Construction and characterization of a recombinant esterase with high activity and enantioselectivity to (S)-ketoprofen ethyl ester

The ester-hydrolyzing enzyme families, including lipase and esterase, mediated a broad range of reactions and, thus, were able to act on a variety of ester compounds that are found naturally or exploited industrially. With the increasing demand for pharmacological use, attempts to produce an enantiomer (S)-ketoprofen from the corresponding ethyl ester have recently been proliferating, but information about the structure and function of related enzymes has not been reported to date in detail. Here, we reported the construction, expression, and one-step purification of a potential esterase in Escherichia coli with a hexahistidine tag at its N-terminus. The expression level of the enzyme was more than 20% of the total protein in E. coli, resulting in approximately 1.2mg of the purified proteins by an affinity resin, Ni-NTA, from a 0.2L of bacterial culture in a single step. As typical properties, its innate traits that revealed favorable reactions at alkaline pH and high activity to the triglycerides composed of short chain fatty acids (99% ee(p)). The small-scale conversion using the recombinant enzyme strongly suggested the enzyme to be useful for enzyme-mediated chiral resolution of (S)-ketoprofen.     

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).     

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.     

Stereoselective cyclooxygenase inhibition in cellular models by the enantiomers of ketoprofen

The pharmacological activity of rac-ketoprofen and its enantiomers was investigated in vitro using different cellular models. The effect of these compounds on arachidonic acid metabolism was assessed by measuring the inhibition of prostanoid generation under the action of several agonists. Thus, we have evaluated the inhibition of (1) thromboxane B₂ synthesis in rabbit platelets and human polymorphonuclear leukocytes (PMNs), (2) prostaglandin E₂ synthesis in three cultured cells, namely human umbilical vein endothelial cells (HUVEC), human keratinocytes, and mouse macrophage-like P388D1 cells. The IC₅₀ values found for (+)-(S)-ketoprofen were in the range between 0.1 nM and 0.8 μM, being slightly lower in all models than those found for rac-ketoprofen (0.4 nM–3 μM). On the other hand, (?)-(R)-ketoprofen showed inhibition of cyclooxygenase only at concentrations two or three orders of magnitude higher than those required for the (+)-(S) enantiomer. These results, obtained with cell types of relevance for inflammatory processes and with compounds of high optical purity, demonstrate that the prostanoid biosynthesis inhibition caused by the drug rac-ketoprofen is exclusively due to its dextrorotatory enantiomer. © 1993 Wiley-Liss, Inc.     

Evaluation of screening methods for demulsifying bacteria and characterization of lipopeptide bio-demulsifier produced by Alcaligenes sp

In this paper, surface tension measurement, oil-spreading test and blood-plate hemolysis test were attempted in the screening of demulsifying bacteria. After the comparison to the screening results obtained in demulsification test, 50 mN/m of surface tension of culture was proposed as a preliminary screening standard for potential demulsifying bacteria. For the identification of efficient demulsifying strains, surface tension level was set at 40 mN/m. The detected strains were further verified in demulsification test. Compared to using demulsification test alone as screening method, the proposed screening protocol would be more efficient. From the screening, a highly efficient demulsifying stain, S-XJ-1, was isolated from petroleum-contaminated soil and identified as Alcaligenes sp. by 16S rRNA gene and physiological test. It achieved 96.5% and 49.8% of emulsion breaking ratio in W/O and O/W kerosene emulsion within 24h, respectively, and also showed 95% of water separation ratio in oilfield petroleum emulsion within 2h. The bio-demulsifier was found to be cell-wall combined. After soxhlet extraction and purification through silicon-gel column, the bio-demulsifier was then identified as lipopeptide biosurfactant by TLC and FT-IR.     

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.     

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.     

Bacteria of the Roseobacter Clade Show Potential for Secondary Metabolite Production

