Biotransformation of alisol G by Penicillium janthinellum and the hCE2 inhibitory effects of its metabolites

Alisol G, also named 25-anhydroalisol A, is a major triterpene obtained from dried rhizomes of Alisma orientalis. This paper investigated the transformation of alisol G by fungi and evaluated the hCE2 inhibitory effects of the transformed products. By screening whole cells of 10 strains of filamentous fungi, Penicillium janthinellum AS 3.510 exhibited a special capability to transform alisol G. Four metabolites were obtained, which were determined to be new compounds on the basis of spectroscopic data, including UV, 1D- and 2D-NMR, and HRESIMS. In addition, in an in vitro bioassay, metabolites 1–4 showed significant hCE2 inhibitory activities with IC₅₀ values of 6.81, 16.66, 3.38, and 6.33 μM, respectively.     

Biotransformation of prednisolone to hydroxy derivatives by Penicillium aurantiacum

Prednisolone, a synthetic adrenal corticosteroid drug, is known to have anti-inflammatory and autoimmune activity. Biotransformation of prednisolone was carried out to obtain more bioactive prednisolone derivatives. Among six different fungi, Penicillium aurantiacum proved to be the best prednisolone hydroxylator. As a result of prednisolone biotransformation by P. aurantiacum, whole cells four different prednisolone derivatives were investigated. 20β-Hydroxyprednisolone (1) and 21,21-dimethoxy-11β-hydroxypregn-1,4-dien-3,20-dione (2) were detected as the main metabolites. These metabolites together with other two metabolites, 11β-hydroxyandrost-1,4-dien-3,17-dione (3) and 11β,17β-dihydroxyandrost-1,4-dien-3- one (4), were purified and assigned by an interpretation of their spectral data using mass spectroscopy (MS), proton nuclear magnetic resonance (¹H-NMR), carbon nuclear magnetic resonance (¹³C-NMR) and infrared spectroscopy (IR) analyses. The best fermentation conditions for production of compounds 1–4 were as follows: medium (3) consisting of (g/l): glucose 20; l-asparagine 0.7; MgSO₄.7H₂O 0.5; KH₂PO₄ 1.52; KCl 0.52; Cu (NO₃)₂ traces; ZnSO₄.7H₂O traces, supplemented with prednisolone concentration of 0.3?mg/ml, inoculated by 10% of microorganism and incubated for 72?h. Under these optimized conditions, ～94.8% of the added prednisolone was converted to aforementioned derivatives, which have the potential to be used in industrial production of important pharmaceutical compounds.     

Oxidant-based anticancer activity of a novel synthetic analogue of capsaicin,capsaicin epoxide

Abstract

Objectives

Plant-derived natural substances, such as capsaicin, with potent antiproliferative activity against cancer cells in vitro are considered to be promising nutraceuticals in anticancer therapy. Nevertheless, the limited systemic bioavailability of phytochemicals may raise questions regarding the physiological relevance of their phytochemical effects in vivo. Thus, the search for novel phytochemical-based substances with more efficient anticancer action is needed.

Methods

In the present study, a capsaicin analogue, namely, capsaicin epoxide, was synthesized, and its cytotoxic potential against cancer cells was evaluated and compared to that of capsaicin through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and multi-caspase assays. The abilities of capsaicin and capsaicin epoxide to induce oxidative stress were estimated using redox-sensitive fluorogenic probes: 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) and dihydroethidium.

Results

The structure and purity of the synthesized product were confirmed by nuclear magnetic resonance spectroscopy, electrospray ionization mass spectrometry, and gas chromatography. Normal human dermal fibroblasts were not susceptible to treatment with the agent, whereas a cancer cell type-specific response was observed. Human breast carcinoma cells were found to be the most sensitive to capsaicin epoxide treatment compared with capsaicin treatment, and the action of capsaicin epoxide was oxidant based.

Discussion

Our data indicate that the antiproliferative activity of capsaicin epoxide is potentiated in vitro, when used at much lower concentrations compared with capsaicin at similar concentrations. Thus, the findings of this study may have implications for phytochemical-based anticancer drug development.     

