Biotransformation of capsaicin by Penicillium janthinellum AS 3.510

Biocatalysis of capsaicin (1) was performed by Penicillium janthinellum AS 3.510. Nine metabolites including four new compounds were afforded, and their structures were elucidated as (8S)-trans-8-hydroxy-8-hydroxymethyl-N-vanillyl-6-nonenamide (2), 6-hydroxy-8-methyl-N-vanillyl-7-nonenamide (3), trans-8-methoxy-8-methyl-N-vanillyl-6-nonenamide (4), 6-methoxy-8-methyl-N-vanillyl-7-nonenamide (5), dihydrocapsaicin (6), ω-1-hydroxydihydrocapsaicin (7), ω-1-hydroxycapsaicin (8), ω-hydroxycapsaicin (9), N-(4-hydroxy-3-methoxybenzyl)-5-[3-(propan-2-yl)oxiran-2-yl]pentanamide (10) by 1D and 2D NMR and HRESIMS spectra. The biotransformation processes include hydroxylation, methylation, reduction, and epoxylation.     

从蛹虫草小麦培养基残渣分离虫草素及提取物对肝癌细胞的抑制效果

从蛹虫草Cordyceps militaris小麦培养基残渣中分离和纯化虫草素,并进行了提取物体外人肝癌细胞Hep G2 (human hepatoma cell line,Hep G2)的抑制作用研究。以蛹虫草小麦培养基残渣为原料,热超声水提取虫草素,探究pH值、温度、浸提时间和超声功率对虫草素的提取效果,用苯乙烯型弱极性大孔吸附树脂(AB-8)柱层析分离、重结晶纯化虫草素,并用高效液相色谱法(high performance liquid chromatography,HPLC)、红外光谱法(infrared spectroscopy,IR)鉴定。虫草素处理肝癌细胞Hep G2后,倒置相差显微镜下观察肝癌细胞形态变化,并用四甲基偶氮唑蓝(methylthiazolyltetrazolium,MTT)法测定细胞生长情况。结果表明：固液比1:50、pH值6.0、温度60 ℃、浸提时间3 h和超声功率300 W时提取效果好。经鉴定,分离提取物的虫草素纯度达到99%以上。不同浓度的虫草素对肝癌细胞Hep G2有明显的抑制作用,且与虫草素浓度和作用时间呈正相关,被抑制的细胞凝集、变圆、碎裂增加。本提取工艺得到的虫草素纯度高,工艺简单易扩大,且提取物对肝癌细胞增殖作用具有明显的抑制作用,为其在功能性食品添加剂方向的应用提供了数据基础。     

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.     

Biotransformation of abietic acid by fungi and biological evaluation of its metabolites

Biotransformation of abietic acid was carried out initially using 28 different microbial strains. Among the evaluated, Mucor ramannianus produced a known metabolite 2α-hydroxy-dehydroabietic acid whereas Neurospora crassa yielded two known metabolites of 7β-hydroxy-dehydroabietic and 1β-hydroxy-dehydroabietic acids in 12.7, 15.5 and 20.1% yields, respectively. The in vitro antimicrobial activities of the metabolites were evaluated against 19 different pathogenic microorganisms, resulting in moderate inhibitory activity when compared to the standards, with MICs > 250 μg/mL. However, in the in vitro anticancer activity studies, 2α-hydroxy-dehydroabietic acid was found to be the most effective derivative against A549 human lung adenocarcinoma cell line with an IC₅₀ value of 320.8 μg/mL and SI (Selectivity index) of 156, respectively. Using the same assay and conditions, 7β-hydroxy-dehydroabietic was found to be the most effective and selective antiproliferative agent against HepG2 cell line with an IC₅₀ value of 196.6 μg/mL and SI of 187, respectively. Contrary to the antimicrobial activity, the biotransformation metabolites showed promising results suggesting selective toxicity against specific cancer cell line where the genotoxicity of the same derivatives were in a negligible range. Furthermore, DNA synthesis inhibition of metabolites were more promising in the A549 cell line while apoptotic effects were better in HepG2 cell line.     

