Comparative Genomic Analysis and BTEX Degradation Pathways of a Thermotolerant Cupriavidus cauae PHS1.

Volatile organic compounds such as benzene, toluene, ethylbenzene, and isomers of xylenes (BTEX) constitute a group of monoaromatic compounds that are found in petroleum and have been classified as priority pollutants. In this study, based on its newly sequenced genome, we reclassified the previously identified BTEX-degrading thermotolerant strain Ralstonia sp. PHS1 as Cupriavidus cauae PHS1. Also presented are the complete genome sequence of C. cauae PHS1, its annotation, species delineation, and a comparative analysis of the BTEX-degrading gene cluster. Moreover, we cloned and characterized the BTEX-degrading pathway genes in C. cauae PHS1, the BTEX-degrading gene cluster of which consists of two monooxygenases and meta-cleavage genes. A genome-wide investigation of the PHS1 coding sequence and the experimentally confirmed regioselectivity of the toluene monooxygenases and catechol 2,3-dioxygenase allowed us to reconstruct the BTEX degradation pathway. The degradation of BTEX begins with aromatic ring hydroxylation, followed by ring cleavage, and eventually enters the core carbon metabolism. The information provided here on the genome and BTEX-degrading pathway of the thermotolerant strain C. cauae PHS1 could be useful in constructing an efficient production host.

Metabolic pathway of 3,6-anhydro-D-galactose in carrageenan-degrading microorganisms.

Complete hydrolysis of κ-carrageenan produces two sugars, D-galactose and 3,6-anhydro-D-galactose (D-AnG). At present, however, we do not know how carrageenan-degrading microorganisms metabolize D-AnG. In this study, we investigated the metabolic pathway of D-AnG degradation by comparative genomic analysis of Cellulophaga lytica LIM-21, Pseudoalteromonas atlantica T6c, and Epulopiscium sp. N.t. morphotype B, which represent the classes Flavobacteria, Gammaproteobacteria, and Clostridia, respectively. In this bioinformatic analysis, we found candidate common genes that were believed to be involved in D-AnG metabolism. We then experimentally confirmed the enzymatic function of each gene product in the D-AnG cluster. In all three microorganisms, D-AnG metabolizing genes were clustered and organized in operon-like arrangements, which we named as the dan operon (3,6-d-anhydro-galactose). Combining bioinformatic analysis and experimental data, we showed that D-AnG is metabolized to pyruvate and D-glyceraldehyde-3-phosphate via four enzyme-catalyzed reactions in the following route: 3,6-anhydro-D-galactose â†’ 3,6-anhydro-D-galactonate â†’ 2-keto-3-deoxy-D-galactonate (D-KDGal) â†’ 2-keto-3-deoxy-6-phospho-D-galactonate â†’ pyruvate + D-glyceraldehyde-3-phosphate. The pathway of D-AnG degradation is composed of two parts: transformation of D-AnG to D-KDGal using two D-AnG specific enzymes and breakdown of D-KDGal to two glycolysis intermediates using two DeLey-Doudoroff pathway enzymes. To our knowledge, this is the first report on the metabolic pathway of D-AnG degradation.

Iron deficiency upregulates Egr1 expression.

Iron-deficient anemia is a prevalent disease among humans. We searched for genes regulated by iron deficiency and its regulated mechanism. cDNA microarrays were performed using Hepa1c1c7 cells treated with 100 µM desferrioxamine (DFO), an iron chelator. Early growth response 1 (Egr1) was upregulated with at least 20-fold increase within 4 h and lasted for 24 h, which was confirmed by qRT-PCR. This activation was not seen by ferric ammonium citrate (FAC). DFO increased the transcriptional activity of Egr1-luc (-604 to +160) and serum response element (SRE)-luc reporters by 2.7-folds. In addition, cycloheximide lowered DFO-induced Egr1 mRNA levels. The upregulation of Egr1 by DFO was accompanied by sustained ERK signals along with phosphorylation of Elk-1. The ERK inhibitor (PD98059) prevented the DFO-induced Egr1 mRNAs. Overexpression of Elk-1 mutant (pElk-1S383A) decreased Egr1 reporter activity. DFO lowered reactive oxygen species (ROS) production and increased caspase 3/7 activity and cell death. DFO-induced iron deficiency upregulates Egr1 in part through transcriptional activation via ERK and Elk-1 signals, which may be important in the regulation of cell death in hepatoma cells. Our study demonstrated that iron depletion controlled the expression of Egr1, which might contribute to decisions about cellular fate in response to iron deficiency.

