Characterization of binding properties of ephedrine derivatives to human alpha-1-acid glycoprotein.

Most drugs, especially those with acidic or neutral moieties, are bound to the plasma protein albumin, whereas basic drugs are preferentially bound to human alpha-1-acid glycoprotein (AGP). The protein binding of the long-established drugs ephedrine and pseudoephedrine, which are used in the treatment of hypotension and colds, has so far only been studied with albumin. Since in a previous study a stereoselective binding of ephedrine and pseudoephedrine to serum but not to albumin was observed, the aim of this study was to check whether the enantioselective binding behavior of ephedrine and pseudoephedrine, in addition to the derivatives methylephedrine and norephedrine, is due to AGP and to investigate the influence of their different substituents and steric arrangement. Discontinuous ultrafiltration was used for the determination of protein binding. Characterization of ligand-protein interactions of the drugs was obtained by saturation transfer difference nuclear magnetic resonance spectroscopy. Docking experiments were performed to analyze possible ligand-protein interactions. The more basic the ephedrine derivative is, the higher is the affinity to AGP. There was no significant difference in the binding properties between the individual enantiomers and the diastereomers of ephedrine and pseudoephedrine.

Pseudoephedrine hydrochloride causes hyperactivity in zebrafish via modulation of the serotonin pathway.

This study aimed to explore behavioral changes of embryonic and larval zebrafish caused by pseudoephedrine hydrochloride (PSE) and its underlying mechanism. Zebrafish embryos were exposed to 0.5 µM, 2 µM, and 8 µM PSE at 4 h post-fertilization (4 hpf) or 22-23 hpf. Mortality, hatching rate, coiling frequency, heart rate, behavior changes, and related gene expression were observed at different developmental stages. PSE below 8 µM did not affect zebrafish mortality, hatching rate, and heart rate compared with the control group. For embryos, PSE caused an increase at 16-32 hpf in zebrafish coiling frequency which could be rescued by serotonin antagonist WAY100635. Similarly, PSE caused an increase in the swimming distance of zebrafish larvae at 120 hpf. PSE also elevated the expression of serotonin (5-HT)-related genes 5-htr1ab and tph2 and dopamine-related gene dbh. Behavioral changes in zebrafish embryos and larvae caused by PSE may be closely associated with increased expression of 5-HT and dopamine-related genes. This may be reflected that the behavioral changes in zebrafish are a possible PSE monitoring indicator.

Ephedra herb reduces adriamycin-induced testicular toxicity by upregulating the gonadotropin-releasing hormone signalling pathway.

OBJECTIVE: We investigated the protective effects of ephedra herb (HEPH) on adriamycin-induced testicular toxicity in rats and explored the potential mechanisms underlying these effects. METHODS: A rat model of adriamycin injury was established, and sperm motility-related indicator and oxidative stress levels in the testis were evaluated. Serum levels of sex hormones and levels of testicular cell apoptosis were detected by enzyme-linked immunosorbent assay and flow cytometry, respectively. Western blotting (WB), immunofluorescence analyses, and reverse transcription-polymerase chain reaction (RT-PCR) were performed to evaluate the gonadotropin-releasing hormone (GnRH) signalling pathway- and meiosis-related genes and proteins. In subsequent in vitro experiments, adriamycin was used to stimulate GC-1 cells, which were treated with HEPH, ephedrine, or pseudoephedrine. Cell viability was assessed using flow cytometry to detect apoptosis and reactive oxygen species, whereas the GnRH signalling pathway and levels of meiosis-related genes and proteins were evaluated by InCell WB, a high-content imaging system, and RT-PCR. RESULTS: Per in vivo experiments, HEPH restored testicular weight and function, sperm characteristics, serum and tissue hormonal levels, and antioxidant defences and significantly activated the GnRH signalling pathway- and meiosis-related protein levels. All protective effects of HEPH against adriamycin-induced injury were antagonised by the GnRH antagonist cetrorelix. In vitro, HEPH, ephedrine, and pseudoephedrine significantly reduced adriamycin-induced GC-1 cell apoptosis and reactive oxygen species levels and increased the expression of GnRH signalling pathway- and meiosis-related proteins. The effect of pseudoephedrine was greater than that of ephedrine, and these findings may be an important basis for understanding the effects of HEPH.

