Isolation and characterization of Acinetobacter sp. ES-1 excreting a lipase with high enantioselectivity for (S)-ketoprofen ethyl ester

A new Acinetobacter sp. ES-1, grown on triolein, tryptone and Triton X-100, excreted a lipase that hydrolyzed 10m M (R,S)-ketoprofen ethyl ester into (S)-ketoprofen. The crude lipase had an activity of 10Uml^-1 and, at 30°C and pH7 over 48h, gave a conversion yield of 35% with an enantiomeric excess for the product 96%.     

拆分获得(R)-酮基布洛芬酯酶基因的筛选、克隆与表达

以自筛选出的具有一定不对称拆分外消旋酮基布洛芬氯乙酯能力的野生菌Bacillus megaterium NK13为材料,通过构建其基因文库,筛选得到一个阳性克隆重组子pUC18-NK-HYD3。分析测序结果发现外源片段中包含一段完整的741 bp的开放阅读框,其编码的蛋白中含有酯酶的GXSXG保守序列。经在NCBI的BLAST系统中比对,证明该酯酶基因属于首次发现(GenBank Accession Number: GU143552)。将酯酶基因克隆到载体pET21b(+)中,转化E. coli BL21(DE3),经IPTG诱导后在宿主菌得到表达。SDS-PAGE电泳检测证明该酯酶蛋白分子量约为28 kDa。TLC和HPLC检测结果显示,该酯酶优先水解(R)-型底物,在重组菌菌液体系,转化率为15%时,酯酶拆分获得(R)-酮基布洛芬的过量值(e.e.%)最高,达62.74％;改用重组菌湿菌体的PBS体系后,在转化率为10%~50%时,酯酶拆分获得(R)-酮基布洛芬的过量值(e.e.%)一直保持在73%~76%之间。     

Simultaneous expression of an arylacetonitrilase from Pseudomonas fluorescens and a (S)-oxynitrilase from Manihot esculenta in Pichia pastoris for the synthesis of (S)-mandelic acid

The arylacetonitrilase of Pseudomonas fluorescens EBC191 catalyzes the conversion of (S)-mandelonitrile to (S)-mandelic acid and (S)-mandeloamide. This biotransformation is optimally performed under acidic pH values because (S)-mandelonitrile rapidly decomposes under neutral conditions. Therefore, the gene encoding the arylacetonitrilase of P. fluorescens EBC191 was integrated and expressed under the control of the AOX1 promoter in the methylotrophic yeast Pichia pastoris which was supposed to act as an acidotolerant expression system. These recombinant strains hydrolyzed (R,S)-mandelonitrile at pH values >or=3 to mandelic acid and mandeloamide and were more acidotolerant than previously constructed Escherichia coli whole cell catalysts synthesizing the same nitrilase activity. Subsequently, recombinant P. pastoris strains were constructed which simultaneously expressed the (S)-oxynitrilase of Manihot esculenta and the arylacetonitrilase of P. fluorescens EBC191 each under the control of individual AOX1 promoters in order to obtain a whole cell catalyst for the synthesis of (S)-mandelic acid from benzaldehyde and cyanide. Resting cells of the recombinant strains converted under acidic conditions benzaldehyde and cyanide initially to mandelonitrile which was immediately converted to mandelic acid and mandeloamide. The chiral analysis of the products formed revealed a high enantiomeric excess for the (S)-enantiomers.     

A chemoenzymatic approach to the synthesis of enantiomerically pure (S)-3-hydroxy-γ-butyrolactone