Identification and characterization of 2-keto-3-deoxy-L-rhamnonate dehydrogenase belonging to the MDR superfamily from the thermoacidophilic bacterium Sulfobacillus thermosulfidooxidans: implications to L-rhamnose metabolism in archaea.

We identified the non-phosphorylated L-rhamnose metabolic pathway (Rha_NMP) genes that are homologous to those in the thermoacidophilic archaeon Thermoplasma acidophilum in the genome of the thermoacidophilic bacterium Sulfobacillus thermosulfidooxidans. However, unlike previously known 2-keto-3-deoxy-L-rhamnonate (L-KDR) dehydrogenase (KDRDH) which belongs to the short chain dehydrogenase/reductase superfamily, the putative KDRDHs in S. thermosulfidooxidans (Sulth_3557) and T. acidophilum (Ta0749) belong to the medium chain dehydrogenase/reductase (MDR) superfamily. We demonstrated that Sulth_3559 and Sulth_3557 proteins from S. thermosulfidooxidans function as L-rhamnose dehydrogenase and KDRDH, respectively. Sulth_3557 protein is an NAD(+)-specific KDRDH with optimal temperature and pH of 50 °C and 9.5, respectively. The K m and V max values for L-KDR were 2.0 mM and 12.8 U/mg, respectively. Sulth_3557 also showed weak 2,3-butanediol dehydrogenase activity. Phylogenetic analysis suggests that Sulth_3557 and its homologs form a new subfamily in the MDR superfamily. The results shown in this study imply that thermoacidophilic archaea metabolize L-rhamnose to pyruvate and L-lactate by using the MDR-family KDRDH similarly to that of the thermoacidophilic bacterium S. thermosulfidooxidans.

Fermentation of soy milk via Lactobacillus plantarum improves dysregulated lipid metabolism in rats on a high cholesterol diet.

We aimed to investigate whether in vitro fermentation of soy with L. plantarum could promote its beneficial effects on lipids at the molecular and physiological levels. Rats were fed an AIN76A diet containing 50% sucrose (w/w) (CTRL), a modified AIN76A diet supplemented with 1% (w/w) cholesterol (CHOL), or a CHOL diet where 20% casein was replaced with soy milk (SOY) or fermented soy milk (FSOY). Dietary isoflavone profiles, serum lipids, hepatic and fecal cholesterol, and tissue gene expression were examined. The FSOY diet had more aglycones than did the SOY diet. Both the SOY and FSOY groups had lower hepatic cholesterol and serum triglyceride (TG) than did the CHOL group. Only FSOY reduced hepatic TG and serum free fatty acids and increased serum HDL-CHOL and fecal cholesterol. Compared to CHOL, FSOY lowered levels of the nuclear forms of SREBP-1c and SREBP-2 and expression of their target genes, including FAS, SCD1, LDLR, and HMGCR. On the other hand, FSOY elevated adipose expression levels of genes involved in TG-rich lipoprotein uptake (ApoE, VLDLR, and Lrp1), fatty acid oxidation (PPARα, CPT1α, LCAD, CYP4A1, UCP2, and UCP3), HDL-biogenesis (ABCA1, ApoA1, and LXRα), and adiponectin signaling (AdipoQ, AdipoR1, and AdipoR2), as well as levels of phosphorylated AMPK and ACC. SOY conferred a similar expression profile in both liver and adipose tissues but failed to reach statistical significance in many of the genes tested, unlike FSOY. Our data indicate that fermentation may be a way to enhance the beneficial effects of soy on lipid metabolism, in part via promoting a reduction of SREBP-dependent cholesterol and TG synthesis in the liver, and enhancing adiponectin signaling and PPARα-induced expression of genes involved in TG-rich lipoprotein clearance, fatty acid oxidation, and reverse cholesterol transport in adipose tissues.

Psychroserpens damuponensis sp. nov., isolated from seawater.

