Enzyme Catalyzed Formation of CoA Adducts of Fluorinated Hexanoic Acid Analogues using a Long-Chain acyl-CoA Synthetase from Gordonia sp. Strain NB4-1Y.

Per and polyfluoroalkyl substances (PFAS) are a large family of anthropogenic fluorinated chemicals of increasing environmental concern. Over recent years, numerous microbial communities have been found to be capable of metabolizing some polyfluoroalkyl substances, generating a range of low-molecular-weight PFAS metabolites. One proposed pathway for the microbial breakdown of fluorinated carboxylates includes ß-oxidation, this pathway is initiated by the formation of a CoA adduct. However, until recently no PFAS-CoA adducts had been reported. In a previous study, we were able to use a bacterial medium-chain acyl-CoA synthetase (mACS) to form CoA adducts of fluorinated adducts of propanoic acid and pentanoic acid but were not able to detect any products of fluorinated hexanoic acid analogues. Herein, we expressed and purified a long-chain acyl-CoA synthetase (lACS) and a A461K variant of mACS from the soil bacterium Gordonia sp. strain NB4-1Y and performed an analysis of substrate scope and enzyme kinetics using fluorinated and nonfluorinated carboxylates. We determined that lACS can catalyze the formation of CoA adducts of 1:5 fluorotelomer carboxylic acid (FTCA), 2:4 FTCA and 3:3 FTCA, albeit with generally low turnover rates (<0.02 s-1) compared with the nonfluorinated hexanoic acid (5.39 s-1). In addition, the A461K variant was found to have an 8-fold increase in selectivity toward hexanoic acid compared with wild-type mACS, suggesting that Ala-461 has a mechanistic role in selectivity toward substrate chain length. This provides further evidence to validate the proposed activation step involving the formation of CoA adducts in the enzymatic breakdown of PFAS.

Semi-rational engineering of CYP153A35 to enhance ω-hydroxylation activity toward palmitic acid.

CYP153A35 from Gordonia alkanivorans was recently characterized as fatty acid ω-hydroxylase. To enhance the catalytic activity of CYP153A35 toward palmitic acid, site-directed saturation mutagenesis was attempted using a semi-rational approach that combined structure-based computational analysis and subsequent saturation mutagenesis. Using colorimetric high-throughput screening (HTS) method based on O-demethylation activity of P450, CYP153A35 D131S and D131F mutants were selected. The best mutant, D131S, having a single mutation on BC-loop, showed 13- and 17-fold improvement in total turnover number (TTN) and catalytic efficiency (k cat/K M) toward palmitic acid compared to wild-type, respectively. However, in whole-cell reaction, D131S mutant showed only 50% improvement in ω-hydroxylated palmitic acid yield compared to the wild type. Docking simulation studies explained that the effect of D131S mutation on the catalytic activity would be mainly caused by the binding pose of fatty acids in the substrate access tunnel of the enzyme. This effect of D131S mutation on the catalytic activity is synergistic with that of the mutations in the active site previously reported.

Oligo(cis-1,4-isoprene) aldehyde-oxidizing dehydrogenases of the rubber-degrading bacterium Gordonia polyisoprenivorans VH2.

The actinomycete Gordonia polyisoprenivorans strain VH2 is well-known for its ability to efficiently degrade and catabolize natural rubber [poly(cis-1,4-isoprene)]. Recently, a pathway for the catabolism of rubber by strain VH2 was postulated based on genomic data and the analysis of mutants (Hiessl et al. in Appl Environ Microbiol 78:2874-2887, 2012). To further elucidate the degradation pathway of poly(cis-1,4-isoprene), 2-dimensional-polyacrylamide gel electrophoresis was performed. The analysis of the identified protein spots by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry confirmed the postulated intracellular pathway suggesting a degradation of rubber via ß-oxidation. In addition, other valuable information on rubber catabolism of G. polyisoprenivorans strain VH2 (e.g. oxidative stress response) was provided. Identified proteins, which were more abundant in cells grown with rubber than in cells grown with propionate, implied a putative long-chain acyl-CoA-dehydrogenase, a 3-ketoacyl-CoA-thiolase, and an aldehyde dehydrogenase. The amino acid sequence of the latter showed a high similarity towards geranial dehydrogenases. The expression of the corresponding gene was upregulated > 10-fold under poly(cis-1,4-isoprene)-degrading conditions. The putative geranial dehydrogenase and a homolog were purified and used for enzyme assays. Deletion mutants for five aldehyde dehydrogenases were generated, and growth with poly(cis-1,4-isoprene) was investigated. While none of the mutants had an altered phenotype regarding growth with poly(cis-1,4-isoprene) as sole carbon and energy source, purified aldehyde dehydrogenases were able to catalyze the oxidation of oligoisoprene aldehydes indicating an involvement in rubber degradation.

