrees C, pH 7.

Degradation of pinene by Bacillus pallidus BR425.

An aerobic thermophile has been isolated from an alpha-pinene enrichment culture. The isolate, which was designated BR425, has been tentatively identified as Bacillus pallidus using 16S ribosomal RNA gene sequencing and organism morphology. Monophasic and biphasic incubations of BR425 cells with alpha-pinene, beta-pinene, and limonene yielded a number of oxidized monoterpene metabolites with carveol as a common metabolite.

Degradation of 3-phenylpropionic acid by Haloferax sp. D1227.

Haloferax sp. D1227, isolated from soil contaminated with highly saline oil brine, is the first halophilic archaeon to demonstrate the utilization of aromatic compounds (i.e.

Gentisate 1,2-dioxygenase from Haloferax sp. D1227.

Gentisate 1,2-dioxygenase from the extreme halophile Haloferax sp. D1227 (Hf. D1227) was purified using a three-step procedure.

Production of alpha-terpineol from Escherichia coli cells expressing thermostable limonene hydratase.

The genes encoding a thermostable limonene hydratase have been located on a cloned fragment in Escherichia coli conferring growth on limonene and production of the monoterpenes perillyl alcohol and alpha-terpineol. Whole cell bioconversion studies at elevated temperature employing both an aqueous phase and neat limonene phase demonstrated significant production of alpha-terpineol with additional production of carvone.

Degradation of 2,4-dichlorophenoxyacetic acid by haloalkaliphilic bacteria.

Three 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial isolates were obtained from the highly saline and alkaline Alkali Lake site in southwestern Oregon contaminated with 2,4-D production wastes. While similar in most respects, the three isolates differed significantly in 2,4-D degradation rates, with the most active strain, I-18, demonstrating an ability to degrade up to 3000 mg 2,4-D I-1 in 3 d. This strain was well adapted to the extreme environment from which it was isolated, growing optimally on 2,4-D at pH 8.

Characterization of the Bacillus stearothermophilus BR219 phenol hydroxylase gene.

The catabolic genes pheA and pheB, coding for the conversion of phenol to catechol and catechol to 2-hydroxymuconic semialdehyde, respectively, have been cloned from Bacillus stearothermophilus BR219 into Escherichia coli. Following its localization on the 11-kb B. stearothermophilus DNA insert by deletion and expression analysis, the phenol hydroxylase gene pheA was subcloned as a 2-kb HindIII fragment, whose transformant expressed the enzyme after phenol induction and even more strongly after o-, m-, and p-cresol induction.

Cloning and expression of a pathway for benzene and toluene from Bacillus stearothermophilus.

Bacillus stearothermophilus strain BR325 demonstrating broad aromatic substrate capability was isolated from petroleum-contaminated soil. The chromosomally-located aromatic pathway from this isolate was cloned into Escherichia coli as a 32 kb insert in cosmid pHC79, conferring growth on benzene, phenol, and toluene as sole carbon sources.

Purification and characterization of lamb pregastric lipase.

Lamb pregastric lipase was purified from a commercial source using delipidation, solubilization with KSCN, acid-precipitation, pepsin-digestion, affinity chromatography with agarose-Cibacron Blue F3GA, gel filtration, and elution from a native 10% (w/v) polyacrylamide gel. The enzyme had a single subunit of 68,000 Da with maximum esterase activity when measured at pH 6.0 and 30 degrees C.

Transfer of Transposon Tn916 from Bacillus subtilis into a Natural Soil Population.

A Bacillus subtilis transconjugant with a Tn916 chromosomal insert was obtained through mating with Escherichia coli carrying the transposon as a plasmid insert. Actinomycetes were identified as frequent transposon recipients following the introduction of the B. subtilis transconjugant into a soil microcosm.

Conjugal transfer of recombinant transposon Tn916 from Escherichia coli to Bacillus stearothermophilus.

A gene for thermostable amylase has been inserted at the BstXI site of Tn916. Mating experiments demonstrated that unlike Tn916, the recombinant transposon, designated Tn916A, could transfer from Escherichia coli to Bacillus stearothermophilus BR219 in broth matings, resulting in chromosomal integration of the transposon and expression of the amylase at significant levels.

Transfer of transposon Tn916 from Bacillus subtilis to Thermus aquaticus.

Broad host range conjugating transposon Tn916 has been introduced into the extreme thermophile Thermus by transposon transformation and transposition into the Bacillus subtilis chromosome followed by broth mating with Thermus aquaticus ATCC27634. Tetracycline resistant Thermus transconjugants were obtained at a frequency of 1.4 X 10(-7) per donor and 1.

Multiple amylase genes in two strains of Bacillus stearothermophilus.

Plasmid DNA fragments from Bacillus stearothermophilus ATCC29609 (BR135) and chromosomal DNA fragments from B. stearothermophilus ATCC31195 (BR132) were cloned into pBR322 and transformed into Escherichia coli strain HB101. Clones were selected which demonstrate extracellular expression of thermostable amylase.

Isolation of phenol-degrading Bacillus stearothermophilus and partial characterization of the phenol hydroxylase.

Bacillus stearothermophilus BR219, isolated from river sediment, degraded phenol at levels to 15 mM at a rate of 0.85 mumol/h (4 x 10(6) cells). The solubilized phenol hydroxylase was NADH dependent, exhibited a 55 degrees C temperature optimum for activity, and was not inhibited by 0.