A novel bacterium, designated strain F051-1(T), isolated from a seawater sample collected from the coast at Damupo beach in Pohang, Korea, was investigated in a polyphasic taxonomic study. Cells were yellow-pigmented, strictly aerobic, Gram-staining-negative and rod-shaped. The temperature, pH and NaCl ranges for growth were 4-30 °C, pH 6.0-9.0 and 1.0-6.0 % (w/v), respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain F051-1(T) belongs to the genus Psychroserpens in the family Flavobacteriaceae. Its closest relatives were Psychroserpens burtonensis ACAM 188(T) (96.8 % 16S rRNA gene sequence similarity) and Psychroserpens mesophilus KOPRI 13649(T) (95.7 %). The major cellular fatty acids were iso-C(15 : 0), iso-C(15 : 1) G and anteiso-C(15 : 0). The polar lipid profile consisted of a mixture of phosphatidylethanolamine, two unidentified aminolipids, one unidentified phospholipid and eight unidentified lipids. The major respiratory quinone was menaquinone-6 and the genomic DNA G+C content of the strain was 33.5 mol%. On the basis of phenotypic, phylogenetic and genotypic data, strain F051-1(T) represents a novel species within the genus Psychroserpens, for which the name Psychroserpens damuponensis sp. nov. is proposed. The type strain is F051-1(T) ( = KCTC 23539(T)  = JCM 17632(T)).

Sagittula marina sp. nov., isolated from seawater and emended description of the genus Sagittula.

A novel bacterium, designated strain F028-2(T), was isolated from seawater at Damupo beach in Pohang, Korea, and investigated in a taxonomic study using a polyphasic approach. This novel strain was strictly aerobic, non-motile, Gram-stain-negative and rod-shaped, and occasionally formed aggregates. The temperature, pH and NaCl ranges for growth were 4-30 °C, pH 6.5-9.0 and 1-7% (w/v), respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain F028-2(T) formed a lineage within the family Rhodobacteraceae of the class Alphaproteobacteria, and was closely related to members of the genera Sagittula and Antarctobacter with 96.3-96.4% sequence similarities. The polar lipid profile of strain F028-2(T) comprised diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids, one unidentified phospholipid and six unidentified lipids. The predominant cellular fatty acids were C18:1ω7c and C12:1 3-OH. The genomic DNA G+C content of strain F028-2(T) was 61.6 mol% and the major respiratory quinone was Q-10. Based on phenotypic, phylogenetic and genotypic data, strain F028-2(T) is considered to represent a novel species in the genus Sagittula, for which the name Sagittula marina sp. nov. is proposed. The type strain is F028-2(T) (=KCTC 23543(T)=JCM 17627(T)). An emended description of the genus Sagittula is also proposed.

Winogradskyella damuponensis sp. nov., isolated from seawater.

A novel bacterium, designated F081-2(T), isolated from seawater from Damupo beach in Pohang, Korea, was investigated using a polyphasic taxonomic approach. Cells were yellow-pigmented, strictly aerobic, motile by gliding, Gram-negative-staining and rod-shaped. The temperature, pH and NaCl ranges for growth were 4-35 °C, pH 5.5-9.5 and 1.0-5.0 %, respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain F081-2(T) belonged to a distinct lineage in the genus Winogradskyella of the family Flavobacteriaceae, sharing 93.7-98.1 % similarity with recognized members of the genus. Low levels of DNA-DNA relatedness values were found between strain F081-2(T) and Winogradskyella eximia KCTC 12219(T) (61.1 %), Winogradskyella thalassocola KCTC 12221(T) (47.0 %), Winogradskyella echinorum KCTC 22026(T) (39.3 %), Winogradskyella rapida CCUG 56098(T) (34.3 %) and Winogradskyella arenosi JCM 17633(T) (33.4 %). The major cellular fatty acids were iso-C(15 : 0) (25.3 %), iso-C(15 : 1) G (14.6 %), iso-C(17 : 0) 3-OH (9.3 %), anteiso-C(15 : 0) (7.8 %) and iso-C(15 : 0) 3-OH (7.6 %). The polar lipid profile was composed of phosphatidylethanolamine, one unidentified aminolipid, one unidentified phospholipid, one unidentified aminophospholipid and six unidentified lipids. The major respiratory quinone was menaquinone-6 and the DNA G+C content of the strain was 32.3 mol%. On the basis of phenotypic, phylogenetic and genotypic data, strain F081-2(T) represents a novel species within the genus Winogradskyella, for which the name Winogradskyella damuponensis sp. nov. is proposed. The type strain is F081-2(T) (=KCTC 23552(T) = JCM 17633(T)).