Direct Biotransformation of Dioscin into Diosgenin in Rhizome of Dioscorea zingiberensis by Penicillium dioscin

Diosgenin is an important precursor for synthesis of more than 200 steroidal hormone medicines. Rhizome of Dioscorea zingiberensis C. H. Wright (RDZ) contained the highest content of diosgenin in Dioscorea plant species. Diosgenin is traditionally extracted by acid hydrolysis from RDZ. However, the acid hydrolysis process produces massive wastewater which caused serious environment pollution. In this study, diosgenin extraction by direct biotransformation with Penicillium dioscin was investigated. The spawn cultivation conditions were optimized as: Czapeks liquid culture medium without sugar and agar (1,000 ml) + 6.0 g dioscin/6.0 g DL, 30 °C, 36 h; solid fermentation of RDZ: mycelia/RDZ of 0.05 g/kg, 30 °C, 50 h; the yield of diosgenin was over 90 %. Spawn cultivation was crucial for the direct biotransformation. In the spawn cultivation, amount and ratio of dioscin/DL were the key factors to promote biotransformation activity of P. dioscin. This biotransformation method was environment-friendly, simple and energy saving, and might be a potential substitute for acid hydrolysis in diosgenin extraction industry.     

Biotransformation of ent-pimaradienoic acid by cell cultures of Aspergillus niger

Microbial transformation stands out among the many possible semi-synthetic strategies employed to increase the variety of chemical structures that can be applied in the search for novel bioactive compounds. In this paper we obtained ent-pimaradienoic acid (1, PA, ent-pimara-8(14),15-dien-19-oic acid) derivatives by fungal biotransformation using Aspergillus niger strains. To assess the ability of such compounds to inhibit vascular smooth muscle contraction, we also investigated their spasmolytic effect, along with another five PA derivatives previously obtained in our laboratory, on aortic rings isolated from male Wistar rats. The microbial transformation experiments were conducted at 30 °C using submerged shaken liquid culture (120 rpm) for 10 days. One known compound, 7α-hydroxy ent-pimara-8(14),15-dien-19-oic acid (2), and three new derivatives, 1β-hydroxy ent-pimara-6,8(14),15-trien-19-oic acid (3), 1α,6β,14β-trihydroxy ent-pimara-7,15-dien-19-oic acid (4), and 1α,6β,7α,11α-tetrahydroxy ent-pimara-8(14),15-dien-19-oic acid (5), were isolated and identified on the basis of spectroscopic analyses and computational studies. The compounds obtained through biotransformation (2–5) did not display a significant antispasmodic activity (values ranging from 0% to 16.8% of inhibition); however the previously obtained diterpene, methyl 7α-hydroxy ent-pimara-8(14),15-dien-19-oate (8), showed to be very effective (82.5% of inhibition). In addition, our biological results highlight the importance to study the antispasmodic potential of a large number of novel diterpenes, to conduct further structure–activity relationship investigations.     

Vanilla Flavour Production Through Biotransformation Using Capsicum frutescens Root Cultures

Normal roots of Capsicum frutescens were excised from tissue-cultured plants into half strength Murashige and Skoog's medium with 2.23 μM naphthalene acetic acid. Maximum growth of cultured roots was 6.5 g fresh weight 40 ml^-1, as recorded on day 20. Even though normal roots were unable to accumulate capsaicin, they contained other phenylpropanoid intermediates and vanillylamine, as detected by HPLC analysis. Normal roots of Capsicum frutescens were treated with ferulic acid and protocatechuic aldehyde in order to study their biotransformation ability. Ferulic acid, which is the nearest precursor to vanillin, when fed at concentrations of 1 and 2 mM led to the accumulation of vanilla flavour metabolites, vanillin being the major one. In cultures treated with 1 and 2 mM ferulic acid, maximum vanillin accumulation of 12.3 and 16.4 μM was observed, on day 6 after precursor addition, respectively. Feeding of ferulic acid and β-cyclodextrin complex (2 mM each) enhanced the accumulation of biotransformed products. Moreover, vanillin accumulation was recorded as 24.7 μM on day 6 after precursor addition, which was 1.5 times higher than in cultures fed with ferulic acid (2 mM) alone. When ferulic acid was fed along with β-cyclodextrin (1 mM each) to cultures growing in a three-litre bubble column bioreactor, the maximum vanillin production of 10.7 μM was obtained; other vanilla flavour metabolites were also formed after 9 days of precursor addition. Root cultures could also biotransform protocatechuic aldehyde wherein a maximum vanillin production of 7.9 μM was recorded on day 6 after precursor addition. The bioconversion efficiency was observed to be 5-7% in case of ferulic acid fed cultures and 3.2% in case of protocatechuic aldehyde fed cultures suggesting the possible channelling of precursors to alternate biosynthetic pathways such as lignin.     

