Molecular cloning, expression and characterization of hyoscyamine 6beta-hydroxylase from hairy roots of Anisodus tanguticus

Anisodus tanguticus, one of the indigenous Chinese ethnological medicinal plants of the Solanaceae, produces anticholinergic alkaloids such as hyoscyamine, 6 beta-hydroxyhyoscyamine and scopolamine. Hyoscyamine 6 beta-hydroxylase (H6H), a key enzyme in the biosynthetic pathway of scopolamine, catalyzes the hydroxylation of hyoscyamine and epoxide formation from 6 beta-hydroxyhyoscyamine to generate scopolamine. A full-length cDNA of H6H has been isolated from A. tanguticus hairy roots. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame of 1035 bp encoding 344 amino acids with high homology to other known H6Hs. The equivalent amino acid sequence shows a typical motif of 2-oxoglutarate-dependent dioxygenase. The A. tanguticus H6H was expressed in Escherichia coli and purified for enzyme function analysis. This study characterized the recombinant AtH6H and showed it could generate scopolamine from hyoscyamine.     

Molecular cloning and expression of putrescine N-methyltransferase from the hairy roots of Anisodus tanguticus

A full-length cDNA encoding putrescine N-methyltransferase (PMT) was isolated from the hairy roots of A. tanguticus. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame of 1017 bp encoding 338 amino acids with high homology to other known PMTs. A. tanguticus PMT was expressed in Escherichia coli. The recombinant AtPMT was purified and exhibited S-adenosyl-methionine-dependent N-methyltransferase activity.     

Papaverine Biotransformation by Silene alba Cell Suspension.

SILENE ALBA cell suspensions were supplied with papaverine hydrochloride. Seven biotransformation compounds were obtained of which four were alkaloidal. The two most important compounds are regioselective demethylation products: 6-monodemethylpapaverine and 4'-monodemethylpapaverine.     

Biotransformation of Tryptamine to Serotonin by Cell Suspension Cultures of Peganum harmala

Cell suspension cultures of P. HARMALA, unable to form serotonin DE-NOVO, hydroxylated fed tryptamine to serotonin independently of the growth cycle up to 20 mg per g dry mass per day. The best biotransformation yield of 80% was obtained with a high cell density (30 g fresh mass/100 ml) and a 5 mM tryptamine concentration in the transformation medium. Under these conditions the cells accumulated serotonin up to 7.5% per unit dry mass, and a maximum yield of 800 mg serotonin/l was achieved. Other indoleethylamines such as alpha-methyltryptamine, N-methyltryptamine, and 6-fluorotryptamine were also transformed to the corresponding 5-hydroxyl compounds, while N,N-dimethyltryptamine, 7-methyltryptamine, tyramine, and phenylethylamine were not hydroxylated.     

Biotransformation of Cannabidiol to Cannabielsoin by Suspension Cultures of Cannabis sativa and Saccharum officinarum

Suspension cultures of CANNABIS SATIVA L. and SACCHARUM OFFICINARUM L. have been shown to biotransform cannabidiol to Cannabielsoin under normal growth conditions. Both Cannabielsoin C-1 stereoisomers were produced. Mass spectral and chromatographic evidence were used in this identification. This is the first reported biologically catalyzed synthesis of Cannabielsoin as well as the first biotransformation of any cannabinoid by plant tissue culture systems. Possible mechanisms and significance of this conversion are discussed.     

Biotransformation of Cannabidiol to Cannabielsoin by Suspension Cultures of Cannabis sativa and Saccharum officinarum.

Suspension cultures of CANNABIS SATIVA L. and SACCHARUM OFFICINARUM L. have been shown to biotransform cannabidiol to Cannabielsoin under normal growth conditions. Both Cannabielsoin C-1 stereoisomers were produced. Mass spectral and chromatographic evidence were used in this identification. This is the first reported biologically catalyzed synthesis of Cannabielsoin as well as the first biotransformation of any cannabinoid by plant tissue culture systems. Possible mechanisms and significance of this conversion are discussed.     