Biotransformation of milbemycin D to milemycin G by Streptomyces lividans conferring avermectin O-methyltransferase activity

Avermectin O-methyltransferase gene was cloned from a cosmid clone covering the first module of polyketide synthase gene cluster of avermectin biosynthesis. Streptomyces lividans transformed with a DNA fragment containing avermectin O-methyltransferase gene efficiently convert milbemycin D to milbemycin G, indicating that biosynthetic genes of milbemycin and avermectin might complement each other. © Rapid Science Ltd. 1998     

Leukemia inhibitory factor inhibits the proliferation of gastric cancer by inducing G1-phase arrest

Leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family, plays a complex role in cancer. LIF inhibits the proliferation and survival of several myeloid leukemia cells but promotes tumor progression and metastasis in many solid tumors. However, the relationship between LIF and gastric cancer has not been well understood. LIF was downregulated in gastric cancer as detected by western blot analysis and immunohistochemistry (IHC). Notably, LIF was downregulated in approximately 70% (56/80) of primary gastric cancers, in which it was significantly associated with advanced clinical stage, lymph node metastasis, and poor overall survival (median 5-year survival = 26 vs. 43 months for patients with high LIF expression and low LIF expression gastric cancer, respectively). To study the potential function of LIF in the downregulation of gastric cancer, we monitored the behavior using proliferation, cell cycle, and flow cytometry analysis. Overexpression of LIF inhibited the gastric cancer cell cycle in the G1 phase. In our experiment, overexpression of LIF by lentivirus upregulated P21 and downregulated cyclin D1. Recombinant human LIF also downregulated P21 and cyclin D1 at various times. A further in vivo tumor formation study in nude mice indicated that overexpression of LIF in gastric cancer significantly delayed the progress of tumor formation. These findings indicate that LIF may serve as a negative regulator of gastric cancer.     

Biotransformation of isoeugenol to vanillin by a newly isolated Bacillus pumilus strain: identification of major metabolites

A bacterial strain S-1 capable of transforming isoeugenol to vanillin was isolated. The strain was identified as Bacillus pumilus based on biochemical tests, cellular fatty acid composition, riboprint pattern and 16S rRNA gene sequence analyses. In the biotransformation of isoeugenol, vanillin was the main product. With the growing culture of B. pumilus S-1, 10 g l⁻¹ isoeugenol was converted to 3.75 g l⁻¹ vanillin in 150 h, with a molar yield of 40.5% that is the highest up to now. Dehydrodiisoeugenol, a dimer of isoeugenol, was separated by preparative thin layer chromatography and identified by gas chromatography–mass spectrometry. Based on the accurate masses obtained from gas chromatography–high resolution mass spectrometry, two key intermediates, isoeugenol-epoxide (IE) and isoeugenol-diol (ID), were identified by mass spectra interpretations. The biotransformation with resting cells showed that vanillin was oxidized to vanillic acid and then to protocatechuic acid before the aromatic ring was broken. These findings suggest that isoeugenol is degraded through an epoxide-diol pathway.     

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.     

Comparative production of cellulases by mutants of Penicillium janthinellum NCIM 1171 and its application in hydrolysis of Avicel and cellulose

Mutants of Penicillium janthinellum NCIM 1171 were evaluated for cellulase production using both submerged fermentation (SmF) and solid state fermentation (SSF). Mutant EU2D-21 gave highest yields of cellulases in both SmF and SSF. Hydrolysis of Avicel and cellulose were compared using SmF and SSF derived enzyme preparations obtained from EU2D-21. Surprisingly, the use of SSF derived preparation gave less hydrolysis compared to SmF derived enzymes. This may be due to inactivation of β-glucosidase at 50 °C in SSF derived enzyme preparations. SmF derived enzyme preparations contained both thermostable and thermosensitive β-glucosidases where as SSF derived enzyme preparations contained predominantly thermosensitive β-glucosidase. This is the first report on less thermostability of SSF derived β-glucosidase which is the main reason for getting less hydrolysis.     