Production of ω-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo.

Bacterial cytochrome P450 enzymes in cytochrome P450 (CYP)153 family were recently reported as fatty acid ω-hydroxylase. Among them, CYP153As from Marinobacter aquaeolei VT8 (CYP153A33), Alcanivorax borkumensis SK2 (CYP153A13), and Gordonia alkanivorans (CYP153A35) were selected, and their specific activities and product yields of ω-hydroxy palmitic acid based on whole cell reactions toward palmitic acid were compared. Using CamAB as redox partner, CYP153A35 and CYP153A13 showed the highest product yields of ω-hydroxy palmitic acid in whole cell and in vitro reactions, respectively. Artificial self-sufficient CYP153A35-BMR was constructed by fusing it to the reductase domain of CYP102A1 (i.e., BM3) from Bacillus megaterium, and its catalytic activity was compared with CYP153A35 and CamAB systems. Unexpectedly, the system with CamAB resulted in a 1.5-fold higher yield of ω-hydroxy palmitic acid than that using A35-BMR in whole cell reactions, whereas the electron coupling efficiency of CYP153A35-BM3 reductase was 4-fold higher than that of CYP153A35 and CamAB system. Furthermore, various CamAB expression systems according to gene arrangements of the three proteins and promoter strength in their gene expression were compared in terms of product yields and productivities. Tricistronic expression of the three proteins in the order of putidaredoxin (CamB), CYP153A35, and putidaredoxin reductase (CamA), i.e., A35-AB2, showed the highest product yield from 5 mM palmitic acid for 9 h in batch reaction owing to the concentration of CamB, which is the rate-limiting factor for the activity of CYP153A35. However, in fed-batch reaction, A35-AB1, which expressed the three proteins individually using three T7 promoters, resulted with the highest product yield of 17.0 mM (4.6 g/L) ω-hydroxy palmitic acid from 20 mM (5.1 g/L) palmitic acid for 30 h.

Synthesis of vanillic acid using whole cell nitrilase of wild and mutant Gordonia terrae.

The resting cells of Gordonia terrae mutant E9 having enhanced nitrilase activity were used for biotransformation of 4-hydroxy-3-methoxybenzonitrile into vanillic acid. The maximum conversion was observed in 0.1 M phosphate buffer (pH 8.0), using 60 mM substrate and 0.75 mgDCW resting cells in 1 mL reaction at 40 °C. Km of the whole cell nitrilase of wild and mutant strains of G. terrae for this substrate were 20 and 16.6 mM, and Vmax were 0.19 and 0.95 Umg(-1)(DCW), respectively. Fed batch reaction for transformation of 4-hydroxy-3-methoxybenzonitrile using whole cell nitrilase of wild G. terrae resulted in 2.36 g of vanillic acid in 5 h with a catalytic and volumetric productivity of 0.78 gg(-1)(DCW) h(-1) and 4.72 gL(-1)h(-1), respectively. The whole cell nitrilase of G. terrae mutant E9 resulted in higher catalytic and volumetric productivity, i.e., 1.68 gg(-1)DCW h(-1) and 10 gL(-1)h(-1). A total 5.04 g of vanillic acid with 99% purity were accumulated in 100 mL of reaction after 5 h.

The effects of putative lipase and wax ester synthase/acyl-CoA:diacylglycerol acyltransferase gene knockouts on triacylglycerol accumulation in Gordonia sp. KTR9.

