Isolation and characterization of indene bioconversion genes from Rhodococcus strain I24

Rhodococcus strain I24 is able to convert indene into indandiol via the actions of at least two dioxygenase systems and a putative monooxygenase system. We have identified a cosmid clone from I24 genomic DNA that is able to confer the ability to convert indene to indandiol upon Rhodococcus erythropolis SQ1, a strain that normally can not convert or metabolize indene. HPLC analysis reveals that the transformed SQ1 strain produces cis-(1R,2S)-indandiol, suggesting that the cosmid clone encodes a naphthalene-type dioxygenase. DNA sequence analysis of a portion of this clone confirmed the presence of genes for the dioxygenase as well as genes encoding a dehydrogenase and putative aldolase. These genes will be useful for manipulating indene bioconversion in Rhodococcus strain I24. Received: 8 December 1998 / Received revision: 26 January 1999 / Accepted: 5 February 1999     

Conversion of indene to cis-(1S),(2R)-indandiol by mutants of Pseudomonas putida F1

Two mutation and selection methods were used to isolate mutants of Pseudomonas putida F1 which convert indene to cis-(1S),(2R)-indandiol in a toluene-independent fashion. Using soybean or silicone oil as a second phase to deliver indene to the culture, cis-(1S),(2R)-indandiol, cis-(1R),(2S)-indandiol, 1,2-indenediol (or the keto-hydroxy indan tautomer), and the monooxygenation products 1-indenol and 1-indanone were produced from indene as a function of time. Similarly the enantiomeric excess of the cis-(1S),(2R)-indandiol produced also increased with increasing time. In addition, mutants were isolated which produced cis-(1S),(2R)-indandiol of lower optical purity which corresponded to reduced levels of 1,2-indenediol. These data suggest this toluene dioxygenase produces cis-(1S),(2R)-indandiol of low optical purity and that cis-glycol dehydrogenase plays a role in resolving the two cis-1,2-indandiol enantiomers. Received 15 November 1996/ Accepted in revised form 09 March 1997     

Directed Evolution of Toluene Dioxygenase from Pseudomonas putida for Improved Selectivity Toward cis-Indandiol during Indene Bioconversion

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S, 2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S, 2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.     

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.     

Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S,2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S,2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.     

cis-Chlorobenzene Dihydrodiol Dehydrogenase (TcbB) from Pseudomonas sp. Strain P51, Expressed in Escherichia coli DH5α(pTCB149), Catalyzes Enantioselective Dehydrogenase Reactions

cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5α(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (−)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (−)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols.     

Desaturation, dioxygenation, and monooxygenation reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp. strain 9816-4.

The stereospecific oxidation of indan and indene was examined with mutant and recombinant strains expressing naphthalene dioxygenase of Pseudomonas sp. strain 9816-4. Pseudomonas sp. strain 9816/11 and Escherichia coli JM109(DE3)[pDTG141] oxidized indan to (+)-(1S)-indanol, (+)-cis-(1R,2S)-indandiol, (+)-(1S)-indenol, and 1-indanone. The same strains oxidized indene to (+)-cis-(1R,2S)-indandiol and (+)-(1S)-indenol. Purified naphthalene dioxygenase oxidized indan to the same four products formed by strains 9816/11 and JM109(DE3)[pDTG141]. In addition, indene was identified as an intermediate in indan oxidation. The major products formed from indene by purified naphthalene dioxygenase were (+)-(1S)-indenol and (+)-(1R,2S)-indandiol. The results show that naphthalene dioxygenase catalyzes the enantiospecific monooxygenation of indan to (+)-(1S)-indanol and the desaturation of indan to indene, which then serves as a substrate for the formation of (+)-(1R,2S)-indandiol and (+)-(1S)-indenol. The relationship of the desaturase, monooxygenase, and dioxygenase activities of naphthalene dioxygenase is discussed with reference to reactions catalyzed by toluene dioxygenase, plant desaturases, cytochrome P-450, methane monooxygenase, and other bacterial monooxygenases.     