固定化细胞转化阿糖尿苷合成阿糖腺苷

采用聚乙烯醇、卡拉胶和聚丙烯酰胺为混合载体固定化埃希菌AEM0812细胞.以8.0％聚乙烯醇、1.0％卡拉胶和10％聚丙烯酰胺作为载体,菌体浓度为30％,在pH 7.0、含4.0％氯化钾的饱和硼酸溶液(成型剂)中处理36h,得相对酶活力为98.17％且较稳定的颗粒型固定化细胞,用来催化转化阿糖尿苷合成阿糖腺苷,在54℃、pH 6.5和缓冲液离子浓度120 mmol/L的最适条件下,阿糖尿苷的转化率为68％.固定化细胞连续5次反应的平均转化率约54.3％.     

利用κ-卡拉胶-魔芋多糖复配胶固定化酵母生产三磷酸腺苷

利用κ-卡拉胶-魔芋多糖复配胶包埋的固定化啤酒酵母细胞,由腺苷(AR)生产三磷酸腺苷(ATP).采用HPLC法分析固定化细胞反应过程核苷(酸)的变化以及用琼脂糖平板电泳法对其工艺条件进行了优化.固定化细胞反应1 h,ATP转化率接近100%.底物溶液灭菌和添加NAD+后,该固定化酵母细胞可反复使用12次,ATP的转化率稳定维持在95%以上.     

Biotransformation of alpha- and 6beta-santonin by fungus and plant cell cultures

One fungus, Abisidia coerulea IFO 4011, and suspended cell cultures of one plant, Asparagus officinalis, were employed to bioconvert alpha- and 6beta-santonin. Incubation of alpha-santonin with the cell cultures of the fungus afforded two products, 11beta-hydroxy-alpha-santonin (1, in 76.5% yield) and 8alpha-hydroxy-alpha-santonin (2, in 2.0% yield). And from 6beta-santonin, four major products (3, 4, 5 and 6) and four minor products (7, 8, 9 and 10) were obtained, including 8alpha-hydroxylated products in trace yields. Very interestingly, a skeletal rearrangement occurred and a guaiane product (13) formed in a very low yield when alpha-santonin incubating with A.officinalis cell cultures, while not in the case of 6beta-santonin as substrate. Among the obtained 15 products, 2, 7, 8, 9, 10 and 12 are new compounds. The fact of 8alpha hydroxylation of santonin enables the formation of 8,12-eudesmanolide instead of 6,12-eudesmanolide and some useful modification at C-8 position. In addition, these reactions would provide evidence for the biogenesis between different types of eudesmane and/or guaiane compounds in the plants in nature.     

裂殖酵母生物合成L-苯基乙酰基甲醇的研究

用裂殖酵母作为生物催化剂，催化苯甲醛与丙酮酸合成L苯基乙酰基甲醇（L-PAC），后者为合成L-麻黄素等多种药物的前体，转化反应的最佳温度为28℃，pH6．0，时间16h，苯甲醛用量160-200mmol／L，丙酮酸120～180mmol／L，LPAC产量可达15g／L。     

Biotransformation of 10-Hydroxygeraniol and Related Compounds by a Cell Suspension Culture of Catharanthus roseus: The Formation of Reduced Products.

The biotransformation of 10-hydroxygeraniol and several closely related compounds by a cell suspension culture of CATHARANTHUS ROSEUS, employing a variety of protocols, was studied. The major products obtained from 10-hydroxygeraniol were: 10-hydroxylinalool, 10-hydroxy-citronellol, 7,8-dihydro-10-hydroxygeraniol, dihydrocitronellol, and 7,8-dihydro-10-hydroxylinalool. The relative proportions of products was dependent on the protocol used. Biotransformation of C-1 dideuterated 10-hydroxygeraniol led to the production of reduced products which were mono-deuterated. Biotransformation of 8-hydroxy-7,8-dihydrogeraniol afforded (-)-8-hydroxy-7,8-dihydrocitronellol while (+)-10-hydroxycitronellol was reduced to afford a mixture of diastereomers.     