Members of the Roseobacter clade are abundant and widespread in marine habitats and have very diverse metabolisms. Production of acylated homoserine lactones (AHL) and secondary metabolites, e.g., antibiotics has been described sporadically. This prompted us to screen 22 strains of this group for production of signaling molecules, antagonistic activity against bacteria of different phylogenetic groups, and the presence of genes encoding for nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS), representing enzymes involved in the synthesis of various pharmaceutically important natural products. The screening approach for NRPS and PKS genes was based on polymerase chain reaction (PCR) with degenerate primers specific for conserved sequence motifs. Additionally, sequences from whole genome sequencing projects of organisms of the Roseobacter clade were considered. Obtained PCR products were cloned, sequenced, and compared with genes of known function. With the PCR approach genes showing similarity to known NRPS and PKS genes were found in seven and five strains, respectively, and three PKS and NRPS sequences from genome sequencing projects were obtained. Three strains exhibited antagonistic activity and also showed production of AHL. Overall production of AHL was found in 10 isolates. Phylogenetic analysis of the 16S rRNA gene sequences of the tested organisms showed that several of the AHL-positive strains clustered together. Three strains were positive for three or four categories tested, and were found to be closely related within the genus Phaeobacter. The presence of a highly similar hybrid PKS/NRPS gene locus of unknown function in sequenced genomes of the Roseobacter clade plus the significant similarity of gene fragments from the strains studied to these genes argues for the functional requirement of the encoded hybrid PKS/NRPS complex. Our screening results therefore suggest that the Roseobacter clade is indeed employing PKS/NRPS biochemistry and should thus be further studied as a potential and largely untapped source of secondary metabolites.     

Saccharomyces cerevisiae in directed evolution: An efficient tool to improve enzymes

Over the past 20 years, directed evolution has been seen to be the most reliable approach to protein engineering. Emulating the natural selection algorithm, ad hoc enzymes with novel features can be tailor-made for practical purposes through iterative rounds of random mutagenesis, DNA recombination and screening. Of the heterologous hosts used in laboratory evolution experiments, the budding yeast Saccharomyces cerevisiae has become the best choice to express eukaryotic proteins with improved properties. S. cerevisiae not only allows mutant enzymes to be secreted but also, it permits a wide range of genetic manipulations to be employed, ranging from in vivo cloning to the creation of greater molecular diversity, thanks to its efficient DNA recombination apparatus. Here, we summarize some successful examples of the use of the S. cerevisiae machinery to accelerate artificial evolution, complementing the traditional in vitro methods to generate tailor-made enzymes.     

A rapid screening method for detecting active compounds against erythromycin-resistant bacterial strains of Finnish origin

A rapid and simple microdilution technique on 96-well microplate based on turbidimetry was optimized and validated for screening of antimicrobial activity against erythromycin-resistant bacterial strains of Streptococcus pyogenes and Staphylococcus simulans isolated from Finnish patients. Using S. pyogenes ATCC 12351 as reference strain the developed method was evaluated by reproducibility measurements and using parameters typically employed for screening methods, i.e. signal-to-background, signal-to-noise and a screening-window coefficient, the Z' factor. The method was further used for screening a group of natural compounds and their synthetic derivatives against resistant bacterial strains. Of these, octyl and dodecyl gallates, and usnic and ursolic acids were the most active. The described method is a rapid, homogeneous, cost-effective and easy-to-perform system for screening of new potential antimicrobial agents in drug discovery.     

A PCR-based method for the screening of bacterial strains with antifungal activity in suppressive soybean rhizosphere

Selection and evaluation of microbial strains for their antifungal activity in natural environments is time- and energy-consuming. We have adapted a PCR-based method to avoid these inconveniences. Soils that are naturally suppressive to plant disease were chosen as a source of antibiotic-producing bacteria. The screening was performed by means of PCR amplification using degenerate primers corresponding to peptide synthetase genes. Amplification fragments were obtained using template DNA from the rhizosphere of three different soybean fields. In order to assay their potential utility in pathogen control, several Bacillus strains were analysed for their in vitro antifungal activity by testing growth inhibition of Sclerotinia sclerotiorum. Four Bacillus sp. isolates gave a positive amplification signal, and three of them had an inhibitory effect on S. sclerotiorum growth, whereas two strains that failed to give an amplification signal did not inhibit fungal growth. These results show that PCR-based techniques could be useful to assess the presence of strains with potential use as biocontrol agents.     

Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media.

Single amino acid substitutions increase the activity and stability of subtilisin E in mixtures of organic solvents and water, and the effects of these mutations are additive. A variant of subtilisin E that exhibits higher activity in mixtures of dimethylformamide (DMF) and water (Q103R) was created by random mutagenesis combined with screening for improved activity (K. Chen and F. H. Arnold, in preparation). Another mutation, N218S, known to improve both the activity and stability of subtilisin BPN', also improves the activity and stability of subtilisin E in the presence of DMF. The effects of the two substitutions on transition-state stabilization are additive. Furthermore, the Q103R mutation that improves activity has no deleterious effect on subtilisin stability. The double mutant Q103R+N218S is 10 times more active than the wild-type enzyme in 20% (v/v) DMF and twice as stable in 40% DMF. Although the effects of single mutations can be impressive, a practical strategy for engineering enzymes that function in nonaqueous solvents will most likely require multiple changes in the amino acid sequence. These results demonstrate the excellent potential for engineering nonaqueous-solvent-compatible enzymes.     