Bioconversion of limonene to α-terpineol by immobilized Penicillium digitatum

Bioconversion of (4R)-(+)-limonene to (4R)-(+)-α-terpineol by immobilized fungal mycelia of Penicillium digitatum was investigated in batch, repeated-batch and continuously fed systems. The fungi were immobilized in calcium alginate beads. These beads remained active for at least 14 days when they were stored at 4 °C. Three different aeration rates were tested. The highest yield was obtained at a dissolved oxygen level of 50.0 μmol/l. α-Terpineol production by this fungus was 12.83 mg (g beads)⁻¹ day⁻¹, producing a 45.81% bioconversion of substrate. Repeated-batch bioconversion showed yield decreases in the second and the third cycles. Regeneration with nutrient media after the third cycle improved the bioconversion yields. With continuous bioconversion, the half-life was dependent on the aeration. The optimum conditions with a continuous reactor were at an aeration rate of 0.3 standard l/min and a dilution rate of 0.0144 h⁻¹. Received: 10 June 1997 / Received revision: 18 August 1997 / Accepted: 11 September 1997     

An extractive membrane biofilm reactor for degradation of 1,3-dichloropropene in industrial waste water

A bacterial biofilm, capable of mineralising a technical mixture of cis- and trans-1,3-dichloropropene (DCPE), was enriched on the biomedium side of an extractive membrane biofilm reactor (EMBR). The membrane separates the biomedium from the industrial waste water, in terms of pH, ionic strength and the concentration of toxic chemicals. The biofilm, attached to a silicone membrane, is able to mineralise DCPE after its diffusion through the membrane. Five bacterial strains with degradation capabilities were isolated from the metabolically active biofilm and further investigated in batch experiments. Two of them, Rhodococcus erythropolis strains EK2 and EK5, can grow with DCPE as the sole carbon source. Pseudomonas sp. EK1 utilises cis-3-chloroallylalcohol and cis-3-chloroacrylic acid, whereas the metabolite trans-3-chloroacrylic acid represents a dead-end product of the pathway of this strain. The other two strains, Delftia sp. EK3 and EK4, although unable to grow with DCPE as the carbon source, can transform DCPE and its upper-pathway intermediates at reasonable conversion rates. They may represent helper functions of the biofilm consortium, which mineralised up to 12.5 mmol DCPE per hour per gram of biomass protein. Higher feed rates in the EMBR (up to 15 mmol per hour per 100-l bioreactor volume) and shock loads corresponding to concentrations up to 1.8 mmol l⁻¹ led to a significant increase in the freely floating bacterial biomass in the reactor medium (OD₅₄₆= 0.2). At the standard operating feed rate of 1.8 mmol h⁻¹, the free biomass concentration was very low (OD₅₄₆= 0.04). Received: 23 April 1999 / Received revision: 1 July 1999 / Accepted: 5 July 1999     

Measurement of strain-dependent toxicity in the indene bioconversion using multiparameter flow cytometry

The bionconversion of indene to cis-(1S,2R)-indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using Rhodococcus, Pseudomonas putida, and Escherichia coli strains. This study reports on the application of multiparameter flow cytometry for the measurement of cytoplasmic membrane integrity and membrane depolarization as indicators of toxic effects of the substrate, product, and by-products using each of these strains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Measurements of the cytoplasmic membrane potential, cell viability, and respiratory activity provided a sensitive set of parameters to assess toxicity in the indene bioconversion and provided the basis for process improvements and strain selection. The toxic concentrations of the substrate, product, and by-products for each strain have been determined. The results show that it is possible to accumulate cis-(1S,2R)-indandiol and cis-1-amino-2-indanol up to 20 g/L without significant negative effects on cell physiology using any of the strains tested. The Gram-negative P. putida (421-5 and GM 730) and E. coli strains were more resistant to indene and the isolated chemicals of the biotransformation than the Gram-positive Rhodoccoccus I24 strain, possibly due to the presence of the outer membrane and efflux pump mechanisms. P. putida GM 730 and the E. coli TDO 123 strains responded similarly to toxic effects, and the E. coli TDO 123 strain was more resistant than the P. putida 421-5 strain. In addition to the recommendations for strain selection, the identified targets for bioprocess improvement include a combination of genetic as well as process engineering approaches.     

Substrate specificity and stereospecificity of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14; an enzyme showing sequential and enantioconvergent substrate conversion

Limonene-1,2-epoxide hydrolase (LEH) from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene degradation pathway of this microlorganism, has a narrow substrate specificity. Of the compounds tested, the natural substrate, limonene-1,2-epoxide, and several alicyclic and 2-methyl-1,2-epoxides (e.g. 1-methylcyclohexene oxide and indene oxide), were substrates for the enzyme. When LEH was incubated with a diastereomeric mixture of limonene-1,2-epoxide, the sequential hydrolysis of first the (1R,2S)- and then the (1S,2R)-isomer was observed. The hydrolysis of (4R)- and (4S)-limonene-1,2-epoxide resulted in, respectively, (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diol as the sole product with a diastereomeric excess of over 98%. With all other substrates, LEH showed moderate to low enantioselectivities (E ratios between 34 and 3).     