Optically pure (S)-3-hydroxy-gamma-butyrolactone, an important chiral building block in the pharmaceutical industry, was synthesized from L: -malic acid by combining a selective hydrogenation and a lipase-catalyzed hydrolysis. Lipase from Candida rugosa was found to be the most efficient enzyme for the hydrolysis of (S)-beta-benzoyloxy-gamma-butyrolactone. The use of organic solvent-aqueous two-phase system was employed to extract benzoic acid generated from enzymatic hydrolysis of the substrate. Tert-butyl methyl ether as an organic solvent was effective to extract the reaction product, benzoic acid, and stably maintained the enzyme activity of Lipase OF immobilized on polymeric supports Amberlite XAD-7. The immobilization made the recovery of the product simpler and prevented the formation of the emulsion. The pH adjustment was unnecessary with the immobilized Lipase OF. The scale-up of the enzymatic hydrolysis of S-BBL at 1,850-kg scale was carried out without problems to give 728.5 kg of S-HGB at 80% isolated yield. The scale-up results are similar to those of bench scale reactions. Racemic (R,S)-beta-benzoyloxy-gamma-butyrolactone was prepared from D-, L: -malic acid and was found to be hydrolyzed nonselectively by the enzyme.     

(2S)-2-Amino-5-chloro-4-hydroxy-5-hexenoic acid,a new chloroamino acid,and related compounds fromAmanita gymnopus

Combining different chromatography systems, unusual nonprotein amino acids were isolated and unequivocally identified from a small amount (less than 100 g fresh weight) ofAmanita gymnopus fruit body. Without obtaining crystals of these amino acids, on the basis of¹H-NMR determination, high resolution mass spectrometry, chlorine analysis and oxidation with L-amino acid oxidase, one of them proved to be a new chloroamino acid, (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid (G2). The other three were (2S)-2-amino-5-hexenoic acid (G1), (2S)-2-amino-4,5-hexadienoic acid (G3) and (2S)-2-amino-5-hexynoic acid (G4). Amino acid (G1) was also encountered for the first time in natural products. Amino acid (G3) has been reported from several kinds of fungi belonging toAmanita, subgenusLepidella. The occurrence of amino acid (G4) was already reported fromCortinarius claricolor.Part 23 in the series Biochemical studies of nitrogen compounds in fungi. Part 22, Hatanaka, S. I. et al. 1985. Trans. Mycol. Soc. Japan26: 61–68.     

The S11 and S13 self incompatibility alleles in Solanum chacoense Bitt. are remarkably similar

A genomic clone of the S11 allele from the self-incompatibility locus (S locus) in Solanum chacoense Bitt. has been isolated by cross-hybridization to the S. chacoense S13 allele and sequenced. The sequence of the S11 allele contains all the features expected for S genes of the Solanaceae, and S11 expression, as assessed by northern blots and RNA-PCR, was similar to that of other S. chacoense S alleles. The S11 protein sequence shares 95% identity with the phenotypically distinct S13 protein of S. chacoense and is the gametophytic S allele with the highest similarity to an existing allele so far discovered. Only 10 amino acid changes differentiate the mature proteins from these two alleles, which sets a new lower limit to the number of changes that can produce an altered S allele specificity. The amino acid substitutions are not clustered, suggesting that an accumulation of random point mutations can generate S allele diversity. The S11 intron is unusual in that it could be translated in frame with the coding sequence, thus suggesting an additional mechanism for the generation of new S alleles.     

Study on the metastable zone width of ketoprofen

With increasing awareness for the need of pure enantiomer drugs, strong emphasis has been focused on the research of chiral drug separation. Compared with other separation methods, crystallization is a simple and economical method, and the metastable zone width (MSZW) is a very important factor for the entire crystallization process. In this paper, the effects of the metastable zones of (R,S)- and (S)-ketoprofen and a 0.94 mole fraction of (S)-ketoprofen in order to enhance the MSZW were studied. Four main factors were studied, namely, temperature, cooling rate, stirring rate, and volume ratio of mixed solvent (water/ethanol). Through the L9 fractional experiment design, it was observed that all samples' MSZWs would increase with an increase in cooling rate and decrease with an increase in the ethanol volume ratio and temperature. The ethanol ratio may have the strongest effect on the process and can greatly enhance the metastable zone, and the other three factors influence the MSZW only slightly. In conclusion, the these four factors for enhancing MSZW have been optimized: water-to-ethanol volume ratio, 1:0.6; temperature, 20 degrees C; stirring rate, 700 rpm; and cooling rate, 12.0 degrees C/h. All of these results will be helpful for the following chiral separation of ketoprofen by crystallization.     