Genome sequence of the unclassified marine Gammaproteobacterium BDW918.

The unclassified marine gammaproteobacterium BDW918, which utilizes volatile fatty acids but not most common carbohydrates and amino acids, was isolated from Dokdo seawater in South Korea. Here we present a draft genome of the strain BDW918, which encodes many putative genes related to fatty acid metabolism and aromatic hydrocarbon degradation.

Characterization of NADP+-specific L-rhamnose dehydrogenase from the thermoacidophilic Archaeon Thermoplasma acidophilum.

Thermoplasma acidophilum utilizes L-rhamnose as a sole carbon source. To determine the metabolic pathway of L-rhamnose in Archaea, we identified and characterized L-rhamnose dehydrogenase (RhaD) in T. acidophilum. Ta0747P gene, which encodes the putative T. acidophilum RhaD (Ta_RhaD) enzyme belonging to the short-chain dehydrogenase/reductase family, was expressed in E. coli as an active enzyme catalyzing the oxidation of L-rhamnose to L-rhamnono-1,4-lactone. Analysis of catalytic properties revealed that Ta_RhaD oxidized L-rhamnose, L-lyxose, and L-mannose using only NADP(+) as a cofactor, which is different from NAD(+)/NADP(+)-specific bacterial RhaDs and NAD(+)-specific eukaryal RhaDs. Ta_RhaD showed the highest activity toward L-rhamnose at 60 °C and pH 7. The K (m) and k (cat) values were 0.46 mM, 1,341.3 min(-1) for L-rhamnose and 0.1 mM, 1,027.2 min(-1) for NADP(+), respectively. Phylogenetic analysis indicated that branched lineages of archaeal RhaD are quite distinct from those of Bacteria and Eukarya. This is the first report on the identification and characterization of NADP(+)-specific RhaD.

Postechiella marina gen. nov., sp. nov., isolated from seawater.

A Gram-staining-negative, strictly aerobic, non-motile, yellow-pigmented bacterium, designated strain M091(T), was isolated from seawater at Damupo beach in Pohang, Republic of Korea, and investigated using a polyphasic taxonomic approach. The novel strain grew optimally at 25 °C, pH 7.0-8.0, and in the presence of 3% (w/v) NaCl. In a phylogenetic analysis based on 16S rRNA gene sequences, strain M091(T) formed a lineage within the family Flavobacteriaceae that was distinct from the most closely related genera of Flaviramulus (95.1% sequence similarity), Algibacter (94.9-93.9%), Mariniflexile (94.8-94.2%), Winogradskyella (94.8-93.2%), Lacinutrix (94.7-93.8%) and Tamlana (94.7-92.9%). The polar lipid profile of the novel strain comprised phosphatidylethanolamine, two unidentified aminolipids, one unidentified phospholipid and seven unidentified lipids. The predominant cellular fatty acids were iso-C(15:0) (20.5%), iso-C(17:0) 3-OH (15.4%), iso-C(15:0) 3-OH (12.4%), C(15:0) (10.9%) and iso-C(15:1) G (9.9%). The genomic DNA G+C content of strain M091(T) was 34.4 mol% and the major respiratory quinone was MK-6. Based on phenotypic and genotypic data, strain M091(T) represents a new genus and novel species in the family Flavobacteriaceae, for which the name Postechiella marina gen. nov., sp. nov. is proposed. The type strain of the type species is M091(T) (=KCTC 23537(T)=JCM 17630(T)).

Tenacibaculum jejuense sp. nov., isolated from coastal seawater.