Multiple-Dose Pharmacokinetics of Ozanimod and its Major Active Metabolites and the Pharmacodynamic and Pharmacokinetic Interactions with Pseudoephedrine, a Sympathomimetic Agent, in Healthy Subjects.

INTRODUCTION: The aims of this study were to characterize the multiple-dose pharmacokinetics (PK) of ozanimod's major active metabolites (CC112273 and CC1084037) and to evaluate the pharmacodynamic and PK interactions with pseudoephedrine (PSE). METHODS: In this phase 1, single-center, randomized, double-blind, placebo-controlled study, 56 healthy adult subjects were randomized to receive either placebo or ozanimod once daily for 30 days (0.23 mg on days 1-4, 0.46 mg on days 5-7, 0.92 mg on days 8-10, and 1.84 mg on days 11-30). On day 30, a single oral dose of PSE 60 mg was co-administered with placebo or ozanimod. Maximum time-matched change in systolic blood pressure (SBP) from baseline (day 29) following PSE administration on day 30 was calculated. Plasma PK parameters for ozanimod, CC112273, CC1084037, and PSE were estimated using noncompartmental methods. RESULTS: Fifty-two subjects (92.9%) completed the study. Following multiple dosing, approximately 94% of circulating total active drug exposure was represented by ozanimod (6%), CC112273 (73%), and CC1084037 (15%). Exposures of CC112273 and CC1084037 were highly correlated. Mean maximum time-matched change from baseline for SBP was not significantly different between ozanimod + PSE and placebo + PSE. Ozanimod also had no effect on the PK of PSE. Co-administration of ozanimod with a single dose of PSE in healthy subjects was generally well tolerated. While CC112273 and CC1084037 selectively inhibited monoamine oxidase (MAO)-B in vitro, both active metabolites do not inhibit platelet MAO-B activity in vivo. CONCLUSION: Concomitant administration of ozanimod with PSE, a sympathomimetic agent, did not potentiate the effects on blood pressure. TRIAL REGISTRATION: NCT03644576.

Biodegradation of Ephedrine Isomers by Arthrobacter sp. Strain TS-15: Discovery of Novel Ephedrine and Pseudoephedrine Dehydrogenases.

The Gram-positive soil bacterium Arthrobacter sp. strain TS-15 (DSM 32400), which is capable of metabolizing ephedrine as a sole source of carbon and energy, was isolated. According to 16S rRNA gene sequences and comparative genomic analysis, Arthrobacter sp. TS-15 is closely related to Arthrobacter aurescens Distinct from all known physiological paths, ephedrine metabolism by Arthrobacter sp. TS-15 is initiated by the selective oxidation of the hydroxyl function at the α-C atom, yielding methcathinone as the primary degradation product. Rational genome mining revealed a gene cluster potentially encoding the novel pathway. Two genes from the cluster, which encoded putative short-chain dehydrogenases, were cloned and expressed in Escherichia coli The obtained enzymes were strictly NAD+ dependent and catalyzed the oxidation of ephedrine to methcathinone. Pseudoephedrine dehydrogenase (PseDH) selectively converted (S,S)-(+)-pseudoephedrine and (S,R)-(+)-ephedrine to (S)- and (R)-methcathinone, respectively. Ephedrine dehydrogenase (EDH) exhibited strict selectivity for the oxidation of the diastereomers (R,S)-(-)-ephedrine and (R,R)-(-)-pseudoephedrine.IMPORTANCEArthrobacter sp. TS-15 is a newly isolated bacterium with the unique ability to degrade ephedrine isomers. The initiating steps of the novel metabolic pathway are described. Arthrobacter sp. TS-15 and its isolated ephedrine-oxidizing enzymes have potential for use in decontamination and synthetic applications.