Previously, we demonstrated triacylglycerol (TAG) accumulation and the in vivo ability to catalyze esters from exogenous short chain alcohol sources in Gordonia sp. strain KTR9. In this study, we investigated the effects that putative lipase (KTR9_0186) and wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT; KTR9_3844) gene knockouts had on TAG accumulation. Gene disruption of KTR9_0186 resulted in a twofold increase in TAG content in nitrogen starved cells. Lipase mutants subjected to carbon starvation, following nitrogen starvation, retained 75 % more TAGs and retained pigmentation. Transcriptome expression data confirmed the deletion of KTR9_0186 and identified the up-regulation of key genes involved in fatty acid degradation, a likely compensatory mechanism for reduced TAG mobilization. In vitro assays with purified KTR9_3844 demonstrated WS/DGAT activity with short chain alcohols and C16 and C18 fatty acid Co-As. Collectively, these results indicate that Gordonia sp. KTR9 has a suitable tractable genetic background for TAG production as well as the enzymatic capacity to catalyze fatty acid esters from short chain alcohols.

Bench scale synthesis of p-hydroxybenzoic acid using whole-cell nitrilase of Gordonia terrae mutant E9.

Mutants of Gordonia terrae were generated using chemical mutagens for better activity, stability and higher substrate/product tolerance of its nitrilase enzyme. Mutant E9 showed two-time increase in activity and tolerated p-hydroxybenzonitrile (p-HBN) up to 50 mM. Response surface methodology and inducer mediation approach further enhanced the production of enzyme to 2.5-fold. The bench scale production of p-hydroxybenzoic acid (p-HBA) was carried out in a fed-batch reaction (500-mL scale) using whole-cell nitrilase of mutant E9 in 0.1 M potassium phosphate buffer (pH 8.0) at 40 °C. Total six feedings each at an interval of 45 min resulted in accumulation of 360 mM (21.6 g) of p-HBA with a purity of 99%. The catalytic and volumetric productivity of bioprocess using mutant G. terrae was improved to 1.8 g h(-1) g DCW (-1) and 43.2 g L(-1), respectively, from 0.78 g h(-1) g DCW (-1) and 28.8 g L(-1) using resting cells of wild strain. K m and V max of purified nitrilase from mutant E9 were 55 U mg(-1) and 1.8 mM for p-HBN with a higher turnover number of 36 s(-1) × 10(-3).

Latex clearing protein-an oxygenase cleaving poly(cis-1,4-isoprene) rubber at the cis double bonds.

Gordonia polyisoprenivorans strain VH2, a potent rubber-degrading actinomycete, harbors two latex clearing proteins (Lcps), which are known to be essential for the microbial degradation of rubber. However, biochemical information on the exact role of this protein in the degradation of polyisoprene was lacking. In this study, the gene encoding Lcp1VH2 was heterologously expressed in strains of Escherichia coli, the corresponding protein was purified, and its role in rubber degradation was examined by measurement of oxygen consumption as well as by chromatographic and spectroscopic methods. It turned out that active Lcp1VH2 is a monomer and is responsible for the oxidative cleavage of poly(cis-1,4-isoprene) in synthetic as well as in natural rubber by the addition of oxygen (O2) to the cis double bonds. The resulting oligomers possess repetitive isoprene units with aldehyde (CHO-CH2-) and ketone (-CH2-CO-CH3) functional groups at the termini. Two fractions with average isoprene contents of 18 and 10, respectively, were isolated, thus indicating an endocleavage mechanism. The activity of Lcp1VH2 was determined by applying a polarographic assay. Alkenes, acyclic terpenes, or other rubber-like polymers, such as poly(cis-1,4-butadiene) or poly(trans-1,4-isoprene), are not oxidatively cleaved by Lcp1VH2. The pH and temperature optima of the enzyme are at pH 7 and 30°C, respectively. Furthermore, it was demonstrated that active Lcp1VH2 is a Cu(II)-containing oxygenase that exhibits a conserved domain of unknown function which cannot be detected in any other hitherto-characterized enzyme. The results presented here indicate that this domain might represent a new protein family of oxygenases.

A novel thermoalkaliphilic xylanase from Gordonia sp. is salt, solvent and surfactant tolerant.