A Gram-staining-negative, strictly aerobic and rod-shaped bacterium, designated strain CNURIC013(T), was isolated from seawater collected on the coast of Jeju Island, South Korea. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain CNURIC013(T) belonged to the genus Tenacibaculum, within the family Flavobacteriaceae. Sequence similarities between the novel strain and the type strains of recognized species of the genus Tenacibaculum were 93.6-96.0 %, the highest value being with Tenacibaculum litopenaei B-I(T) (96 %). The DNA G+C content of the novel strain was 34.5 mol% and the major respiratory quinone was menaquinone-6. The major fatty acids were summed feature 3 (comprising C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH; 26.0 %), iso-C(15 : 0) (24.4 %), iso-C(15 : 1) G (18.5 %) and iso-C(17 : 0) 3-OH (8.1 %). The polar lipids consisted of phosphatidylethanolamine, one unknown aminophospholipid and nine unknown polar lipids. On the basis of the phenotypic, phylogenetic and genotypic data, strain CNURIC013(T) represents a novel species within the genus Tenacibaculum, for which the name Tenacibaculum jejuense sp. nov. is proposed. The type strain is CNURIC013(T) ( = KCTC 22618(T) = JCM 15975(T)).

Algoriphagus jejuensis sp. nov., isolated from seawater.

A gram-negative, pink-pigmented, non-motile, strictly aerobic rod, designated CNU040(T), was isolated from seawater from the coast of Jeju Island in Korea. The temperature, pH and NaCl ranges for growth were 4-30 °C, pH 5.5-10.0 and 0-5.0 % NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CNU040(T) belonged to a distinct lineage in the genus Algoriphagus and exhibited high sequence similarity with Algoriphagus terrigena DS-44(T) (98.3 %) and Algoriphagus alkaliphilus AC-74(T) (96.6 %) and lower sequence similarity (<96.0 %) with all other members of the genus Algoriphagus. DNA-DNA relatedness between strain CNU040(T) and A. terrigena KCTC 12545(T) was 44.5 %. The DNA G+C content of the isolate was 48.5 mol% and the major respiratory quinone was menaquinone-7. The major cellular fatty acids were iso-C(15 : 0) (28.6 %) and summed feature 3 (consisting of iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c; 24.0 %). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, one unknown amino lipid, one unknown aminophospholipid and three unknown polar lipids. On the basis of phenotypic, phylogenetic and genotypic data, strain CNU040(T) represents a novel species within the genus Algoriphagus, for which the name Algoriphagus jejuensis sp. nov. is proposed. The type strain is CNU040(T) ( = KCTC 22647(T)  = JCM 16112(T)).

Fractal analysis of mandibular trabecular bone: optimal tile sizes for the tile counting method.

PURPOSE: This study was performed to determine the optimal tile size for the fractal dimension of the mandibular trabecular bone using a tile counting method. MATERIALS AND METHODS: Digital intraoral radiographic images were obtained at the mandibular angle, molar, premolar, and incisor regions of 29 human dry mandibles. After preprocessing, the parameters representing morphometric characteristics of the trabecular bone were calculated. The fractal dimensions of the processed images were analyzed in various tile sizes by the tile counting method. RESULTS: The optimal range of tile size was 0.132 mm to 0.396 mm for the fractal dimension using the tile counting method. The sizes were closely related to the morphometric parameters. CONCLUSION: The fractal dimension of mandibular trabecular bone, as calculated with the tile counting method, can be best characterized with a range of tile sizes from 0.132 to 0.396 mm.

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of glucuronic acid dehydrogenase from Chromohalobacter salexigens.

Glucuronic acid dehydrogenase (GluUADH), the product of the Csal-2474 gene from the halophilic bacterium Chromohalobacter salexigens DSM 3043, is an enzyme with potential use in the conversion of glucuronic acid in seaweed biomass to fuels and chemicals. GluUADH is an enzyme that catalyzes the oxidation of glucuronic acid (GluUA) and galacturonic acid (GalUA) and has a preference for NAD(+) rather than NADP(+) as a cofactor. Recombinant GluUADH was crystallized in the presence of 0.2 M calcium acetate, 0.1 M Tris-HCl pH 7.0 and 20% PEG 3000 at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 Å. The GluUADH crystal belonged to space group P6(3), with unit-cell parameters a = b = 122.58, c = 150.49 Å, γ = 120°. With one molecule per asymmetric unit, the crystal volume per unit protein weight (V(M)) is 2.78 Å(3) Da(-1). The structure was solved by the single anomalous dispersion method and structure refinement is in progress.

Soluble expression of archaeal proteins in Escherichia coli by using fusion-partners.