Vanilla Flavour Production Through Biotransformation Using Capsicum frutescens Root Cultures

Normal roots of Capsicum frutescens were excised from tissue-cultured plants into half strength Murashige and Skoog's medium with 2.23 μM naphthalene acetic acid. Maximum growth of cultured roots was 6.5 g fresh weight 40 ml^-1, as recorded on day 20. Even though normal roots were unable to accumulate capsaicin, they contained other phenylpropanoid intermediates and vanillylamine, as detected by HPLC analysis. Normal roots of Capsicum frutescens were treated with ferulic acid and protocatechuic aldehyde in order to study their biotransformation ability. Ferulic acid, which is the nearest precursor to vanillin, when fed at concentrations of 1 and 2 mM led to the accumulation of vanilla flavour metabolites, vanillin being the major one. In cultures treated with 1 and 2 mM ferulic acid, maximum vanillin accumulation of 12.3 and 16.4 μM was observed, on day 6 after precursor addition, respectively. Feeding of ferulic acid and β-cyclodextrin complex (2 mM each) enhanced the accumulation of biotransformed products. Moreover, vanillin accumulation was recorded as 24.7 μM on day 6 after precursor addition, which was 1.5 times higher than in cultures fed with ferulic acid (2 mM) alone. When ferulic acid was fed along with β-cyclodextrin (1 mM each) to cultures growing in a three-litre bubble column bioreactor, the maximum vanillin production of 10.7 μM was obtained; other vanilla flavour metabolites were also formed after 9 days of precursor addition. Root cultures could also biotransform protocatechuic aldehyde wherein a maximum vanillin production of 7.9 μM was recorded on day 6 after precursor addition. The bioconversion efficiency was observed to be 5–7% in case of ferulic acid fed cultures and 3.2% in case of protocatechuic aldehyde fed cultures suggesting the possible channelling of precursors to alternate biosynthetic pathways such as lignin.     

生物转化法制备L-天冬酰胺

探索生物转化法制备L-天冬酰胺的技术与工艺。通过分子生物学方法,克隆来源于大肠杆菌(Escherichia coli, E.coli)JM109的天冬酰胺合成酶A基因asnA,并于E. coli BL21(DE3)中表达,利用构建的E.coli基因工程菌E.coli BL21(DE3)/pET28a(+)-asnA全细胞高密度催化L-天冬氨酸生产L-天冬酰胺,以PITC柱前衍生-高效液相检测底物和产物。表达的蛋白质分子质量约为37kDa,与预期大小相符,比酶活力为1786.6U/g。L-天冬氨酸转化率为95.8%,L-天冬酰胺产量可达126.5g/L,生产速率为15.81g/(L·h)。结果表明,已成功构建高效表达天冬酰胺合成酶A基因工程菌株,并用于催化L-天冬氨酸转化生产L-天冬酰胺,解决了L-天冬酰胺生物转化生产工艺中ATP成本过高的难题,为L-天冬酰胺制备提供新的绿色途径。     

Optimization of pyrene oxidation by Penicillium janthinellum using response-surface methodology

At present, there is little information on the optimization of the degradation of polycyclic aromatic hydrocarbons (PAH) by deuteromycete filamentous fungi, a reaction catalyzed by cytochrome P450 monooxygenases. We utilized response-surface methodology to determine the optimal growth conditions for the oxidation of the PAH pyrene by Penicillium janthinellum SFU403, with respect to the variables glucose concentration, nitrate concentration and bioconversion time. Models were derived for the relationship between the variables tested and the level of the pyrene oxidation products, 1-pyrenol (1-PY) and pyrenequinones (PQ). Production of 1-PY and PQ were optimized by the same glucose and nitrate concentrations: 2.5% glucose and 1.5% sodium nitrate. The optimized 1-PY and PQ bioconversion times were 71 h and 73 h respectively. These conditions improved the yield of 1-PY by fivefold and PQ were more than 100-fold higher than the baseline levels obtained in this study. The optimized PQ yield represented 95% of the initial pyrene, thus the total optimised pyrene bioconversion to 1-PY and PQ was approximately 100%. Concentrations of glucose exceeding 4.0% repressed pyrene hydroxylation. Pyrene hydroxylation occurred almost exclusively during the deceleration phase of culture growth. Received: 20 July 1998 / Received revision: 7 December 1998 / Accepted: 10 January 1999     