Two aerobic bacterial consortia namely Con T and Con R were developed by enrichment technique from termite gut and cow dung respectively, using xylan as a sole carbon source. Molecular characterization of Con R based on 16S rRNA sequence analysis showed the presence of Pannonibacter sp. R-3 and Pseudoxanthomas sp. R-5. On the other hand, Con T showed the presence of Pseudoxanthomas sp. T-5, Cellulosimicrobium sp. T-21, and Gordonia sp. T-30. Being the maximum xylanase producer among the five isolates and being a novel xylanase producing bacterial genus, Gordonia sp. T-30 was selected. Xylanase produced by Gordonia sp. T-30 showed optimum activity at 60 °C and pH 9. Xylanase was 95% stable for 120 min at pH 9.0 and 98% stable at 60 °C for 90 min. Xylanase activity was stimulated in the presence of organic solvents such as petroleum ether, acetone, diethyl ether, n-hexane, and benzene. Detergent like cetyltrimethylammonium bromide and presence of NaCl also accelerated the xylanase function. Comparative evaluation was studied between sterilized and non-sterilized solid fermentation to produce xylanase by Gordonia sp. T-30 using various agricultural residues as growth substrate in cost effective manner. Industrially important features endowed by this xylanase make it a very promising candidate for food, feed, and fuel industry.

Physiological characterization of lipid accumulation and in vivo ester formation in Gordonia sp. KTR9.

Previous work has demonstrated the feasibility of in vivo biodiesel synthesis in Escherichia coli, however, ethyl ester formation was dependent on an external fatty acid feedstock. In contrast to E. coli, actinomycetes may be ideal organisms for direct biodiesel synthesis because of their capacity to synthesize high levels of triacylglcerides (TAGs). In this study, we investigated the physiology and associated TAG accumulation along with the in vivo ability to catalyze ester formation from exogenous short chain alcohol sources in Gordonia sp. KTR9, a strain that possesses a large number of genes dedicated to fatty acid and lipid biosynthesis. Total lipid fatty acids content increased by 75 % and TAG content increased by 50 % under nitrogen starvation conditions in strain KTR9. Strain KTR9 tolerated the exogenous addition of up to 4 % methanol, 4 % ethanol and 2 % propanol in the media. Increasing alcohol concentrations resulted in a decrease in the degree of saturation of recovered fatty acid alcohol esters and a slight increase in the fatty acid chain length. A linear dose dependency in fatty alcohol ester synthesis was observed in the presence of 0.5-2 % methanol and ethanol compared to control KTR9 strains grown in the absence of alcohols. An inspection of the KTR9 genome revealed the presence of several putative wax ester synthase/acyl-coenzyme A : diacylglycerol acyltransferase (WS/DGAT) enzymes, encoded by atf gene homologs, that may catalyze the in vivo synthesis of fatty acid esters from short chain alcohols. Collectively, these results indicate that Gordonia sp. KTR9 may be a suitable actinomycete host strain for in vivo biodiesel synthesis.

[Catalase activity of hydrocarbon-oxidizing bacteria].

The dynamics of catalase activity of the hydrocarbon-oxidizing bacteria Cordona terrae, Rhodococcus rubropertinctus, and Rhodococcus erythropolis during petroleum product destruction has been studied. A direct relationship between decreasing catalase activity of hydrocarbon-oxidizing microorganisms and the intensity of petroleum product destruction has been established experimentally. The revealed dependence allows one to consider the catalase activity of bacteria as an indicator of the initial stage of petroleum product oxidation and may be used for choosing destructor strains to construct biopreparations suitable for natural ecosystem remediation.

Stress proteins, nonribosomal peptide synthetases, and polyketide synthases regulate carbon sources-mediated bio-demulsifying mechanisms of nitrate-reducing bacterium Gordonia sp. TD-4.

