Enantiomeric Resolution of 1-Phenyl-2-propanol by Pseudomonas cepacia

Pseudomonas cepacia hydrolyzed rac-1-phenyl-2-propyl acetate and propionate asymmetrically, affording R(?)-1-phenyl-2-propanol and the ester of S(+)-l-phenyl-2-propanol.     

Microbial kinetic resolution of 2-substituted-1-propanol

Micro-organisms that assimilate 2-phenylethanol were screened from soil and strains that enantioselectively oxidized 2-phenyl-1-propanol to 2-phenylpropanoic acid were selected. By optimizing the cultural conditions as well as the reaction conditions, various enantiomerically enriched 2-substituted propanoic acids and 2-aryl-1-propanols were obtained.     

Preparation of Optically Active Secondary Alcohols Related to Synthetic Pyrethroids by Microbial Asymmetric Hydrolysis of the Corresponding Acetates

Asymmetric hydrolysis of the acetates of racemic secondary alcohols related to synthetic pyrethroids by Bacillus subtilis var. niger (IFO 3108) yielded optically active acetates and alcohols of varying optical purities.     

Asymmetric Hydrolysis of Racemic 2-Oxazolidinone Esters with Lipases

The enzymatic hydrolysis of (R, S)-5-acyloxymethyl-3-alkyl-oxazolidin-2-one I and the behavior of (S)-I for extraction with an organic solvent were examined so as to extend the biological resolution to racemates, and to learn about more appropriate combinations of substrates with lipases on the asymmetric hydrolysis. The combination of (R, S)-5-hexanoyloxymethyl-3-tert-butyl-oxazolidin-2-one 4 with lipoprotein lipase Amano 3 (L. P. L. Amano 3, origin; Pseudomonas aeruginosa) and that of (R, S)-5-octanoyloxymethyl-3-isopropyl-oxazolidin-2-one 14 with L. P. L. Amano 3 efficiently gave (S)-5-hydroxymethyl-3-tert-butyl-oxazolidin-2-one (S)-lla (99% e.e.) and (S)-5-hydroxymethyl-3-isopropyl-oxazolidin-2-one (S)-IIb (99% e.e.),respectively. (S)-IIa and (S)-IIb could be considered to be favorable intermediates for preparing optically active β-blockers.     

Microbial Resolution of Racemic 2- and 3-Alkylcyclohexanols

By using microorganisms (their esterase), (±)-trans and cis-2-methylcyclohexyl acetates were asymmetrically hydrolyzed to (?)-trans-2-methylcyc]ohexanol with (+)-trans-2-methyl-cyclohexyl acetate and (?)-cis-2-methylcyclohexanol with (+)-cis-2-methylcyclohexyl acetate. Similarly (±)-trans and cis-3-methylcyclohexyl acetates were hydrolyzed by the same microorganisms to give (+)-trans-3-methylcyclohexanol with (?)-trans-3-methylcyclohexyl acetate and (?)-cis-3-methylcyclohexanol with (+)-cis-3-methylcyclohexyl acetate.     

Enzymatic Preparation of (R)-3-Hydroxy-2-Methylpropyl Butyrate by Asymmetric Hydrolysis

(R)-3-Hydroxy-2-methylpropyl butyrate was formed by asymmetric hydrolysis of the corresponding prochiral diester with lipase P (Amano) in high enantiomeric excess. Various physical and chemical reaction parameters were altered in order to optimize the stereoselectivity of the enzymatic reaction; low temperature (0d`C) combined with the application of salting-in salts or (polyhydric) alcohols turned out to be the most suitable systems providing the monobutyrate in 96% ee. Attempts towards chiral monobutyrate by enzymatic esterification of the corresponding prochiral diol were unsuccessful.     