原儿茶酸的生物转化

【背景】原儿茶酸(Protocatechuic acid,PCA)是一些植物的主要活性成分,可作为许多聚合物和药物的前体物质,目前PCA的主要来源是利用化学法从植物中提取,然而该法提取率低且对环境造成一定程度的破坏。【目的】克隆对羟基苯甲酸-3-羟化酶基因ρ-HBA-3H并进行异源表达,利用该酶催化实现原儿茶酸的生物转化。【方法】以红球菌R04基因组DNA为模板,PCR扩增得到对羟基苯甲酸-3-羟化酶基因ρ-HBA-3H,构建重组基因工程菌BL21(DE3)/pET21a(+)-ρ-HBA-3H,诱导表达对羟基苯甲酸-3-羟化酶,在底物对羟基苯甲酸(ρ-Hydroxybenzoicacid,ρ-HBA)存在下进行PCA的生物转化,并对生物转化的条件进行优化。【结果】对羟基苯甲酸-3-羟化酶基因在大肠杆菌中实现了高效表达。通过生物转化PCA产量可达1.156 g/L。优化实验表明Mg2+、Triton X-100对转化效率无影响,增加反应体系的溶氧量及添加适量的吐温-80能够促进转化反应的进行。细胞连续转化基础上适量补充葡萄糖可以有效增加工程菌的转化效率,减少PCA的消耗。【结论】通过生物酶催化法实现了PCA的高效率、绿色生产,为其他重要发酵产品的工业化生产提供理论研究基础。     

Microbial biomass production from rice straw by fermentation with Penicillium janthinellum

Production of microbial biomass through fermentation of pretreated rice straw using Penicillium janthinellum (St-F-3B) is reported, with emphasis on the use of non-effluent generating pretreatment procedures. The fungus readily metabolized a rice straw substrate that had been subjected to alkali pretreatment by steaming at atmospheric pressure followed by the neutralization of the alkali. The crude protein content of the microbial biomass averaged 15–20%. The fermentation could be carried out in aerated-agitated fermenters using fertilizer grade nutrient salts to produce a biomass with 17.5% crude protein. When operated on a semicontinuous basis using 20% of the previous batch as inoculum, successive batches produced a biomass product with 12–19% crude protein content in 48 h. The problems of developing a technology for protein from agricultural residues with particular reference to developing countries are discussed.     

Biotransformation of monoterpenes by Oocystis pusilla

The biotransformation of several monoterpenes by the locally isolated unicellular microalga, Oocystis pusilla was investigated. The metabolites were identified by thin layer chromatography and GC/MS. The results showed that O. pusilla had the ability to reduce the C=C double bond in (+)-carvone to yield trans-dihydrocarvone and traces of cis-dihydrocarvone. O. pusilla also converted (+)-limonene to trans-carveol, as the main product, and yielded carvone and trans-limonene oxide. Furthermore, (−)-linalool was converted to trans-furanoid and trans-pyranoid linalool oxide, thymol was converted to thymoquinone, (−)-carveol was converted to carvone and trans-dihydrocarvone, (−)-menthone and (+)-pulegone were converted to menthol, (L)-citronellal was converted to citronellol, and (+)-β-pinene was converted to trans-pinocarveol.     

Action of crystalline acid carboxypeptidase from Penicillium janthinellum.

Acid carboxypeptidase (EC 3.4.12.-) crystallized from culture filtrate of Penicillium janthinellum has been investigated for its use in carboxy-terminal sequence determination of Z-Gly-Pro-Leu-Gly, Z-Gly-Pro-Leu-Gly-Pro, angiotensin I, native lysozyme, native ribonuclease T1, and reduced S-carboxy-methyl-lysozyme. The examination indicated that proline and glycine were liberated from Z-Gly-Pro-Leu-Gly-Pro. At high enzyme concentration, the enzyme catalyzed complete sequential release of amino acids from the carboxy-terminal leucine to the amino-terminal aspartic acid of angiotensin I. The enzyme released the carboxy-terminal leucine from native lysozyme, however, no release of the threonine from native ribonuclease T1 was observed after a prolonged period of incubation with the enzyme. The sequence of the first nine carboxy-terminal residues of denatured lysozyme, leucine, arginine, S-carboxymethyl-cysteine, glycine, arginine, isoleucine, tryptophane, alanine, and glutamine, could be deduced unequivocally from a time release plot of an incubation mixture with the enzyme.     