Biotransformation of podophyllotoxin to picropodophyllin by microbes

Biotransformation of podophyllotoxin (PT) by several microbial species has been investigated. Among the fungi tested, it was found that Penicillium strains can isomerize PT to picropodophyllin (PPT) in 8% yield and other strains also transform the substrate into the same product but with lower yield.     

商陆培养细胞对6β-santonin的生物转化

目的利用商陆培养细胞对6β-santonin (1)进行生物转化，以获得多种利于进一步进行结构改造的衍生物。方法将底物1与商陆培养细胞共孵化，采用色谱分离技术得到产物，通过理化性质和光谱数据对产物进行结构鉴定。结果与商陆培养细胞共孵化7 d后，底物1转化为5个产物(2～6)，其中3为新化合物。结论利用商陆培养细胞可对6β-santonin进行选择性还原及羟基化反应，可为其进一步化学结构改造提供有价值的中间体。     

用固定化氧化葡萄糖酸菌转化6-(N-羟乙基)胺基-6-脱氧-L-山梨呋喃糖

目的探索用固定化氧化葡萄糖酸菌转化6-(N-羟乙基)胺基-6-脱氧-L-山梨呋喃糖的最优条件。方法以海藻酸钠为载体把氧化葡萄糖酸菌固定化,考察固定化细胞在不同转化条件时对底物的转化率。结果通过条件优化,确定固定化细胞转化时间为24 h,底物质量浓度为10 g.L-1,采用的海藻酸钠质量浓度为40g.L-1,菌体包埋量为每g凝胶包埋3.1 g菌体,最适pH为7.0,最适温度为28℃,摇床转速要求高于100r.min-1;用60mmol.L-1的MnCl2替代原转化液中20mmol.L-1的MgSO4;固定化细胞与原游离细胞工艺相比,24h内底物转化率由原来的47%上升到93%,连续转化10次后,转化率仍为38%。结论用固定化氧化葡萄糖酸菌转化6-(N-羟乙基)胺基-6-脱氧-L-山梨呋喃糖,转化率提高46%并能连续转化约10次。     

Accumulation and Biotransformation of Chromenes and Benzofurans in a Cell Suspension Culture of Ageratina adenophora

A cell suspension culture of AGERATINA ADENOPHORA was shown to yield several novel chromene and benzofuran derivatives in minute amounts that were different to the compounds found in seedlings of the same species. The structure elucidation of the new compounds is described. When two of the seedling chromenes (demethoxyencecalin and demethylencecalin) were fed to the cell suspension culture, one biotransformation product each was obtained in high yields (80%) that originated from a hydroxylation at one of the geminal methyl groups of the chromene heterocycle. These products accumulated largely in the growth media even though the presence of cells was necessary for the biotransformations to occur. When the third seedling chromene (encecalin) was fed to the cell auspension culture, no significant biotransformation was noted but several of the benzofurans present as cell culture metabolites showed a significantly increased accumulation in the growth media of the treated cultures. This increased accumulation of benzofurans was found to be inducible also by adding yeast extract to the cell culture. The metabolism of chromenes and bezofurans in the cell suspension culture is discussed.     

固定化嗜热脂肪芽孢杆菌生产1,6-二磷酸果糖的研究

采用卡拉胶包埋嗜热脂肪芽孢杆菌，制成块状固定化细胞，对固定化细胞包埋方法及其用于生产1，6－二磷酸果糖（FDP）的工艺条件作了探索，在固定化细胞包埋前，包埋后及用于生产FDP 10批次后采用活化培养工艺，均可提高固定化细胞生产FDP的能力，转化培养液中的FDP产量达到40－65mg/ml，固定化细胞用于生产FDP的批次可达到20次左右。     