Optimization of the hydroxylation of 2-cyclopentylbenzoxazole with Cunninghamella blakesleeana DSMZ 1906

Biohydroxylation of 2-cyclopentyl-1,3-benzoxazole with the filamentous fungus Cunninghamella blakesleeana DSMZ 1906 was studied in a 15-l stirred tank reactor. The aim of the work was to avoid substrate limitation through sub-optimal mixing by formation of pellets with a uniform pellet size distribution of 250–500 μm, obtained at an inoculum concentration of 10⁷ spores ml⁻¹ and an agitation rate of 390 rpm. Due to the high toxicity of the educt, 2-cyclopentyl-1,3-benz- oxazole, on the fungus, the medium composition, the time of educt addition, and the educt starting concentration were optimized to reach high educt tolerance and hydroxylation activity. A good maintenance of biotransformation capacity was obtained without excessive loss of activity of the biocatalyst by addition of 30 mg 2-cyclopentyl-1,3-benzoxazole/g biomass (cell dry mass) during the stationary phase in a medium which was optimized in batch fermentations with experimental designs. An increase in product yield and quality (enantiomeric excess) was achieved by developing feeding strategies combining the educt and medium components. The resulting fermentation broth contained 450 mg l⁻¹ of the product (1S,3S)-3-(benz-1,3-oxazol- 2-yl)cyclopentan-1-ol with an enantiomeric excess of 95%, which represents a 48% increase over former reported results. Received: 8 July 1999 / Accepted: 2 October 1999     

Production of single cell protein from rice polishings using Candida utilis

Microbial protein was produced from defatted rice polishings using Candida utilis in shake-flasks and a 14-l fermentor to optimize fermentation conditions before producing biomass in a 50-l fermentor. The organism supported maximum values of 0.224 h⁻¹, 0.94, 1.35, 1.75, 2.12 g l⁻¹ h⁻¹, 0.62 g cells g⁻¹ substrate utilized and 0.38 g g⁻¹ for specific growth rate, true protein productivity, crude protein productivity, cell mass productivity, substrate consumption rate, cell yield, crude protein yield, respectively in 50-l fermentor studies using optimized cultural conditions. Maximum values compared favourably or were superior to published data in literature. The biomass protein in the 50-l fermentor contained 22.3, 27.8, 19.2, 9.5, 38.12, 8.5 and 0.27% true protein, crude protein, crude fibre, ash, carbon, cellulose and RNA content, respectively. The dried biomass showed a gross metabolizable energy value of 2678 kcal kg⁻¹ and contained all essential and non-essential amino acids. Yeast biomass as animal feed may replace expensive feed ingredients currently being used in poultry feed and may improve the economics of feed produced in countries like Pakistan. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cardiovascular control via angiotensin II and circulating catecholamines in the spiny dogfish, Squalus acanthias

The contributions of circulating angiotensin II (Ang II) and catecholamines to cardiovascular control in the spiny dogfish were investigated by monitoring the effects of exogenous and endogenous dogfish [Asn¹, Pro³, Ile⁵]-Ang II (dfAng II) on plasma catecholamine levels and blood pressure regulation. Bolus intravenous injections of dfAng II (30–1200 pmol kg⁻¹) elicited dose-dependent increases in plasma adrenaline and noradrenaline concentrations, caudal artery pressure (P _CA), and systemic vascular resistance (R _S), and a decrease in cardiac output (Q). Similar injections of Ang II in dogfish pre-treated with the α-adrenoceptor antagonist yohimbine (4 mg kg⁻¹) also elicited dose-dependent increases in plasma catecholamine levels yet the cardiovascular effects were abolished. Dogfish treated with yohimbine were hypotensive and had elevated levels of plasma Ang II and catecholamines. Intravenous injection of the smooth muscle relaxant papaverine (10 mg kg⁻¹) elicited a transient decrease in P _CA and R _S, and increases in plasma Ang II and catecholamine levels. In dogfish first treated with lisinopril (10⁻⁴ mol kg⁻¹), an angiotensin converting enzyme inhibitor, papaverine treatment caused a more prolonged and greater decrease in P _CA and R _S, an attenuated increase in plasma catecholamines, and no change in plasma Ang II. By itself, lisinopril treatment had little effect on P _CA, and no effect on R _S, plasma Ang II or catecholamines. In yohimbine-treated dogfish, papaverine treatment elicited marked decreases in P _CA, R _S, and Q, and increases in plasma Ang II and catecholamines. Among the three papaverine treatments, there was a positive linear relationship between plasma Ang II and catecholamine concentrations, and the cardiovascular and hormonal changes were most pronounced in the yohimbine + papaverine treatment. Therefore, under resting normotensive conditions, while Ang II does not appear to be involved in cardiovascular control, catecholamines play an important role. However, during a hypotensive stress elicited by vascular smooth muscle relaxation, Ang II indirectly contributes to cardiovascular control by dose-dependently stimulating catecholamine release. Accepted: 24 February 1999     