Design, synthesis and evaluation of racemic 1-(4-hydroxyphenyl)-2-[3-(substituted phenoxy)-2-hydroxy-1-propyl]amino-1-propanol hydrochlorides as novel uterine relaxants

Novel 1-(4-hydroxyphenyl)-2-[3-(substituted phenoxy)-2-hydroxy-1-propyl]amino-1-propanol hydrochlorides were designed based on the pharmacophore for potent uterine relaxant activity and by utilizing the principles of structural hybridization. The designed molecules were synthesized as racemates by a novel route and were evaluated for uterine relaxant activity in vitro on isolated rat uterus and in vivo in pregnant rats. Their cAMP-releasing potential was studied using rat uterus tissue homogenates by the cAMP [(3)H] assay, and cardiac stimulant potential was evaluated on isolated guinea pig right atrium. All compounds exhibited potent uterine relaxant activity in vitro and produced a significant delay in the onset of labour in pregnant rats; their cAMP-releasing potential was slightly less, while their cardiac stimulant potential was insignificant as compared to isoxsuprine hydrochloride.     

Hydrolysis of synthetic chromogenic substrates by HIV-1 and HIV-2 proteinases

Kinetic constants (Km,Kcat) are derived for the hydrolysis of a number of chromogenic peptide substrates by the aspartic proteinase from HIV-2. The effect of systematic replacement of the P2 residue on substrate hydrolysis by HIV-1 and HIV-2 proteinases is examined.     

Site-Specific and Asymmetric Hydrolysis of Prochiral 2-Phenyl-1,3-propanediol Diacetate by a Bacterial Esterase from an Isolated Strain

Bacillus cereus 809A and Burkholderia sp. 711C were isolated from soil. These strains demonstrate hydrolysis activity towards prochiral 2-phenyl-1,3-propanediol diacetate and accumulated the corresponding chiral monoacetates into the reaction mixture. When 2-phenyl 1,3-propanediol diacetate was used as a substrate, the produced monoacetates with Burkholderia sp. 711C were obtained in a racemic form but that produced by Bacillus cereus 809A showed an excess of the (S)-form. The resting cell reaction revealed that for Bacillus cereus 809A, there was an enrichment of one of the enantiomers of the monoacetate such that the enantiomeric excess (e.e.) of the (S)-form was over 95%. The purified enzyme from Bacillus cereus 809A hydrolyzed diacetate to monoacetate, and the e.e. value of the (S)-form increased by prolonged reaction in a way similar to the resting cell reaction. From N-terminal amino acids, this esterase is conserved in some strains of Bacillus for which the genomic sequences have been reported.     

Asymmetric synthesis of (S)-3-chloro-1-phenyl-1-propanol using Saccharomyces cerevisiae reductase with high enantioselectivity

3-Chloro-1-phenyl-1-propanol is used as a chiral intermediate in the synthesis of antidepressant drugs. Various microbial reductases were expressed in Escherichia coli, and their activities toward 3-chloro-1-phenyl-1-propanone were evaluated. The yeast reductase YOL151W (GenBank locus tag) exhibited the highest level of activity and exclusively generated the (S)-alcohol. Recombinant YOL151W was purified by Ni-nitrilotriacetic acid (Ni-NTA) and desalting column chromatography. It displayed an optimal temperature and pH of 40°C and 7.5–8.0, respectively. The glucose dehydrogenase coupling reaction was introduced as an NADPH regeneration system. NaOH solution was occasionally added to maintain the reaction solution pH within the range of 7.0–7.5. By using this reaction system, the substrate (30 mM) could be completely converted to the (S)-alcohol product with an enantiomeric excess value of 100%. A homology model of YOL151W was constructed based on the structure of Sporobolomyces salmonicolor carbonyl reductase (Protein Data Bank ID: 1Y1P). A docking model of YOL151W with NADPH and 3-chloro-1-phenyl-1-propanone was then constructed, which showed that the cofactor and substrate bound tightly to the active site of the enzyme in the lowest free energy state and explained how the (S)-alcohol was produced exclusively in the reduction process.     