汉滩病毒囊膜糖蛋白G1、G2腺病毒载体的表达及免疫分析

为了研究利用腺病毒载体表达汉滩病毒囊膜糖蛋白G1、G2的可行性及免疫原性。通过克隆76-118株G1、G2基因至腺病毒表达载体pAdTrackCMV,得到阳性克隆padTrackCMV-G1、G2。PmeI线性化的阳性克隆与腺病毒骨架载体pAdeasy-1共转化BJ5183宿主菌,经同源重组后得到重组病毒rAdeasy-G1、rAdeasy-G2。重组病毒经PacI线性化后,脂质体介导转染293细胞,使重组病毒得到扩增。将重组病毒免疫Balb／c小鼠,并通过ELISA和间接免疫荧光对免疫小鼠血清进行了分析。结果表明,rAdeasy—G1组六只免疫小鼠、rAdeasy—G2组4只免疫小鼠均产生了能与汉滩病毒抗原发生反应的特异抗体。该研究为进一步研制以腺病毒为活载体的汉坦病毒工程疫苗奠定了基础。     

汉滩病毒囊膜糖蛋白G1、G2腺病毒载体的表达及免疫分析

为了研究利用腺病毒载体表达汉滩病毒囊膜糖蛋白G1、G2的可行性及免疫原性.通过克隆76-118株G1、G2基因至腺病毒表达载体pAdTrackCMV,得到阳性克隆pAdTrackCMV-G1、G2.PmeI线性化的阳性克隆与腺病毒骨架载体pAdeasy-1共转化BJ5183宿主菌,经同源重组后得到重组病毒rAdeasy-G1、rAdeasy-G2.重组病毒经PacI线性化后,脂质体介导转染293细胞,使重组病毒得到扩增.将重组病毒免疫Balb/c小鼠,并通过ELISA和间接免疫荧光对免疫小鼠血清进行了分析.结果表明,rAdeasy-G1组六只免疫小鼠、rAdeasy-G2组4只免疫小鼠均产生了能与汉滩病毒抗原发生反应的特异抗体.该研究为进一步研制以腺病毒为活载体的汉坦病毒工程疫苗奠定了基础.     

Direct targeting of sEH with alisol B alleviated the apoptosis,inflammation, and oxidative stress in cisplatin-induced acute kidney injury

Acute kidney injury (AKI) is a pathological condition characterized by a rapid decrease in glomerular filtration rate and nitrogenous waste accumulation during hemodynamic regulation. Alisol B, from Alisma orientale, displays anti-tumor, anti-complement, and anti-inflammatory effects. However, its effect and action mechanism on AKI is still unclear. Herein, alisol B significantly attenuated cisplatin (Cis)-induced renal tubular apoptosis through decreasing expressions levels of cleaved-caspase 3 and cleaved-PARP and the ratio of Bax/Bcl-2 depended on the p53 pathway. Alisol B also alleviated Cis-induced inflammatory response (e.g. the increase of ICAM-1, MCP-1, COX-2, iNOS, IL-6, and TNF-α) and oxidative stress (e.g. the decrease of SOD and GSH, the decrease of HO-1, GCLC, GCLM, and NQO-1) through the NF-κB and Nrf2 pathways. In a target fishing experiment, alisol B bound to soluble epoxide hydrolase (sEH) as a direct cellular target through the hydrogen bond with Gln384, which was further supported by inhibition kinetics and surface plasmon resonance (equilibrium dissociation constant, K_D = 1.32 μM). Notably, alisol B enhanced levels of epoxyeicosatrienoic acids and decreased levels of dihydroxyeicosatrienoic acids, indicating that alisol B reduced the sEH activity in vivo. In addition, sEH genetic deletion alleviated Cis-induced AKI and abolished the protective effect of alisol B in Cis-induced AKI as well. These findings indicated that alisol B targeted sEH to alleviate Cis-induced AKI via GSK3β-mediated p53, NF-κB, and Nrf2 signaling pathways and could be used as a potential therapeutic agent in the treatment of AKI.     

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.     

Biotransformation of the SDG in defatted flaxseed into END co-cultured by three single bacterial colonies

Enterodiol (END) possesses the estrogenic and antiestrogenic activities, which could prevent the development of breast cancer and prostate cancer, as well as menopausal syndrome.Previous studies in our laboratory set up a bio-transformation method for largely yielding secoisolariciresinol (SECO) from the substrate of defatted flaxseeds by strain Bacteroides uniformis ZL-I. In this research, another two single colonies, designated as strain ZL-II and strain ZL-III, were isolated, which were closely related to Eubacterium limosum species (ZL-II), and Eggerthella lenta species (ZL-III) on the basis of 16S rRNA gene sequence data. Under the combining actions of strains ZL-(I + II + III), END could be produced from defatted flaxseed directly, ZL-II was proved to possess the activities of demethylation, while ZL-III had the activities of dehydroxylation. Secoisolariciresinol diglucoside (SDG) existed in the form of oligomeric with 3-hydroxy-3-methyl glutaric acid in flaxseed could be efficiently transformed into END under the co-culture of strains ZL-(I + II + III), with the conversion rate of more than 90%. The method for mass-producing END from defatted flaxseed reported here is meaningful not only for the medicinal values of END but also for the resource utilization of flaxseed materials.     