Screening and characterization of a novel esterase from a metagenomic library

Metagenomes from various environmental soils were screened using alpha-naphthyl acetate and Fast Blue RR for a novel ester-hydrolyzing enzyme on Escherichia coli. Stepwise fragmentations and subcloning of the initial insert DNA (30-40 kb) using restriction enzymes selected to exclude already known esterases with subsequent screenings resulted in a positive clone with a 2.5-kb DNA fragment. The cloned sequence included an open reading frame consisting of 1089 bp, designated as est25, encoding a protein of 363 amino acids with a molecular mass of about 38.3 kDa. Amino acid sequence analysis revealed only moderate identity (< or = 48%) to the known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases, such as HGGG (residues 124-127), GxSxG (residues 199-203), and the putative catalytic triad composed of Ser201, Asp303, and His333. Est25 was functionally overexpressed in a soluble form in E. coli with optimal activity at pH 7.0 and 25 degrees C. The purified Est25 exhibited hydrolyzing activity toward p-nitrophenyl (NP)-fatty acyl esters with short-length acyl chains (< or = C6) with the highest activity toward p-NP-acetate (Km=1.0 mM and Vmax = 63.7 U/mg), but not with chain lengths > or = C8, demonstrating that Est25 is an esterase originated most likely from a mesophilic microorganism in soils. Est25 efficiently hydrolyzed (R,S)-ketoprofen ethyl ester with Km of 16.4 mM and Vmax of 59.1 U/mg with slight enantioselectivity toward (R)-ketoprofen ethyl ester. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzymes from a metagenome.     

Enantioselective pharmacodynamics of the nonsteroidal antiinflammatory drug ketoprofen: in vitro inhibition of human platelet cyclooxygenase activity.

The pharmacological activity of ketoprofen enantiomers was investigated in humans by an in vitro method. The antiplatelet effect of ketoprofen was assessed by measuring the inhibition of platelet thromboxane B2 (TXB2) generation during the controlled clotting of whole blood obtained from each of four healthy volunteers. Ketoprofen was added separately to whole blood as a range of concentrations of (1) predominantly (S)-ketoprofen, (2) racemic ketoprofen, and (3) predominantly (R)-ketoprofen. (S)-Ketoprofen was found to be solely active at inhibiting human platelet TXB2 production; (R)-ketoprofen was devoid of such activity and did not modify the potency of its optical antipode. A relationship between the percentage inhibition of TXB2 generation and the unbound concentration of (S)-ketoprofen in serum was modelled according to a sigmoidal Emax equation. The mean (+/- SD) serum unbound concentration of (S)-ketoprofen required to inhibit platelet TXB2 generation by 50% (EC50) was 0.320 (+/- 0.062) ng/ml. This value for ketoprofen is considerably lower than previously reported values for (S)-ibuprofen and (S)-naproxen.     

Prevalence and diversity of tetracycline resistant lactic acid bacteria and their tet genes along the process line of fermented dry sausages

In order to study the prevalence and diversity of tetracycline resistant lactic acid bacteria (Tc(r) LAB) along the process line of two different fermented dry sausage (FDS) types, samples from the raw meat, the meat batter and the fermented end product were analysed quantitatively and qualitatively by using a culture-dependent approach. Both the diversity of the tet genes and their bacterial hosts in the different stages of FDS production were determined. Quantitative analysis showed that all raw meat components of both FDS types (FDS-01 and FDS-08) contained a subpopulation of Tc(r) LAB, and that for FDS-01 no Tc(r) LAB could be recovered from the samples after fermentation. Qualitative analysis of the Tc(r) LAB subpopulation in FDS-08 included identification and typing of Tc(r) LAB isolates by (GTG)5-PCR fingerprinting, plasmid profiling, protein profiling and a characterization of the resistance by PCR detection of tet genes. Two remarks can be made when the results of this analysis for the different samples are compared. (i) The taxonomic diversity of Tc(r) LAB varies along the process line, with a higher diversity in the raw meat (lactococci, lactobacilli, streptococci, and enterococci), and a decrease after fermentation (only lactobacilli). (ii) Also the genetic diversity of the tet genes varies along the process line. Both tet(M) and tet(S) were found in the raw meat, whereas only tet(M) was found after fermentation. A possible relationship was found between the disappearing of species other than lactobacilli and tet(S), because tet(S) was only found in lacotocci, enterococci, and streptococci. These data suggest that fermented dry sausages are among those food products that can serve as vehicles for Tc(r) LAB and that the raw meat already contains a subpopulation of these bacteria. Whether these results reflect the transfer of resistant bacteria or of bacterial resistance genes from animals to man via the food chain is difficult to ascertain and may require a combination of cultivation-dependent and cultivation-independent approaches.     

A stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation

S-Adenosyl-L-methionine (AdoMet) which is biologically synthesized by AdoMet synthetase bears an S configuration at the sulfur atom. The chiral sulfonium spontaneously racemizes to form a mixture of S and R isomers of AdoMet under physiological conditions or normal storage conditions. The chirality of AdoMet greatly affects its activity; the R isomer is not accepted as a substrate for AdoMet-dependent methyltransferases. We report a stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on an enzyme-coupled reaction in which (S,S)-AdoMet reacts with 2-nitro-5-thiobenzoic acid to form AdoHcy and 2-nitro-5-methylthiobenzoic acid. The transformation is catalyzed by recombinant human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) and is associated with a large spectral change at 410 nm. Accumulation of the S-adenosylhomocysteine (AdoHcy) product, a feedback inhibitor of TPMT, slows the assay. AdoHcy nucleosidase (EC 3.2.2.9) irreversibly cleaves AdoHcy to adenine and S-ribosylhomocysteine, significantly shortening the assay time to less than 10 min. The assay is linear from 5 to at least 60 microM (S,S)-AdoMet.     

Pharmacokinetics of ketoprofen enantiomers in monkeys following single and multiple oral administration

Pharmacokinetic studies are reported after single oral administration of 3 mg/kg of stereochemically pure (S)-ketoprofen [(S)-KP] and (R)-ketoprofen [(R)-KP] to three male Cynomolgus monkeys and after repeated administration for 6 months of 3, 15 and 75 mg/kg/day of (S)-KP to both male and female monkeys. A high-performance liquid chromatographic (HPLC) analysis was performed without derivatization of the samples, using a chiral column. The pharmacokinetic parameters for (S)-KP after administration of (S)-KP and for (R)-KP after administration of (R)-KP were, respectively, elimination half-life 2.32 ± 0.36 and 1.64 ± 0.40 h; oral clearance 3.50 ± 0.66 and 7.50 ± 3.20 ml/min/kg; apparent volume of distribution 0.74 ± 0.24 and 1.16 ± 0.76 liter/kg; mean residence time 1.79 ± 0.77 and 1.41 ± 0.65 h; area under the concentration/time curve 14.16 ± 2.93 and 7.31 ± 2.98 μg·h/ml. Forty-nine percent unidirectional bioinversion of (R)-KP to (S)-KP was observed in this species and the pharmacokinetic parameters for the (S)-KP resulting from this inversion were also calculated. In the study of 6-month repeated administration of (S)-KP, linear pharmacokinetic behavior and no evidence of drug accumulation were observed at the three dose levels. © 1994 Wiley-Liss, Inc.     

A novel design of simulated moving bed (SMB) chromatography for separation of ketoprofen enantiomer

A simulated moving bed (SMB) chromatography system is a powerful tool for preparative scale separation, which can be applied to the separation of chiral compound. We have designed our own lab-scale SMB chromatography using 5 HPLC pumps, 6 stainless steel columns and 4 multi-position valves, to separate a racemic mixture of ketoprofen in to its enantiomers. Our design has the characteristics of the low cost for assembly for the SMB chromatography and easy repair of the unit, which differs from the designs suggested by other investigators. It is possible for the flow path through each column to be independently changed by computer control, using 4 multi-position rotary valves and 5 HPLC solvent delivery pumps. In order to prove the operability of our SMB system, attempts were made to separate the (S)-ketoprofen enantiomer from a ketoprofen racemic mixture. The operating parameters of the SMB chromatography were calculated for ketoprofen separation from a batch chromatography experiment as well as by the triangle theory. With a feed concentration of 1 mg/mL, (S)-ketoprofen was obtained with a purity of 96% under the calculated operating conditions.     