Determination of plasma protein binding for sympathomimetic drugs by means of ultrafiltration.

Ephedrine and its diastereomer pseudoephedrine have long been used in therapy and are part of over-the-counter combination drugs against colds or allergies. Nonetheless, there is scarcely any information on their plasma protein binding in literature. Plasma protein binding is an important parameter from a pharmacokinetic and pharmacodynamic point of view and can play a crucial role in therapy. The aim of this study was to determine the extent of plasma protein binding using ultrafiltration and different types of plasma proteins like human (HSA) and bovine serum albumin (BSA) and human serum. Two orthogonal methods - continuous and discontinuous ultrafiltration - were used to confirm our findings. To get some structural and stereochemical insights into binding affinity towards plasma proteins, all four stereoisomers of ephedrine and pseudoephedrine were included in this study. Since all four stereoisomers exhibit a low affinity towards albumin, other sympathomimetic drugs of structural similarity were tested to investigate the influence of the basic character of the Ephedra alkaloids in their binding to plasma proteins.

An N-methyltransferase from Ephedra sinica catalyzing the formation of ephedrine and pseudoephedrine enables microbial phenylalkylamine production.

Phenylalkylamines, such as the plant compounds ephedrine and pseudoephedrine and the animal neurotransmitters dopamine and adrenaline, compose a large class of natural and synthetic molecules with important physiological functions and pharmaceutically valuable bioactivities. The final steps of ephedrine and pseudoephedrine biosynthesis in members of the plant genus Ephedra involve N-methylation of norephedrine and norpseudoephedrine, respectively. Here, using a plant transcriptome screen, we report the isolation and characterization of an N-methyltransferase (NMT) from Ephedra sinica able to catalyze the formation of (pseudo)ephedrine and other naturally occurring phenylalkylamines, including N-methylcathinone and N-methyl(pseudo)ephedrine. Phenylalkylamine N-methyltransferase (PaNMT) shares substantial amino acid sequence identity with enzymes of the NMT family involved in benzylisoquinoline alkaloid (BIA) metabolism in members of the higher plant order Ranunculales, which includes opium poppy (Papaver somniferum). PaNMT accepted a broad range of substrates with phenylalkylamine, tryptamine, ß-carboline, tetrahydroisoquinoline, and BIA structural scaffolds, which is in contrast to the specificity for BIA substrates of NMT enzymes within the Ranunculales. PaNMT transcript levels were highest in young shoots of E. sinica, which corresponded to the location of NMT activity yielding (pseudo)ephedrine, N-methylcathinone, and N-methyl(pseudo)ephedrine, and with in planta accumulation of phenylalkylamines. Co-expression of recombinant genes encoding PaNMT and an ω-transaminase (PP2799) from Pseudomonas putida in Escherichia coli enabled the conversion of exogenous (R)-phenylacetylcarbinol (PAC) and (S)-PAC to ephedrine and pseudoephedrine, respectively. Our work further demonstrates the utility of plant biochemical genomics for the isolation of key enzymes that facilitate microbial engineering for the production of medicinally important metabolites.

Stabilization of Ephedrine Alkaloid Content in Ephedra sinica by Selective Breeding and Stolon Propagation.

The Ephedra herb, which has been used in Kampo medicines, originates from terrestrial stems of Ephedra species. It is important to establish cultivation methods and cultivars to secure a stable supply of the Ephedra herb that would meet the quality standards for the ephedrine alkaloids content. In this study, we first grew Ephedra sinica plants derived from seeds in the field for 5 years. Then, for selective breeding of cultivars that could meet the quality standards for the ephedrine alkaloids content, we measured the content of total alkaloids (TAs), ephedrine (Eph), and pseudoephedrine (PEph) in individual plants derived from seedlings and grown for 4 years in the field. The range of the TA content in each individual plant was narrower than that among individual plants grown in the field. Therefore, individual plants were selected according to their TA content, Eph/PEph ratio, and stolon-formation capability. The selected individuals were propagated using stolons, and their TA content was studied for 2 years. In the second year, the TA content in terrestrial stems derived from stolons of the selected individuals was as high as that of their parents. Therefore, it was confirmed that the selected individuals that were propagated using stolons could produce TA reproducibly. This study suggested that selective breeding using stolon propagation is effective for stabilizing Ephedra herb TA content.