Carbon sources have been reported to determine the bio-demulsifying performance and mechanisms. However, the genetic regulation of carbon sources-mediated bio-demulsification remains unclear. Here, the effects of ß-oxidation, stress response, and nitrate metabolism on the demulsification of alkaline-surfactant-polymer flooding produced water by Gordonia sp. TD-4 were investigated. The results showed that competitive adsorption-derived demulsification was mediated by oil-soluble carbon sources (paraffin). Surface-active lipopeptides responsible for competitive adsorption-derived demulsification could be biosynthesized by the nonribosomal peptide synthetases and polyketide synthases using oil-soluble carbon sources. Bio-flocculation-derived demulsification was mediated by water-soluble carbon sources. Water-soluble carbon sources (sodium acetate and glucose) mediated the process of the dissimilatory reduction of nitrate to ammonia, which resulted in the variable accumulation of nitrite. The accumulated nitrite (>180 mg-N/L) stimulated stress response and induced the upregulation of chaperone-associated genes. The upregulation of chaperonins increased the cell surface hydrophobicity and the cation-dependent bio-flocculating performance, which were responsible for bio-flocculation-derived demulsification. The ß-oxidation of fatty acids significantly affected both competitive adsorption-derived demulsification and bio-flocculation-derived demulsification. This study illustrates the synergistic effects of nitrogen sources and carbon sources on the regulation of bio-demulsifying mechanisms of TD-4 and identifies two key functional gene modules responsible for the regulation of bio-demulsifying mechanisms.

Involvement of an alkane hydroxylase system of Gordonia sp. strain SoCg in degradation of solid n-alkanes.

Enzymes involved in oxidation of long-chain n-alkanes are still not well known, especially those in gram-positive bacteria. This work describes the alkane degradation system of the n-alkane degrader actinobacterium Gordonia sp. strain SoCg, which is able to grow on n-alkanes from dodecane (C(12)) to hexatriacontane (C(36)) as the sole C source. SoCg harbors in its chromosome a single alk locus carrying six open reading frames (ORFs), which shows 78 to 79% identity with the alkane hydroxylase (AH)-encoding systems of other alkane-degrading actinobacteria. Quantitative reverse transcription-PCR showed that the genes encoding AlkB (alkane 1-monooxygenase), RubA3 (rubredoxin), RubA4 (rubredoxin), and RubB (rubredoxin reductase) were induced by both n-hexadecane and n-triacontane, which were chosen as representative long-chain liquid and solid n-alkane molecules, respectively. Biotransformation of n-hexadecane into the corresponding 1-hexadecanol was detected by solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME/GC-MS) analysis. The Gordonia SoCg alkB was heterologously expressed in Escherichia coli BL21 and in Streptomyces coelicolor M145, and both hosts acquired the ability to transform n-hexadecane into 1-hexadecanol, but the corresponding long-chain alcohol was never detected on n-triacontane. However, the recombinant S. coelicolor M145-AH, expressing the Gordonia alkB gene, was able to grow on n-triacontane as the sole C source. A SoCg alkB disruption mutant that is completely unable to grow on n-triacontane was obtained, demonstrating the role of an AlkB-type AH system in degradation of solid n-alkanes.

Isolation and preliminary characterization of a respiratory nitrate reductase from hydrocarbon-degrading bacterium Gordonia alkanivorans S7.

Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors. The respiratory nitrate reductase (Nar) from this organism has been isolated using ion exchange chromatography and gel filtration, and then preliminarily characterized. PAGE, SDS-PAGE and gel filtration chromatography revealed that Nar consisted of three subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40 degrees C. K(m) values for NO(3)(-) (110 microM) and for ClO(3)(-) (138 microM) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from Gordonia species.

Enhancing the synthesis of latex clearing protein by different cultivation strategies.

In this study, we improved the synthesis of the latex clearing protein from Gordonia polyisoprenivorans VH2 (Lcp1VH2), a key enzyme for the initial cleavage of the rubber backbone. Cultivations using a recombinant strain of Escherichia coli were optimized to overcome poor solubility of Lcp1VH2 and improve the production yields. Different cultivation temperatures and agitation rates were evaluated in the process to demonstrate their impact on the solubility of Lcp1VH2. A specific maximum production rate of 28.3 mg Lcp1VH2 g-1 cell dry weight h-1 was obtained at 25 °C and at agitation rates between 200-300 rpm. The activity of Lcp1VH2 was strongly influenced by variations in the cultivation temperature with a specific maximum activity of 0.81 U mg-1 in cultures incubated at 30 °C. Besides cultivation-based optimization, also the strategy of fusion protein expression with NusA was successfully applied. The in vivo solubility of the Lcp1VH2 fusion protein was calculated to be 73.1%, which means an enhancement of 5.7-fold in comparison to the solubility of the native Lcp1VH2. The fusion protein of Lcp1VH2 and NusA still exhibited oxygenase activity with polyisoprene latex as a substrate. In fact, NusA-His-Lcp1VH2 reached a 4-fold higher volumetric activity in comparison to Lcp1VH2. Oligo(cis-1,4-isoprene) molecules were produced as degradation products due to the cleavage of the polymer backbone by NusA-His-Lcp1VH2. The formation of oligo-isoprenoid molecules with molecular weights between 236 and 984 Da were confirmed by electrospray ionization-mass spectrometry analysis.