Preparation of S-( )-ketoprofen by coupling the enantioselective hydrolysis of ketoprofen methyl ester with the photo-oxidation of methanol

A novel method for preparation of S-(+)-ketoprofen is presented involving coupling enantioselective hydrolysis of ketoprofen methyl ester catalyzed by a surfactant-coated-lipase with the photo-oxidation of methanol in a water-saturated organic solvent. The effect of photocatalytic conversion of methanol into water and carbon dioxide on the hydrolysis of ketoprofen methyl ester and the stability of the enzyme was investigated. The photo-oxidation of methanol shifted the equilibrium of the hydrolysis toward the formation of ketoprofen, increasing the equilibrium conversion ratio and improving the enantioselectivity. Because the surfactant-coated lipase and ketoprofen methyl ester dissolved in the organic solvent and ketoprofen was absorbed on the TiO₂ photocatalyst particles, the separation procedures could be simplified and the stability of the enzyme was increased.     

Plasma protein binding of ketoprofen enantiomers in man: method development and its application.

The protein binding of ketoprofen enantiomers was investigated in human plasma at physiological pH and temperature by ultrafiltration. 14C-labelled (RS)-ketoprofen was synthesized and purified by high-performance liquid chromatography and utilized as a means of quantifying the unbound species. In vitro studies were conducted with plasma obtained from six healthy volunteers. The plasma was spiked with (R)-ketoprofen alone, (S)-ketoprofen alone, and (RS)-ketoprofen in the enantiomeric concentration range of 1.0 to 19.0 micrograms/ml. The plasma protein binding of ketoprofen was nonenantioselective. At a racemic drug concentration of 2.0 micrograms/ml the mean (+/- SD) percentage unbound of (R)-ketoprofen was 0.80 (+/- 0.15)%. The corresponding value for (S)-ketoprofen, 0.78 (+/- 0.18)%, was not statistically different (P greater than 0.05). At this racemic drug concentration (2.0 micrograms/ml) the percentage unbound of each enantiomer was unaffected (P greater than 0.05) by the presence of the glucuronoconjugates of ketoprofen (10 micrograms/ml) in plasma. At clinically relevant concentrations, the plasma binding of ketoprofen did not exhibit enantioselectivity or concentration dependence nor was the binding of either enantiomer influenced by its optical antipode (P greater than 0.05).     

Simultaneous determination of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in biological samples using solid-phase extraction and high-performance liquid chromatography

A sensitive and rapid method for measuring simultaneously adenosine, S-adenosylhomocysteine and S-adenosylmethionine in renal tissue, and for the analysis of adenosine and S-adenosylhomocysteine concentrations in the urine is presented. Separation and quantification of the nucleosides are performed following solid-phase extraction by reversed-phase ion-pair high-performance liquid chromatography with a binary gradient system. N⁶-Methyladenosine is used as the internal standard. This method is characterized by an absolute recovery of over 90% of the nucleosides plus the following limits of quantification: 0.25–1.0 nmol/g wet weight for renal tissue and 0.25–0.5 μM for urine. The relative recovery (corrected for internal standard) of the three nucleosides ranges between 98.1±2.6% and 102.5±4.0% for renal tissue and urine, respectively (mean±S.D., n=3). Since the adenosine content in kidney tissue increases instantly after the onset of ischemia, a stop freezing technique is mandatory to observe the tissue levels of the nucleosides under normoxic conditions. The resulting tissue contents of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in normoxic rat kidney are 5.64±2.2, 0.67±0.18 and 46.2±1.9 nmol/g wet weight, respectively (mean±S.D., n=6). Urine concentrations of adenosine and S-adenosylhomocysteine of man and rat are in the low μM range and are negatively correlated with urine flow-rate.     