Pseudoephedrine and circadian rhythm interaction on neuromuscular performance.

This study analyzed the effects of pseudoephedrine (PSE) provided at different time of day on neuromuscular performance, side effects, and violation of the current doping cut-off threshold [World Anti-Doping Agency (WADA)]. Nine resistance-trained males carried out bench press and full squat exercises against four incremental loads (25%, 50%, 75%, and 90% one repetition maximum [1RM]), in a randomized, double-blind, cross-over design. Participants ingested either 180 mg of PSE (supra-therapeutic dose) or placebo in the morning (7:00 h; AM(PLAC) and AM(PSE)) and in the afternoon (17:00 h; PM(PLAC) and PM(PSE)). PSE enhanced muscle contraction velocity against 25% and 50% 1RM loads, only when it was ingested in the mornings, and only in the full squat exercise (4.4-8.7%; P < 0.05). PSE ingestion raised urine and plasma PSE concentrations (P < 0.05) regardless of time of day; however, cathine only increased in the urine samples. PSE ingestion resulted in positive tests occurring in 11% of samples, and it rose some adverse side effects such us tachycardia and heart palpitations. Ingestion of a single dose of 180 mg of PSE results in enhanced lower body muscle contraction velocity against low and moderate loads only in the mornings. These mild performance improvements are accompanied by undesirable side effects and an 11% risk of surpassing the doping threshold.

Immobilised histidine tagged ß2-adrenoceptor oriented by a diazonium salt reaction and its application in exploring drug-protein interaction using ephedrine and pseudoephedrine as probes.

A new oriented method using a diazonium salt reaction was developed for linking ß2-adrenoceptor (ß2-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between ß2-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting ß2-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×103/M for ephedrine and (3.80±0.02) ×103/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10-4 M. Thermodynamic studies showed that the binding of the two compounds to ß2-AR was a spontaneous reaction with exothermal processes. The ΔGθ, ΔHθ and ΔSθ for the interaction between ephedrine and ß2-AR were -(22.33±0.04) kJ/mol, -(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were -(21.17±0.02) kJ/mol, -(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to ß2-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.

In vitro, in vivo, and clinical studies of tedizolid to assess the potential for peripheral or central monoamine oxidase interactions.

Tedizolid phosphate is a novel oxazolidinone prodrug whose active moiety, tedizolid, has improved potency against Gram-positive pathogens and pharmacokinetics, allowing once-daily administration. Given linezolid warnings for drug-drug and drug-food interactions mediated by monoamine oxidase (MAO) inhibition, including sporadic serotonergic toxicity, these studies evaluated tedizolid for potential MAO interactions. In vitro, tedizolid and linezolid were reversible inhibitors of human MAO-A and MAO-B; the 50% inhibitory concentration (IC50) for tedizolid was 8.7 µM for MAO-A and 5.7 µM for MAO-B and 46.0 and 2.1 µM, respectively, with linezolid. Tedizolid phosphate was negative in the mouse head twitch model of serotonergic activity. Two randomized placebo-controlled crossover clinical studies assessed the potential of 200 mg/day tedizolid phosphate (at steady state) to enhance pressor responses to coadministered oral tyramine or pseudoephedrine. Sensitivity to tyramine was determined by comparing the concentration of tyramine required to elicit a ≥ 30-mmHg increase in systolic blood pressure (TYR30) when administered with placebo versus tedizolid phosphate. The geometric mean tyramine sensitivity ratio (placebo TYR30/tedizolid phosphate TYR30) was 1.33; a ratio of ≥ 2 is considered clinically relevant. In the pseudoephedrine study, mean maximum systolic blood pressure was not significantly different when pseudoephedrine was coadministered with tedizolid phosphate versus placebo. In summary, tedizolid is a weak, reversible inhibitor of MAO-A and MAO-B in vitro. Provocative testing in humans and animal models failed to uncover significant signals that would suggest potential for hypertensive or serotonergic adverse consequences at the therapeutic dose of tedizolid phosphate. Clinical studies are registered at www.clinicaltrials.gov as NCT01539473 (tyramine interaction study conducted at Covance Clinical Research Center, Evansville, IN) and NCT01577459 (pseudoephedrine interaction study conducted at Vince and Associates Clinical Research, Overland Park, KS).