Resolution of rac-ketoprofen esters by enzymatic reactions in organic media

Enzyme-catalyzed reactions in organic media of rac-ketoprofen esters with different nucleophiles such as alcohols, amines, and water have been studied. Among the parameters optimized are the enzyme, the activated substrate, and the solvent. With the enzymes used in this study the preferred substrate was the trifluoroethyl ester of rac-ketoprofen (rac- 2 ), whose (R)-enantiomer reacted preferentially. The enzyme of choice was the lipase M-AP-10 from Mucor miehei and best results were obtained with diisopropyl ether as solvent. Three different methods have been scaled-up for the resolution of 75–150 g of substrate: transesterification with 1-butanol (90% yield of (S)-ketoprofen, 88% ee), transesterification with 2-(2-pyridyl)ethanol (94% yield, 92% ee), and hydrolysis in wet organic solvent (93% yield, 97% ee). Despite the comparable chemical and optical yields obtained with these three methods, the use of 2-(2-pyridyl)ethanol and the hydrolysis allowed a much easier work-up and isolation of the desired (+)-(S)-ketoprofen. © 1993 Wiley-Liss, Inc.     

Novel strains of Bacillus,isolated from compost and compost-amended soils,as biological control agents against soil-borne phytopathogenic fungi

A stepwise screening strategy made it possible to identify five new Bacillus spp. strains for biocontrol of Rhizoctonia solani, Sclerotinia minor and Fusarium solani. In vitro and in vivo biocontrol activity and M13-PCR DNA-fingerprinting led to the selection of these valuable biological control agents (BCAs) from a wide collection of over 250 candidates. At the end of this selection, the highest potential antagonists were identified at species level by 16S-rRNA gene sequence analysis, and results assigned them to Bacillus subtilis group as Bacillus amyloliquefaciens- and Bacillus methylotrophicus-related strains. In the current study, spore-forming bacteria provided substantial biocontrol of telluric diseases on cress and other different host plants. The strains named 15S and 09C were effective in disease control on Brassica oleracea/R. solani pathosystem, whereas Sclerotinia drop of lettuce was reduced by treatments with the strains 17S and 08C. Finally, the strains 17S and 12S were equally effective to control potato Fusarium rot. The evident zone of inhibition seen in dual culture plates suggested antibiosis-like antagonisms as the main mechanisms used by these bacterial isolates in interaction with the pathogens. Additionally, the API-ZYM method revealed constitutive activity of certain extracellular enzymes that could be involved in plant fortification. Bacillus strains isolated from compost and compost-amended soils are promising BCAs that have potential for practical application as biofungicides.     

Grape marcs as unexplored source of new yeasts for future biotechnological applications

In recent years the potential of using microbes as biotechnological sources of industrially relevant enzymes has stimulated a renewed interest in the exploration of new unconventional habitats like trove of natural biodiversity. In this work, grape marcs was selected as extreme environment because of its limited nutrients, exposure to solar radiation, temperature fluctuations and ethanol. One hundred and eighty non-Saccharomyces yeasts and two hundred and twenty Saccharomyces cerevisiae strains were screened for the production of extracellular amylases, cellulases, lipases, pectinases, proteases and xylanases. Two non-Saccharomyces strains were found effective for the hydrolysis of cellulose and starch while eleven S. cerevisiae isolates were described as proficient pectinase producers. For the first time, thirteen S. cerevisiae strains, potentially able to use starch as the sole carbon source, were reported and their potential amylolytic phenotype was found to be related to a non extracellular alpha-amylase. This study encourages the selection of yeasts isolated from grape marcs as sources of unusual and industrially interesting enzymes for future biotechnological applications.     

Partially purified Carica papaya lipase: a versatile biocatalyst for the hydrolytic resolution of (R,S)-2-arylpropionic thioesters in water-saturated organic solvents

With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioesters in water-saturated isooctane as a model system, improvements of the specific lipase activity and thermal stability were found when a crude Carica papaya lipase (CPL) was partially purified and employed as the biocatalyst. The partially purified Carica papaya lipase (PCPL) was furthermore explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-profen thioesters in water-saturated organic solvents. The kinetic analysis in water-saturated isooctane indicated that both acyl donor and acyl acceptor have profound influences on the lipase activity, E-value, and enantioselectivity. Inversion of the enantioselectivity from (S)- to (R)-thioester was found for (R,S)-fenoprofen and (R,S)-ketoprofen thioesters that contained a bulky substituent at the meta-position of 2-phenyl moiety of the acyl part. Kinetic constants for the acylation step were furthermore estimated for elucidating the kinetic data and postulating an active site model. The thermodynamic analysis indicated that the enantiomer discrimination was driven by the difference of activation enthalpy (DeltaDeltaH) and that of activation entropy (DeltaDeltaS), yet the latter was dominated for most of the reacting systems. The postulated active site model was supported from the variation of DeltaDeltaH and DeltaDeltaS with the acyl moiety, in which a good linear enthalpy-entropy compensation relationship was also illustrated. A comparison of the performances between Candida rugosa lipase (CRL) and PCPL indicated that PCPL was superior to CRL in terms of the better thermal stability, similar or better lipase activity for the fast-reacting substrate, time-course-stability, and lower enzyme cost.     