柚皮苷转化生成红花素及异红花素的研究

摘要：目的 利用一株日本曲霉,以柚皮苷为底物,转化生成红花素和异红花素并提高产物的发酵单位。方法 对发酵培养基中氮源种类进行筛选并确定最适的添加浓度,考察微量元素对发酵的影响并确定最适添加浓度,降低溶解底物的乙醇浓度并采用二次添加底物的方式等。结果 红花素发酵单位由66 μg/mL提高至389 μg/mL,异红花素发酵单位由263 μg/mL提高至946 μg/mL。热豆粉是最佳氮源,添加浓度为0.3%;添加硫酸亚铁和氯化锰能够分别促进红花素和异红花素的生成,其最适添加浓度分别为0.02%和0.01%;溶解底物的乙醇浓度由50%降低至26.6%,降低了乙醇对转化的不利影响;底物添加次数由1次变为2次,有利于发酵单位的提高。结论 首次发现日本曲霉可直接转化柚皮苷生成红花素和异红花素,且柚皮苷生物转化为红花素和异红花素的发酵单位较高。     

Scopolamine prevents tolerance to the effects of caffeine on rotational behavior in 6-hydroxydopamine-denervated rats

Continuous administration of caffeine has been shown to induce tolerance to its psychostimulant effects. In this study, using unilateral 6-hydroxydopamine nigrostriatal denervated rats, we tested the hypothesis that the muscarinic receptor antagonist, scopolamine, would prevent the tolerance to caffeine-induced contralateral rotational behavior. For that purpose we administered either caffeine (40 mg/kg) plus saline or scopolamine (5, 10 and 20 mg/kg) plus saline, as well as caffeine in combination with the various doses of scopolamine for 7 consecutive days, and measured ipsilateral and contralateral rotational behavior. The results showed that acute injections of scopolamine plus saline produced similar levels of both ipsilateral and contralateral turning, while caffeine produced more contralateral than ipsilateral turning. Tolerance to caffeine-induced contralateral turning was observed as of the second administration, while scopolamine plus saline injections did not produce significant changes in rotational behavior with repeated treatment. Scopolamine co-administered with caffeine significantly attenuated the increased contralateral turning produced by acute injections of caffeine plus saline, but significantly prevented the tolerance effects with repeated administration. These findings strongly suggest that muscarinic cholinergic processes may be involved in tolerance to caffeine-induced contralateral turning. The results are interpreted in terms of the possible interactions between dopamine, adenosine and acetylcholine neurotransmitter systems within the basal ganglia circuitry involved in motor behavior.     

Biotransformation of ginsenosides F4 and Rg6 in zebrafish

Abstract

Ginsenosides F₄ and Rg₆ (GF₄ and GRg₆), two main active components of steamed notoginseng or red ginseng, are dehydrated disaccharide saponins. In this work, biotransformation of ginsenosides F₄ and Rg₆ in zebrafish was investigated by qualitatively identifying their metabolites and then proposing their possible metabolic pathways. The prediction of possible metabolism of ginsenosides F₄ and Rg₆ using zebrafish model which can effectively simulate existing mammals model was early and quickly performed. Metabolites of ginsenosides F₄ and Rg₆ after exposing to zebrafish for 24 h were identified by Ultraperformance Liquid Chromatography/Quadrupole-Time-of-Flight Mass Spectrometry. A total of 8 and 6 metabolites of ginsenosides F₄ and Rg₆ were identified in zebrafish, respectively. Of these, 7 and 5, including M1, M3?M5, M7?M9 and N1 (N5), N2, N4 (N9), N7?N8 were reported for the first time as far as we know. The mechanisms of their biotransformation involved were further deduced to be desugarization, glucuronidation, sulfation, dehydroxylation, loss of C-17 and/or C-23 residue pathways. It was concluded that loss of rhamnose at position C-6 and glucuronidation at position C-3 in zebrafish were considered as the main physiologic and metabolic processes of ginsenosides F₄ and ginsenosides Rg₆, respectively.