Regional-dependent intestinal permeability and BCS classification: elucidation of pH-related complexity in rats using pseudoephedrine.

Based on its lower Log P value relative to metoprolol, a marker for the low/high-permeability (P(eff)) class boundary, pseudoephedrine was provisionally classified as BCS low-permeability compound. On the other hand, following oral administration, pseudoephedrine fraction dose absorbed (F(abs)) and systemic bioavailability approaches 100%. This represents a challenge to the generally recognized P(eff)-F(abs) correlation. The purpose of this study was to elucidate the underlying mechanisms behind the confusion in pseudoephedrine's BCS classification. Pseudoephedrine's BCS solubility class was determined, and its physicochemical properties and intestinal permeability were thoroughly investigated, both in vitro and in vivo in rats, considering the complexity of the whole of the small intestine. Pseudoephedrine was found to be unequivocally a high-solubility compound. All of the permeability studies revealed similar phenomenon; at any given intestinal segment/pH, the permeability of metoprolol was higher than that of pseudoephedrine, however, as the intestinal region becomes progressively distal, and the pH gradually increases, pseudoephedrine's permeability rises above that of metoprolol in the former segment. This unique permeability pattern likely explains pseudoephedrine's complete absorption. In conclusion, pseudoephedrine is a BCS Class I compound; no discrepancy between P(eff) and F(abs) is involved in its absorption. Rather, it reflects the complexity behind P(eff) when considering the whole of the intestine. We propose to allow high-permeability classification to drugs with P(eff) that matches/exceeds the low/high class benchmark anywhere throughout the intestinal tract and not restricted necessarily to the jejunum.

Biosynthesis of amphetamine analogs in plants.

Amphetamine analogs are produced by plants in the genus Ephedra and by Catha edulis, and include the widely used decongestants and appetite suppressants pseudoephedrine and ephedrine. A combination of yeast (Candida utilis or Saccharomyces cerevisiae) fermentation and subsequent chemical modification is used for the commercial production of these compounds. The availability of certain plant biosynthetic genes would facilitate the engineering of yeast strains capable of de novo pseudoephedrine and ephedrine biosynthesis. Chemical synthesis has yielded amphetamine analogs with myriad functional group substitutions and diverse pharmacological properties. The isolation of enzymes with the serendipitous capacity to accept novel substrates could allow the production of substituted amphetamines in synthetic biosystems. Here, we review the biology, biochemistry and biotechnological potential of amphetamine analogs in plants.

Determination of urinary concentrations of pseudoephedrine and cathine after therapeutic administration of pseudoephedrine-containing medications to healthy subjects: implications for doping control analysis of these stimulants banned in sport.

Due to its stimulatory effects on the central nervous system, and its structural similarity to banned stimulants such as ephedrine and methamphetamine, pseudoephedrine (PSE) at high doses is considered as an ergogenic aid for boosting athletic performance. However, the status of PSE in the International Standard of the Prohibited List as established under the World Anti-Doping Code has changed over the years, being prohibited until 2003 at a urinary cut-off value of 25 µg/ml, and then subsequently removed from the Prohibited List during the period 2004-2009. The re-consideration of this position by the World Anti-Doping Agency (WADA) List Expert Group has led to the reintroduction of PSE in the Prohibited List in 2010. In this manuscript, we present the results of two WADA-sponsored clinical studies on the urinary excretion of PSE and its metabolite cathine (CATH) following the oral administration of different PSE formulations to healthy individuals at therapeutic regimes. On this basis, the current analytical urinary threshold for the detection of PSE as a doping agent in sport has been conservatively established at 150 µg/ml

[Asymmetric biosynthesis of d-pseudoephedrine by recombinant Bacillus subtilis].