Pyrene is metabolized to bound residues by Penicillium janthinellum SFU403

We have previously shown that the filamentous fungus, Penicillium janthinellum SFU403 (SFU403) oxidizes pyrene to pyrene 1,6- and 1,8-quinones and that the level of pyrenequinones (PQs) subsequently declines suggesting that PQs are not terminal metabolites. The purpose of this study was to determine the fate of PQs in SFU403. First, we compared the fate of ¹⁴C-pyrene in SFU403 and a non-pyrene-oxidizing fungus, a Paecilomyces sp. After 7 days of incubation, more than 80% of the radioactivity was cell-associated in both fungi; however, while 90% of the ¹⁴C could be extracted from the Paecilomyces sp. as unmetabolized pyrene, 65–80% of the bound radioactivity remained inextractable from SFU403. Further evidence that pyrene oxidation to PQs was required for irreversible binding was obtained by comparing the extent of ¹⁴C bound to SFU403 when it was grown for 21 days under conditions that resulted in differing amounts of ¹⁴C-pyrene oxidation. The results showed that 40% of the inextractable products were bound residues derived from pyrene metabolites. The balance (60%) could be attributed to strong sorption of unreacted pyrene. We used electron paramagnetic resonance spectroscopy and oxygen consumption studies to demonstrate that both NADPH and glutathione can reduce PQs by one electron to their corresponding semiquinone anion radicals in vitro. These studies demonstrate that PQs are metabolized by SFU403 to bound residues, possibly via semiquinone intermediates.     

微紫青霉菌Penicillium janthinellum菌株GXCR吸附废水中Cd2+的研究

研究了5种不同预处理方式对丝状真菌微紫青霉菌Penicillium janthinellum菌株 GXCR的Cd2+吸附的影响。结果表明,高温（80℃）、去离子水中的匀浆化、匀浆+碱化（NaOH,0.5mol/L）（简称匀浆碱化）和匀浆+30%二甲基亚砜处理均能提高菌体的吸附率,其中匀浆碱化处理后菌体的吸附效果最佳,吸附增量达到117.96%;匀浆+酸化（H2SO4, 0.5mol/L）处理则导致菌体的Cd2+吸附能力显著下降。匀浆碱化菌体吸附符合典型的Langmuir方程,表明该菌对Cd2+的吸附可能是以表面吸附为主的吸附行为。在吸附-解吸附循环4次后匀浆碱化菌体的Cd2+的吸附效率为58.01%。红外光谱分析显示匀浆碱化处理主要影响菌体表面分子的–OH和C=O基团,其中与Cd2+结合的主要基团是–OH。结果也表明,匀浆碱化菌体具有处理电镀废水的潜能。     

青蒿素在露水草毛状根中的生物转化

露水草毛状根培养系中加入青蒿素培养８ｄ后,青蒿素转化去氧青蒿素。根据光谱数据,对去氧青蒿素的结构进行了鉴定。结果表明,通过水草毛状根能将青蒿素进行选择性还原为去氧青蒿素。     

Biotransformation of phenolic compounds by the cultured cells of Catharanthus roseus

The cultured cells of Catharanthus roseus were able to convert 2-, 3-, and 4-hydroxybenzyl alcohols into their corresponding hydroxybenzyl-β- -glucopyranosides or β- -glucopyranosylbenzyl alcohols, and then convert 2- and 3-hydroxybenzyl-β- -glucopyranosides into primeverosides and vicianosides. Further, the C. roseus cells were capable of hydroxylation of 2-hydroxybenzoic acid to afford 2,5-dihydroxybenzoic acid and then glucosylation of the newly introduced phenolic hydroxyl group.     

基因工程菌对阿特拉津的生物转化及其影响因素

研究考察了基因工程菌转化阿特拉津的共代谢碳源、转化动力学和影响因素。结果表明,作为共代谢碳源,葡萄糖优于乙酸盐,碳源浓度对转化影响不大,对工程菌生长影响显著。阿特拉津比转化速率与工程菌初始密度无关,与阿特拉津初始浓度有关,用Monod方程拟合转化动力学,求得方程参数为V_(max)=0．168/h,Ks= 30．49mg/L。降低温度会显著降低阿特拉津比转化速率;偏碱性的条件下,阿特拉津转化率较高,酸性条件严重抑制阿特拉津转化;盐度在一定范围内不影响转化活性;Co~(2 )、Fe~(2 )、Fe~(3 )和Zn~(2 )促进阿特拉津转化,Mn~(2 )、Ni~(2 )和Cu~(2 )抑制阿特拉津转化。菌体细胞对阿特拉津的吸附和转化作用呈正相关关系。     

Biotransformation of dicarboxylic acid by immobilized Cryptococcus cells

Altering the cell permeability by treating Cryptococcus neoformans with 1% (v/v) hexane stimulated the yield of transformation of n-pentadecane to the corresponding dioic acid, tridecane 1,13-dicarboxylic acid (DC-15); however, the biotransformation process was inhibited by the elevated levels of DC-15. To avoid product inhibition, a continuous process with immobilized cells was performed, and the result showed that the yield of DC-15 production was increased up to fivefold as compared with the batch type of DC-15 production. To integrate the product recovery process with the biotransformation, Amberlite XAD-2 resin was used for adsorbing DC-15 and configured as an external in situ product recovery system. The continuous process described in this study is adaptable for large-scale production of DC-15.