Biodegradation of Structurally Diverse Phthalate Esters by a Newly Identified Esterase with Catalytic Activity toward Di(2-ethylhexyl) Phthalate.

Herein, we report a double enzyme system to degrade 12 phthalate esters (PAEs), particularly bulky PAEs, such as the widely used bis(2-ethylhexyl) phthalate (DEHP), in a one-pot cascade process. A PAE-degrading bacterium, Gordonia sp. strain 5F, was isolated from soil polluted with plastic waste. From this strain, a novel esterase (GoEst15) and a mono(2-ethylhexyl) phthalate hydrolase (GoEstM1) were identified by homology-based cloning. GoEst15 showed broad substrate specificity, hydrolyzing DEHP and 10 other PAEs to monoalkyl phthalates, which were further degraded by GoEstM1 to phthalic acid. GoEst15 and GoEstM1 were heterologously coexpressed in Escherichia coli BL21 (DE3), which could then completely degrade 12 PAEs (5 mM), within 1 and 24 h for small and bulky substrates, respectively. To our knowledge, GoEst15 is the first DEHP hydrolase with a known protein sequence, which will enable protein engineering to enhance its catalytic performance in the future.

A simple, rapid and cost-effective process for production of latex clearing protein to produce oligopolyisoprene molecules.

Aiming at finding feasible alternatives for rubber waste disposal, the partial enzymatic degradation of poly(cis-1,4-isoprene)-containing materials represents a potential solution. The use of rubber-degrading enzymes and the biotransformation of rubber into new materials is limited by the high costs associated with the production and purification of the enzyme and the complexity of the process. This study presents a simple and low-cost procedure to obtain purified latex clearing protein (Lcp), an enzyme capable of cleaving the double bonds of poly(cis-1,4-isoprene) in presence of oxygen to produce different size of oligomers with terminal aldehyde and ketone groups, respectively. The gene coding for Lcp1VH2 from Gordonia polyisoprenivorans strain VH2 was overexpressed in Escherichia coli C41 (DE3), and by using an auto-induction medium high protein yields were obtained. The cultivation process was described and compared with an IPTG-inducible medium previously used. Purification of the enzyme was performed using salting out precipitation with ammonium sulfate. Different salt concentrations and pH were tested in order to find the optimal for purification, obtaining a concentration of 60mg Lcp per l. The enzymatic activity of the purified enzyme was measured by an oxygen consumption assay in the presence of polyisoprene latex. Volumetric activities of 0.16Uml-1 were obtained at optimal conditions of temperature and pH. The results showed an active and partial purified fraction of Lcp1VH2, able to cleave the backbone of poly(cis-1,4-isoprene) and to produce degradation products that were identified with staining methodologies (Schiff reagent for aldehyde groups and 2,4-DNPH for carbonyl groups) and characterized using nuclear magnetic resonance (NMR). Thirteen different storage conditions were tested for the purified enzyme analyzing the enzymatic activity after 1 and 3 months. Lcp1VH2, as an ammonium sulfate precipitate, was stable, easy to handle and sufficiently active for further analysis. The described methodology offers the possibility to upscale the process and to produce large amounts of this protein.

Phylogenetic analysis of members of the metabolically diverse genus Gordonia based on proteins encoding the gyrB gene.