Expression of archaeal proteins in soluble form is of importance because archaeal proteins are usually produced as insoluble inclusion bodies in Escherichia coli. In this study, we investigated the use of soluble fusion tags to enhance the solubility of two archaeal proteins, d-gluconate dehydratase (GNAD) and 2-keto-3-deoxy-D-gluconate kinase (KDGK), key enzymes in the glycolytic pathway of the thermoacidophilic archaeon Sulfolobus solfataricus. These two proteins were produced as inclusion bodies in E. coli when polyhistidine was used as a fusion tag. To reduce inclusion body formation in E. coli, GNAD and KDGK were fused with three partners, thioredoxin (Trx), glutathione-S-transferase (GST), and N-utilization substance A (NusA). With the use of fusion-partners, the solubility of the archaeal proteins was remarkably enhanced, and the soluble fraction of the recombinant proteins was increased in this order: Trx>GST>NusA. Furthermore, In the case of recombinant KDGKs, the enzyme activity of the Trx-fused proteins was 200-fold higher than that of the polyhistidine-fusion protein. The strategy presented in this work may contribute to the production of other valuable proteins from hyperthermophilic archaea in E. coli.

In situ monitoring of cell concentration in a photobioreactor using image analysis: comparison of uniform light distribution model and artificial neural networks.

Light intensity is a very important factor that determines the growth of photosynthetic cells. In this study, the light distribution in a photobioreactor was analyzed by processing the images captured with a digital camera. The contour images obtained by filtering the original images clearly showed the effects of the cell concentration and external light intensity on the light distribution. Image-processing techniques were then applied to predict the cell density in the photobioreactor. To correlate the cell concentration with the light intensity in the photobioreactor, the captured images were processed using two different approaches. The first method involved the use of an average gray value after deriving a simplified model equation that could be related to the cell density. The second method involved the use of local points instead of a representative value. In this case, an artificial neural network model was adopted to infer the cell density from the information of the local points. By using these two methods, it was possible to relate the image data to the cell concentration. Finally, we compared these two methods with regard to their accuracy, easiness, and effectiveness.

Rare codon clusters at 5'-end influence heterologous expression of archaeal gene in Escherichia coli.

Proteins from hyperthermophilic microorganisms are attractive candidates for novel biocatalysts because of their high resistance to temperature extremes. However, archaeal genes are usually poorly expressed in Escherichia coli because of differences in codon usage. Genes from the thermoacidophilic archaea Sulfolobus solfataricus and Thermoplasma acidophilum contain high proportions of rare codons for arginine, isoleucine, and leucine, which are recognized by the tRNAs encoded by the argU, ileY, and leuW genes, respectively, and which are rarely used in E. coli. To examine the effects of these rare codons on heterologous expression, we expressed the Sso_gnaD and Tac_gnaD genes from S. solfataricus and T. acidophilum, respectively, in E. coli. The Sso_gnaD product was expressed at very low levels when the open reading frame (ORF) was cloned in pRSET and expressed in E. coli BL21(DE3), and was expressed at much higher levels in the E. coli BL21(DE3)-CodonPlus RIL strain, which contains extra copies of the argU, ileY, and leuW tRNA genes. In contrast, Tac_gnaD was expressed at similar levels in both E. coli strains. Comparison of the Sso_gnaD and Tac_gnaD gene sequences revealed that the 5'-end of the Sso_gnaD sequence was rich in AGA(arg) and ATA(Ile) codons. These codons were replaced with the codons commonly used in E. coli by polymerase chain reaction-mediated site-directed mutagenesis. The results of expression studies showed that a non-tandem repeat of rare codons is critical in the observed interference in heterologous expression of this gene. We concluded that the level of heterologous expression of Sso_gnaD in E. coli was limited by the clustering of the rare codons in the ORF, rather than on the rare codon frequency.

Adverse effect of chloride impurities on lipase-catalyzed transesterifications in ionic liquids.

The adverse influence of chloride impurities on the lipase-catalyzed transesterification in ionic liquid is described. The activity of lipase from Rhizomucor miehei exponentially decreased with increasing Cl(-) content in 1-octyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl] amide, [Omim][Tf(2)N], and the activity of lipase in [Omim][Tf(2)N] mixture containing 2% [Omim] [Cl] was only about 2% of the activity in pure [Omim][Tf(2)N]. The activity of lipase from Candidantarctica linearly decreased at about 5% with every 1% increase in [Omim][Cl] with there being no activity in [Omim][Tf(2)N] containing about 20% [Omim][Cl].