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。     

Isolation,identification, and bioactivity of microbial metabolites of cyclopamine and its congeners

The biotransformation of cyclopamine (1) and its congeners (2, 3 and 4) by Cunninghamella echinulata (ACCC 30369) was investigated. The chemical structures of two new congeners (2 and 4) and nine new metabolites were elucidated by 1D NMR (¹H, ¹³C and DEPT), 2D NMR (COSY, HMBC, HSQC and NOESY) and HRESIMS analyses and further confirmed by X-ray diffraction studies. Among these compounds, 2 and 4 showed moderate cytotoxicity in HepG2 cells (2, IC₅₀ = 8.03 ± 1.92 μM) and A549 cells (4, IC₅₀ = 10.19 ± 2.18 μM). Conversely, the cytotoxicity of the nine metabolites was sharply reduced. Similar to 1, compound 4 induced a cyclopia phenotype in zebrafish embryos at 20 μM. Moreover, compound 4 was more stable than 1 in an acidic environment.     

Fungal transformation of dydrogesterone and inhibitory effect of its metabolites on the respiratory burst in human neutrophils

The biotransformation of the synthetic hormone dydrogesterone (1) by a number of fungal strains - including Cephalosporium aphidicola, Rhizopus stolonifer, Cunninghamella elegans, and Fusarium lini - afforded ten different metabolites, compounds 2-11. From a structural point of view, these transformations involved a combination of hydroxylation, oxidation, reduction, and/or epoxidation. The pregnane-based metabolites 10 and 11 are new compounds. All the known compounds were obtained for the first time from 1 by fungal transformation. The metabolites 3, 5, and 8 showed more-potent respiratory-burst inhibitory activity than the substrate 1 in a neutrophil-based cellular assay (Table 3).     

基因组步移法克隆小菜蛾CYP9G2基因的上游序列

利用4种产生平端切头的限制性内切酶消化小菜蛾(Plutella xylostella)的基因组DNA,然后利用DNA连接酶的催化作用,在4种不同平端切头的小菜蛾基因组DNA上连接一个氨基化的基因组步移衔接头序列,针对衔接头及已克隆的CYP9G2基因的序列,设计两对PCR上、下游引物,进行PCR扩增、T-A克隆和阳性克隆的巢式PCR验证,通过测序克隆到了小菜蛾CYP9G2基因上游未知序列约1.8 kb.通过对该基因的上游序列进行信息分析,发现1个可能的节肢动物动物转录起始子(Inr),3个CAAT样盒及1个抗氧化剂样反应因子,共5个可能的顺式调控元件.研究还表明,利用基因组步移方法可以快速地克隆已知序列的上游未知序列,实验操作经济、简便,对于已知cDNA序列或部分基因组序列的基因,其上游调控序列的克隆,基因组步移具有较高的实用价值.     

Biotransformation of terpenes by fungi: Study of the pathways involved

The biotransformation of the pure terpene alcohols geraniol and nerol, the mixture of the alcohols, ‘citrol’, and the mixture of the aldehydes, citral, to 6-methyl-5-hepten-2-one by sporulated surface cultures of Penicillium digitatum was compared. It was found that citral was converted faster than the alcohols but gave a lower overall yield of ≈76%, whereas the pure alcohols and their mixture, ‘citrol’, gave a yield of ≈83%. It was also established that the bioconversion over prolonged periods was possible with an overall yield of 80–90% depending on the substrate used. The bioconversion of nerol to 6-methyl-5-hepten-2-one by a spore suspension was also shown. The pathways involved in the biotransformation of geraniol and citral to 6-methyl-5-hepten-2-one are also discussed.     

Biotransformation of aflatoxin B1 and its conjugated metabolites by rat gastrointestinal microfloras

Rat cecal microflora from high- and low-fiber-fed animals hydrolyzed aflatoxin conjugates to metabolites indistinguishable from aflatoxin B1 and aflatoxin P1, but aflatoxicol was not a transformation product.