In order to successfully express the carbonyl reductase gene mldh in Bacillus subtilis and complete coenzyme regeneration by B. subtilis glucose dehydrogenase, the promoter PrpsD and the terminator TrpsD from B. subtilis rpsD gene were used as the expression cassette to be a recombinant plasmid pHY300plk-PrpsD-TrpsD. After that, the carbonyl reductase gene mldh was inserted into the previous plasmid and a plasmid pHY300plk-PrpsD-mldh-TrpsD was achieved, followed by transformed into B. subtilis Wb600 to obtain a recombinant B. subtilis Wb600 (pHY300plk-PrpsD-mldh-TrpsD). Subsequently, the results for whole-cell biotransformation from recombinant B. subtilis showed that it could be used to catalyze MAK (1-phenyl- 1-keto-2-methylaminopropane) to d-pseudoephedrine in the presence of glucose. The yield of d-pseudoephedrine could be up to 97.5 mg/L and the conversion rate of MAK was 24.1%. This study indicates the possibility of biotransformation production of d-pseudoephedrine from recombinant B. subtilis.

A systematic review of methamphetamine precursor regulations.

AIMS: To assess the effectiveness of methamphetamine precursor regulations in reducing illicit methamphetamine supply and use. METHODS: A systematic review of 12 databases was used to identify studies that had evaluated the impact of methamphetamine precursor regulations on methamphetamine supply and/or use. The guidelines of the Effective Practice and Organization of Care Group (EPOC) of The Cochrane Collaboration were used to determine which study designs were included and assess their quality. RESULTS: Ten studies met the inclusion criteria. These studies evaluated 15 interventions (13 regulations and two related interdiction efforts), all of which were located in North America. Interventions had consistent impacts across various indicators of methamphetamine supply and use. Seven of the 15 interventions produced reductions in methamphetamine indicators (ranging from 12% to 77%). Two of the largest impacts were seen following interdiction efforts, involving the closure of rogue pharmaceutical companies. There was no evidence of a shift into other types of drug use, or injecting use, although the impact on the synthetic drug market was not examined. Null effects were related largely to the existence of alternative sources of precursor chemicals or the availability of imported methamphetamine. CONCLUSIONS: Methamphetamine precursor regulations can reduce indicators of methamphetamine supply and use. Further research is needed to determine whether regulations can be effective outside North America, particularly in developing countries, and what impact they have on the broader synthetic drug market. Improved data on precursor diversion are needed to facilitate the evaluation of precursor regulations.

Genetic analysis around aminoalcohol dehydrogenase gene of Rhodococcus erythropolis MAK154: a putative GntR transcription factor in transcriptional regulation.

NADP(+)-dependent aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 catalyzes the reduction of (S)-1-phenyl-1-keto-2-methylaminopropane ((S)-MAK) to d-pseudoephedrine, which is used as a pharmaceutical. AADH is suggested to participate in aminoalcohol or aminoketone metabolism in this organism because it is induced by the addition of several aminoalcohols, such as 1-amino-2-propanol. Genetic analysis of around the aadh gene showed that some open reading frames (ORFs) are involved in this metabolic pathway. Four of these ORFs might form a carboxysome-like polyhedral organelle, and others are predicted to encode aminotransferase, aldehyde dehydrogenase, phosphotransferase, and regulator protein. OrfE, a homologous ORF of the FadR subfamily of GntR transcriptional regulators, lies downstream from aadh. To investigate whether or not orfE plays a role in the regulation of aadh expression, the gene disruption mutant of R. erythropolis MAK154 was constructed. The ΔorfE strain showed higher AADH activity than wild-type strain. In addition, a transformed strain, which harbored multi-orfE, showed no AADH activity even in the induced condition with 1-amino-2-propanol. These results suggest that OrfE is a negative regulator that represses aadh expression in the absence of 1-amino-2-propanol.