Isolation and characterization of highly (R)-specific N-acetyl-1-phenylethylamine amidohydrolase, a new enzyme from Arthrobacter aurescens AcR5b

A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S )-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8 and 50 °C, respectively. The enzyme was stable in the range of pH 7–9 and at temperatures up to 30 °C. The enzyme activity was inhibited by Cu²⁺, Co²⁺, Ni²⁺, and Zn²⁺, and this inhibition was reversed by EDTA.M Received: 20 September 1996 / Received version: 23 December 1996 / Accepted: 30 December 1996     

Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

Enantioselective synthesis of (R)-(+)- and (S)-(-)-higenamine and their analogues with effects on platelet aggregation and experimental animal model of disseminated intravascular coagulation

Optically active tetrahydroisoquinoline alkaloids, (R)-(+)-higenamine (1R) and (S)-(−)-higenamine (1 S), and their optically active 1-naphthylmethyl analogues (2 and 3), were synthesized by enantioselective hydrogenation of the corresponding dihydroisoquinoline intermediates 7 as a key step. The evaluation of the platelet anti-aggregation effect demonstrated clearly that the (S)-(−)-enantiomers, 1S, 2S, and 3S, had higher inhibitory potency than the corresponding (R)-(+)-antipodes, 1R, 2R, and 3R, respectively, to platelet aggregation induced by epinephrine. 1S enantiomer was superior to the corresponding 1R enantiomer in attenuating all of the disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) parameters tested, while the S enantiomers 2S and 3S ameliorated some of the DIC and MOF parameters more effectively than the corresponding antipodes 2R and 3R.     

Determination of enantiomeric concentrations of a 2,5-diaryltetrahydrofuran (L-668,750), a platelet-activating factor receptor antagonist, in rat plasma using a chiral α1-acid glycoprotein high-performance liquid chromatographic column

Racemic sulfonylated 2,5-diaryltetrahydrofuran [L-668,750, (±)-trans-2-[3-methoxy-5-(2-hydroxy)ethylsulfonyl-4-n-propoxy]-phenyl-5-(3,4,5-trimethoxyphenyl)-tetrahydrofuran, I] is a potent, specific and orally active platelet-activating factor (PAF) receptor antagonist. Its (—)-(2S,5S) enantiomer [L-680,573, (S)-I] exhibited higher PAF antagonistic potency than the (+)-(2R,5R) enantiomer [L-680,574, (R)-I] in vitro and in animal models. For assay of drug concentrations in plasma of rats dosed intravenously or orally with tritium-labeled I, we have developed a high-performance liquid chromatographic (HPLC) method which directly resolved the two enantiomers. The column contained α₁-acid glycoprotein as the chiral stationary phase and was eluted with phosphate buffer, methanol and ethanol at neutral pH. The concentration of each enantiomer in the plasma was then determined by reverse isotope dilution assay. Results showed that the plasma clearance rate of the more potent (S)-I enantiomer was more than ten-fold faster than that of the (R)-I enantiomer; the enantioselective clearance resulted in nearly ten-fold higher concentrations of the latter in plasma at all time points regardless of the dosing route. This paper describes the HPLC chiral resolution method and its application in plasma analysis.     

Production of (R )-3-pentyn-2-ol through stereospecific hydrolysis of racemic 3-pentyn-2-ol esters with microbial enzymes

Microorganisms and commercial enzymes were screened for their ability to produce (R)-3-pentyn-2-ol from racemic 3-pentyn-2-ol esters through stereospecific hydrolysis. Among the esters formed with acetic acid, propionic acid, hexanoic acid and benzoic acid, the acetate was most effectively hydrolyzed by microbial cells and commercial lipases with high stereospecificity. Rhodococcus rubropertinctus AKU NOC082 was a good catalyst for (R)-3-pentyn-2-ol production through the hydrolytic resolution of racemic 3-pentyn-2-yl acetate. With 15%, 25% and 50% (v/v) racemic 3-pentyn-2-yl acetate as the substrate, 42.6%, 40.8% and 40.0% was hydrolyzed in 5 h, 10 h and 98 h respectively, under the optimized conditions (pH 7.0, 30 °C, 7.5% wet cell concentration), the (R) enantiomer of 3-pentyn-2-ol being formed with an optical purity of 97.8%, 98.0% and 94.2% respectively. Received: 2 June 1998 / Received revision: 3 August 1998 / Accepted: 3 September 1998     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998