Distribution of <Emphasis Type="Italic">S</Emphasis> haplotypes and its relationship with restorer–maintainers of self-incompatibility in cultivated <Emphasis Type="Italic">Brassica napus</Emphasis>

Brassica napus (AACC, 2n = 38) is a self-compatible amphidiploid plant that arose from the interspecies hybridization of two self-incompatible species, B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18). Self-incompatibility (S) haplotypes in one self-incompatible line and 124 cultivated B. napus lines were detected using S-locus-specific primers, and their relationships with restorer-maintainers were investigated. Two class I (S-I ( SLG ) a and S-I ( SLG ) b) and four class II (S-II ( SLG ) a, S-II ( SLG ) b, S-II ( SP11 ) a and S-II ( SP11 ) b) S haplotypes were observed, of which S-II ( SP11 ) b was newly identified. The nucleotide sequence of SP11 showed little similarity to the reported SP11 alleles. The lines were found to express a total of eleven S genotypes. The self-incompatible line had a specific genotype consisting of S-II ( SP11 ) a, similar to B. rapa S-60, and S-II ( SLG ) a, similar to B. oleracea S-15. Restorers expressed six genotypes: the most common genotype contained S-I ( SLG ) a, similar to B. rapa S-47, and S-II ( SLG ) b, similar to B. oleracea S-15. Maintainers expressed nine genotypes: the predominant genotype was homozygous for two S haplotypes, S-II ( SLG ) a and S-II ( SP11 ) b. One genotype was specific to restorers and four genotypes were specific to maintainers, whereas five genotypes were expressed in both restorers and maintainers. This suggests that there is no definitive correlation between the distribution of S genotypes and restorer-maintainers of self-incompatibility. The finding that restorers and maintainers express unique genotypes, and share some common genotypes, would be valuable for detecting the interaction of S haplotypes in inter- or intra-genomes as well as for developing markers-assisted selection in self-incompatibility hybrid breeding.     

Genetic mapping and molecular characterization of the self-incompatibility (S) locus in Petunia inflata

Gametophytic self-incompatibility (SI) possessed by the Solanaceae is controlled by a highly polymorphic locus called the S locus. The S locus contains two linked genes, S-RNase, which determines female specificity, and the as yet unidentified pollen S gene, which determines male specificity in SI interactions. To identify the pollen S gene of Petunia inflata, we had previously used mRNA differential display and subtractive hybridization to identify 13 pollen-expressed genes that showed S -haplotype-specific RFLP. Here, we carried out recombination analysis of 1205 F2 plants to determine the genetic distance between each of these S -linked genes and S-RNase. Recombination was observed between four of the genes (3.16, G211, G212, and G221) and S-RNase, whereas no recombination was observed for the other nine genes (3.2, 3.15, A113, A134, A181, A301, G261, X9, and X11). A genetic map of the S locus was constructed, with 3.16 and G221 delimiting the outer limits. None of the observed crossovers disrupted SI, suggesting that all the genes required for SI are contained in the chromosomal region defined by 3.16 and G221. These results and our preliminary chromosome walking results suggest that the S locus is a huge multi-gene complex. Allelic sequence diversity of G221 and 3.16, as well as of 3.2, 3.15, A113, A134 and G261, was determined by comparing two or three alleles of their cDNA and/or genomic sequences. In contrast to S-RNase, all these genes showed very low degrees of allelic sequence diversity in the coding regions, introns, and flanking regions.     

Preparation of S-(+)-ketoprofen by coupling the enantioselective hydrolysis of ketoprofen methyl ester with the photo-oxidation of methanol

A novel method for preparation of S-(+)-ketoprofen is presented involving coupling enantioselective hydrolysis of ketoprofen methyl ester catalyzed by a surfactant-coated-lipase with the photo-oxidation of methanol in a water-saturated organic solvent. The effect of photocatalytic conversion of methanol into water and carbon dioxide on the hydrolysis of ketoprofen methyl ester and the stability of the enzyme was investigated. The photo-oxidation of methanol shifted the equilibrium of the hydrolysis toward the formation of ketoprofen, increasing the equilibrium conversion ratio and improving the enantioselectivity. Because the surfactant-coated lipase and ketoprofen methyl ester dissolved in the organic solvent and ketoprofen was absorbed on the TiO₂ photocatalyst particles, the separation procedures could be simplified and the stability of the enzyme was increased.