Enzyme System Involved in the Decomposition of Phenyl Mercuric Acetate by Mercury-resistant Pseudomonas

The enzymatic decomposition of phenyl mercuric acetate (PMA) to metallic mercury by a mercury-resistant Pseudomonas has been studied. The formation of the system involved in the decomposition was found to be inducible. Glucose dehydrogenase (D-glucose: NAD oxidoreductase), arabinose dehydrogenase (L-arabinose: NADP oxidoreductase), cytochrome c and the “decomposing enzyme” which catalyzes the splitting of the C–Hg linkage, were separated from cell free extract by gel filtration on a column of Sephadex G–150. The cytochrome fraction was further separated into two types, c-I and c-II, by chromatography on a column of CM-Sephadex. Each of these cytochromes showed absorption peaks at 416, 519 and 547 mμ in the reduced form, but they were different in the molecular weight; cytochrome c-I was estimated to be about 26,000, and cytochrome c-II about 14,000. The reconstruction of these enzymes demonstrated that a reduced NAD(P) generating system, glucose dehydrogenase or arabinose dehydrogenase, cytochrome c-I and the “decomposing enzyme” were required for the decomposition of PMA. A hypothetical scheme for the decomposition of PMA was proposed and the decomposition mechanism was discussed.     

Microbial Resolution of alpha-Hydroxy Acids by Enantiospecifically Dehydrogenating Bacteria from Soil

Thirty-three bacterial strains were isolated from soil, utilizing optically asymmetric degradation of dl-2-hydroxy-4-methylpentanoic acid (dl-HMPA) as the screening probe. Those strains were distributed in the following group and genera: Coryneform and Bacillus, Pseudomonas, and Streptomyces. Among them, the most potent strains, Bacillus freudenreichii NRS-137KH20B and Brevibacterium albidum NRS-130KH20B, could perform the resolution of more than 30 g of dl-HMPA per liter within 4 to 5 days of fermentation. Optically pure l- and d-HMPA enantiomers were obtained in more than 80% theoretical yield, whereas the transformed enantiomer was almost quantitatively recovered as 2-oxo-4-methyl-pentanoic acid in the culture broth. The enantiospecific dehydrogenation responsible for this resolution reaction had a rather wide substrate specificity on straight or branched aliphatic C(4) to C(16) 2-hydroxy acids, exhibiting the optima at chain lengths of either C(7) or C(5), although the enantiospecificity was not changed by chain length. The process was thus successfully extended to the preparation of optically pure C(5) to C(9) 2-hydroxy acids.     

Stereospecific Reduction of 2-Oxo-4-phenylbutanoate to (R)-2-Hydroxy-4-phenylbutanoate with Microbial Cells

A method for analyzing the EGCg concentration in human serum was developed by using high-performance liquid chromatography with electrochemical detection. EGCg was detected in human serum after the ingestion of 5 g of green tea powder (matsu-cha) dissolved in 200 ml of hot water. The concentration of EGCg in the serum reached the highest level about 2 h after ingesting the green tea, and then decreased.     

磷酸三(1-氯-2-丙基)酯降解菌筛选及其降解特性

【背景】磷酸三(1-氯-2-丙基)酯[tris-(1-chloro-2-propyl)phosphate,TCIPP]作为全球广泛关注的新兴有机污染物,具有环境赋存含量高、不易生物降解等特点,亟须开发TCIPP的高效去除技术。【目的】获得具有较高TCIPP降解效率并可用于TCIPP污染修复的新菌株。【方法】利用梯度提高无机盐培养基中TCIPP浓度的方法,从TCIPP污染土壤中筛选出1株能够降解液体中高浓度TCIPP (100 mg/L)的菌株,根据16S rRNA基因序列分析对其进行鉴定,并首次对其降解液体中TCIPP的特性进行研究。【结果】所筛选的TCIPP降解菌株DT-6为苍白杆菌(Ochrobactrum sp.),它能够利用TCIPP作为唯一碳源和能源;当TCIPP初始浓度为50 mg/L、培养时间为7 d时DT-6的生物量最大,对TCIPP的降解率也达到最高,为34.6%;蔗糖的加入能够显著促进DT-6的生长,但却抑制了其对TCIPP的降解。【结论】本研究报道了一株TCIPP高效降解菌Ochrobactrum sp. DT-6,能够为环境中TCIPP污染的生物修复提供新的种质...     