Members of the metabolically diverse genus Gordonia were isolated from various biotopes including pristine and polluted sites around Taiwan. Identification, comparison and diversity assessment based on the gyrB gene were carried out using a newly developed primer pair for gyrB. The 16S rRNA gene was also sequenced for comparison. A 1.2-kb fragment of the gyrB gene of 17 Gordonia strains including type strains was determined by direct sequencing of PCR amplified fragments. A total of 25 strains (8 of which were retrieved from a public database) of the genus Gordonia form a distinct phyletic line in the GyrB-based tree and are separated from other closely related species of genera of the suborder Corynebacterineae. Sequence similarity of the gyrB sequence from twelve Gordonia type strains ranged from 79.3 to 97.2%, corresponding to between 270 and 41 nucleotide differences, while there was only a 0.3-3.8% difference in 16S rRNA gene sequence similarity at the interspecies level. Phylogenetic analysis based on the GyrB sequence deduced from the gyrB gene is consistent with that of DNA-DNA hybridization results and provides a better discrimination within the species of Gordonia compared to the 16S rRNA gene. The present study demonstrates that gyrB gene analysis will aid in describing novel species belonging to the genus Gordonia.

Re-characterization of mono-2-ethylhexyl phthalate hydrolase belonging to the serine hydrolase family.

A novel bacterium assimilating di-2-ethylhexyl phthalate as a sole carbon source was isolated, and identified as a Rhodococcus species and the strain was named EG-5. The strain has a mono-2-ethylhexyl phthalate (MEHP) hydrolase (EG-5 MehpH), which exhibits some different enzymatic features when compared with the previously reported MEHP hydrolase (P8219 MehpH) from Gordonia sp. These differences include different pH optimum activity, maximal reaction temperature and heat stability. The Km and Vmax values of EG-5 MehpH were significantly higher than those of P8219 MehpH. The primary structure of EG-5 MehpH showed the highest sequence identity to that of P8219 MehpH (39%) among hydrolases. The phylogenetic tree suggested that EG-5 MehpH and P8219 MehpH were categorized in different groups of the novel MEHP hydrolase family. Mutation of a conserved R(109) residue of EG-5 MehpH to a hydrophobic residue resulted in a dramatic reduction in the Vmax value towards MEHP without affecting the Km value. These results indicate that this residue may neutralize the negative charge of a carboxylate anion of MEHP, and thus inhibit the catalytic nucleophile from attacking the ester bond. In other words, the R residue blocks inhibition from the carboxylate anion of MEHP. Recently, registered hypothetical proteins exhibiting 98% or 99% identities for EG-5 MehpH or for P8219 MehpH were found from some pathogens belonging to Actinomycetes. The protein may have other activities besides MEHP hydrolysis and function in other physiological reactions in some Actinomycetes.

Substrate specificity of ß-glucosidase from Gordonia terrae for ginsenosides and its application in the production of ginsenosides Rg3, Rg2, and Rh1 from ginseng root extract.

A ß-glucosidase from Gordonia terrae was cloned and expressed in Escherichia coli. The recombinant enzyme with a specific activity of 16.4 U/mg for ginsenoside Rb1 was purified using His-trap chromatography. The purified enzyme specifically hydrolyzed the glucopyranosides at the C-20 position in protopanaxadiol (PPD)-type ginsenosides and hydrolyzed the glucopyranoside at the C-6 or C-20 position in protopanaxatriol (PPT)-type ginsenosides. The reaction conditions for the high-level production of Rg3 from Rb1 by the enzyme were pH 6.5, 30°C, 20 mg/ml enzyme, and 4 mg/ml Rb1. Under these conditions, G. terrae ß-glucosidase completely converted Rb1 and Re to Rg3 and Rg2, respectively, after 2.5 and 8 h, respectively. Moreover, the enzyme converted Rg1 to Rh1 at 1 h with a molar conversion yield of 82%. The enzyme at 10 mg/ml produced 1.16 mg/ml Rg3, 1.47 mg/ml Rg2, and 1.17 mg/ml Rh1 from Rb1, Re, and Rg1, respectively, in 10% (w/v) ginseng root extract at pH 6.5 and 30°C after 33 h with molar conversion yields of 100%, 100%, and 77%, respectively. The combined molar conversion yield of Rg2, Rg3, and Rh1 from total ginsenosides in 10% (w/v) ginseng root extract was 68%. These above results suggest that this enzyme is useful for the production of ginsenosides Rg3, Rg2, and Rh1.