基于活性和基因从海洋微生物中筛选烯二炔类抗生素

【目的】高质量的海洋微生物菌种库及其天然产物库是新药开发的重要来源。在本研究中我们通过筛选新的烯二炔类抗生素来对已经构建的海洋微生物天然产物库进行质量评价。【方法】首先我们根据烯二炔类抗生素能引起DNA断裂的活性构建了活性筛选模型,并对我们的天然产物库进行筛选;其次根据合成烯二炔核心结构的独特且保守的重复I型聚酮合成酶设计了引物,通过PCR扩增的方法对海洋微生物库进行序列筛选。【结果】通过活性筛选从我们的海洋微生物天然产物库中获得一个阳性的发酵产物。对该阳性菌(LS481)的系统发育学分析表明该菌属于能产生烯二炔类化合物—Dynemicin的Micromonospora chersina,对其发酵产物TLC分析证明该菌确实产生Dynemicin类化合物。通过基因筛选得到了2个具备合成烯二炔核心结构聚酮合成酶的菌株,16S rRNA基因分析显示其中一个很可能为灰色链霉菌(MS098),另外一株菌则同Streptomyces vinaceus NBRC 13425T和Streptomyces cirratus NRRLB-3250T最相近。【结论】我们的活性筛选模型能够有效获得烯二炔类物质,结合基因筛选能够进一步获得可能产生烯二炔物质的菌株。初筛结果也再一次验证了我们海洋微生物天然产物库的质量较好。     

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 10 mM (R,S)-ketoprofen ethyl ester into (S)-ketoprofen. The crude lipase had an activity of 10 U ml(-1) and, at 30 degrees C and pH 7 over 48 h, gave a conversion yield of 35% with an enantiomeric excess for the product 96%.     

Development of a screening method for the indentification of a novelSaccharomyces cerevisiae mutant over-expressingTrichoderma reesei cellobiohydrolase II

In a previous study we showed that the fusion of the cellulose-binding domain (CBD2) fromTrichoderma reesei cellobiohydrolase II to a β-glucosidase (BGL1) enzyme fromSaccharomycopsis fibuligera significantly hindered its expression and secretion inSaccharomyces cerevisiae. This suggests that the possible low secretion of heterologous cellulolytic enzymes inS. cerevisiae could be attributed to the presence of a cellulose-binding domain (CBD) in these enzymes. The aim of this study was to increase the extracellular production of the chimeric CBD2-BGL1 enzyme (designated CBGL1) inS. cerevisiae. To achieve this, CBGL1 was used as a reporter enzyme for screening mutagenisedS. cerevisiae strains with increased ability to secrete CBD-associated enzymes such as cellulolytic enzymes. A mutant strain ofS. cerevisie, WM91-CBGL1, which exhibited up to 200 U L^?1 of total activity, was isolated. Such activity was approximately threefold more than that of the parental host strain. Seventy-five per cent of the activity was detected in the extracellular medium. The mutant strain transformed with theT. resei CBH2 gene produced up to threefold more cellobiohydrolase enzyme than the parental strain, but with 50% of the total activity retained intracellularly. The cellobiohydrolase enzymes from the parent and mutant strains were partially purified and the characteristic properties analysed.     

Isolation of an esterase-producing Trichosporon brassicae and its catalytic performance in kinetic resolution of ketoprofen

A yeast strain CGMCC 0574, identified as Trichosporon brassicae, was selected from 92 strains for its high (S) selectivity in the hydrolysis of ketoprofen ethyl ester. The effective strains of the microorganisms were isolated from soil samples with the ester as the sole carbon source. The ethyl ester proved to be the best substrate for resolution of ketoprofen among several ketoprofen esters examined. The resting cells of CGMCC 0574 could catalyze the hydrolysis of ketoprofen ethyl ester with an enantiomeric ratio of 44.9, giving (S)-ketoprofen an enantiomeric excess of 91.5% at 42% conversion.