Improved 2-methyl-1-propanol production in an engineered Bacillus subtilis by constructing inducible pathways

High-level constitutive gene expression can result in cellular metabolic imbalance and limit production. To circumvent these problems, a P _alsSD -controlled auto-inducible 2-ketoisovalerate biosynthetic pathway and a P _spac -controlled IPTG-inducible Ehrlich pathway were constructed in Bacillus subtilis to modulate gene expression. Based on the precise gene expression characteristics of the two inducible pathways, the optimal IPTG induction time point and dose for 2-methyl-1-propanol biosynthesis were determined as 9.5?h and 300?μM, respectively. Under the optimized conditions, strain BSUΔL-03 with inducible pathways produced up to 3.83?±?0.46?g 2-methyl-1-propanol/l, which was about 60?% higher than BSUL04 with constitutive pathways.     

Formation of D-1-amino-2-propanol from L-threonine by enzymes from Escherichia coli K-12

Escherichia coli K-12 cells contain two dehydrogenases which in sequence catalyze the net conversion of L-threonine to the D-isomer of 1-amino-2-propanol. These two enzymes are L-threonine dehydrogenase (L-threonine + NAD⁺ → aminoacetone + CO₂ + NADH + H⁺) and D-1-amino-2-propanol dehydrogenase (aminoacetone + NADH + H⁺D-1-amino-2-propanol + NAD⁺). Each enzyme has been obtained in purified form free of the other; the nature of the reaction catalyzed by the latter dehydrogenase alone and in a coupled system with the former enzyme has been studied. The results provide an explanation on the enzymological level for the utilization of L-threonine by cell suspensions of certain microorganisms for the biosynthesis of the D-1-amino-2-propanol moiety of Vitamin B₁₂.     

Determination of 1-methoxy-2-propanol and its metabolite 1,2-propanediol in rat and mouse plasma by gas chromatography

A method utilizing capillary GC and flame ionization detection was developed for the simultaneous determination of 1-methoxy-2-propanol (propylene glycol monomethyl ether; PGME) and its metabolite 1,2-propanediol (propylene glycol; PG) in rat and mouse plasma. The calibration graphs for rat and mouse plasma were linear with correlation coefficients at>0.997 over the range 2–700 μg/ml. The limit of quantification was ca. 2 μg/ml (2 ng on-column) for both compounds in plasma of each species. The ranges of the precision and accuracy for PGME were 2.8–8.8% and 3.2–13%, respectively, and for PG were 11–26% and 10–25%, respectively. The recovery of PGME from rat and mouse plasma was ca. 73% and for PG it was ca. 65 and 31% from rat and mouse plasma, respectively. The method was used to study the oral absorption and metabolism of PGME in mice. PGME was readily absorbed and metabolized to PG following oral gavage administration at 90 mg/kg. The maximum concentrations of PGME and PG in plasma were attained at 20 and 30 min following dosing, respectively.     

Stereoselective Synthesis of Ectocarpene and Its Antipode via Microbiological Asymmetric Hydrolysis

Biological asymmetric hydrolysis of ethyl (±)-cycloheptadienecarboxylate with Rhodotorula minuta var. texensis IFO 1102 and chemical resolution of the corresponding carboxylic acid with (?)-quinine provided (R)-(+)-ethyl 2,5-cycloheptadienecarboxylate (78% e.e.) and (S)-(+)-2,5-cycloheptadienecarboxylic acid (95% e.e.), respectively. The (R)-(+)-carboxylate was converted to (R)-(?)-2,5-cycloheptadienylcarbaldehyde and the (S)-(+)-carboxylic acid to (S)-(+)-2,5-cycloheptadienylcarbaldehyde. Ectocarpene (78% e.e.), male-gamete attractant of marine brown alga, and its antipode (95% e.e.) were synthesized by stereoselective Wittig reaction between the (R)-(?)- and (S)-(+)-aldehydes and propyltriphenylphosphonium bromide in a liquid-solid two phase system using 18-crown ether-t-BuOK, respectively.