Transfer of Ephedra genomic DNA to yeasts by ion implantation.

The genomic DNA from Ephedra glauca was randomly transferred to Saccharomyces cerevisiae and Hansenula anomala by argon and nitrogen ion implantation. Through repeated subculturing and using reversed phase high-performance liquid chromatography analysis to quantify the concentrations of the secondary metabolites, l-ephedrine and d-pseudoephedrine, 12 recombinant strains of genetically stable yeast were obtained, each using glucose as a carbon source, NaNO3 as a nitrogen source and producing l-ephedrine and/or d-pseudoephedrine. After culturing in liquid medium for 72 h, extracellular l-ephedrine and d-pseudoephedrine concentrations of 18.85 and 4.11 mg/L, respectively, were detected. Using l-ephedrine and d-pseudoephedrine as the target products, the transformation efficiencies of the genomic DNA from E. glauca transferred to S. cerevisiae and H. anomala were 1.15% (1/87) and 2.13% (8/376), respectively. The addition of the amino acid, L-Phe, to culture media substantially changed the amount of l-ephedrine and/or d-pseudoephedrine produced by the recombined yeasts. However, the change in metabolite production was not consistent among strains, rising in some, while dropping to nondetectable levels in others. After random amplification of polymorphic DNA (RAPD) analysis, four RAPD primers were obtained from the initial 100 RAPD primers, each amplifying different fragments with the recombined yeast Ar_Han0458 genome. Using one primer as polymerase chain reaction primer, the result showed that the recombined yeast Ar_Han0458 genome matched E. glauca genomic DNA at 150 bp, indicating a successful transfer of genetic information, facilitated by ion implantation.

[Production of l-ephedrine and d-pseudoephedrine in recombined yeasts obtained by argon ion implantation mediated Ephedra genome DNA transformation].

The genome DNA from Ephedra glauca was randomly transferred into Hansenula anomala, respectively, by argon ion bombardment. Then, after screening by the motheds of bromothymol blue indicator selection, slant cultivation, l-ephedrine and d-pseudoephedrine copper chromic salt qualitative test and RP-HPLC determination, 3 strains, producing recombined yeasts were obtained, which can use glucose as a carbon source, NaNO3 as nitrogen source and be genetically stable. After cultivated in liquid medium for 72 hours and analyzed by the RP-HPLC, the recombined strains can produce l-ephedrine 11.87 mg/L and d-pseudoephedrine 4.11 mg/L excellular, d-pseudoephedrine 294.86 mg/g dry cell incellular, but l-ephedrine not detected incellular. The transformation efficiency of Ephedra genome DNA transferred into yeasts via argon ion bombardment was 0.65%. The effects of Ephedra genome DNA macromolecule integrity on yeast transformation system were discussed. The results shown that DNA macromolecule with integrated structure used as exogenous donor can obtain higher transformation efficiency than DNA macromolecule random fragments by ion implantation mediated DNA transformation. It was inferred that biosynthesis of l-ephedrine and the d-pseudoephedrin were controlled by linked together genes or gene clusters.

[Purification and characterization of carbonyl enantioselective reductase from Morganella morganii J-8].

The purification and the characteristics of an enzyme from Morganella morganii J-8, which could produce d-pseudoephedrine from 1-phenyl-2-methylamine-acetone, were performed in this study. In this research, first, cells were disrupted by ultrasonic treatment at 4 degrees C. The carbonyl enantioselective reductase was purified with a combination of ammonium precipitation, Phenyl Superose hydrophobic chromatography, DEAE anion exchange, and native polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme subunit was estimated to be 42.5kD on sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). The native molecular mass of the enzyme that was analyzed by high-performance liquid chromatography was found out to be 84.1 kD, which indicated that the enzyme was a dimmer. The purified enzyme was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the result showed that the purified enzyme had high homology with leucine dehydrogenase.