原人参二醇型皂苷水解酶及制备人参皂苷Compound K的研究进展

人参皂苷Compound K (CK)是一种具有抗癌抗炎等药理活性的化合物。目前在天然人参中暂未鉴定出,工业上主要通过原人参二醇型皂苷的去糖基化进行制备。相对于传统的物理、化学的去糖基化法,利用原人参二醇型皂苷水解酶制备CK具有特异性强、绿色环保和高效稳定的优点。本文根据水解酶作用的糖基连接碳原子的差异将原人参二醇型皂苷水解酶分成了3类,发现大多数能制备CK的水解酶为Ⅲ型原人参二醇型皂苷水解酶。此外,对水解酶在制备CK中的应用进行了总结评估,旨在为人参皂苷CK的大规模制备及其在食品和药品行业中的开发提供参考。     

一种真菌对人参皂苷Rg3的转化

[目的]筛选长白山人参土壤中的活性微生物,转化人参总皂苷及单体人参皂苷产生稀有抗肿瘤成份.[方法]从长白山人参根际土壤中分离各类菌株,对人参总皂苷及单体人参皂苷进行微生物转化,并通过硅胶柱层析等方法对转化产物进行分离纯化,采用波谱解析及理化常数对其进行结构鉴定;结合菌落形态、产孢结构、孢子形态特征以及菌株ITS rDNA核酸序列分析,对活性菌株进行鉴定.[结果]从长白山人参根际土壤中分离各类真菌菌株68株,有12株菌株对人参总皂苷有转化活性,其中菌株SYP2353对二醇组人参皂苷Rg3具有较强的转化活性.[结论]阳性菌株SYP2353被鉴定为疣孢漆斑菌(Myrothecium verrucaria),能将人参皂苷Rg3转化为稀有人参皂苷Rh2及二醇组人参皂苷苷元PPD,为稀有人参皂苷Rh2的制备提供了新的方法.     

酵母菌转化人参皂苷Rb1的动力学

在分批发酵中,对酵母菌转化人参皂苷的动力学进行了研究。应用Logistic方程和Luedeking-Piret方程,得到了描述分批发酵过程中,酵母菌体生长、底物消耗和产物合成的动力学模型及模型参数,并对实验数据与模型进行了验证比较,模型计算值与实验值拟合良好,所建模型能很好地描述酵母菌转化人参皂苷的动力学特征。为进一步研究和预测酵母菌生物转化过程奠定理论基础。     

Simple and efficient preparation of ginsenoside (S)-Rg2 from ginsenoside Re by biotransformation with Cellulosimicrobium sp. 21

A novel ginsenoside-hydrolyzing strain was isolated from ginseng-cultivation soil in Changbai Mountain (China). The strain was identified as Cellulosimicrobium sp. 21 by 16S rDNA sequencing. Using the β-glucosidases secreted from Cellulosimicrobium sp. 21, protopanaxatriol-type ginsenoside Re was converted to the highly active neuroprotective molecule (S)-Rg2 by removal of the C-20-glucopyranosyl residue. The α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranose at the C-6 position of Rg2 was not further attacked by Cellulosimicrobium sp. 21, so the transformation shows high specificity. To simplify the transformation and product-preparation process, a simple and efficient transformation system was developed in a phosphate buffer system instead of organic media. The optimum conditions for transforming ginsenoside Re into Rg2 by Cellulosimicrobium sp. 21 were determined through single-factor experiments and response surface methodology. Under the optimized conditions: transformation buffer, 50 mM phosphate buffer, at pH: 7.00; temperature: 27.6°C; substrate concentration: 0.50 mg/ml; biotransformation period: 12 h; the biotransformation efficiency reached 89.8% (molar ratio) in 2-L reaction system. This simple biotransformation with high specificity and efficiency has potential for use in Rg2 preparation in the pharmaceutical industry.     

人参皂苷Rb3糖基水解酶的纯化及其反应特性

人参皂苷Rb3是三七茎叶皂苷的主要成分。为了充分利用廉价的三七茎叶皂苷,该研究以微生物Aspergillus sp. P90r菌为对象,综合运用生物转化的方法,经过提取、分离纯化和酶活力测定等步骤,最终以确定酶反应途径的方式得到了所产的特异性人参皂苷Rb3糖基水解酶的相关性质和动力学等反应特性。结果表明:该酶比Absidia sp. GRB3-X8r菌产酶活力高15%~25%,SDS-PAGE电泳结果测得分子量约为65.6ku,纯化后酶蛋白的含量为0.237 mg·mL~(-1),蛋白比活力可达到169 U·mg~(-1),纯化倍数为13.70,回收率为9.39%。人参皂苷Rb3糖基水解酶在pH=5.0的偏酸性环境下酶活力很高,最适反应条件:pH=3.0~5.0,温度45℃,其中在pH=4.0~6.0范围内相对稳定。该酶在20 min时进入混合级反应,酶反应米氏常数Km值为8.77 mmol·L~(-1),V_(max)为57.44 mmol·L~(-1)·h~(-1),在60 min时反应速度达到最大,Vmax趋于稳定,为66.63mmol·L~(-1)·h~(-1)。通过对酶的催化特性研究表明,该酶先水解Rb3的20-O-木糖基,其次水解3-O-葡萄糖基,最终催化反应产物中有F2和C-K生成。综上结果,微生物Aspergillus sp. P90r菌酶具有能水解人参皂苷Rb3木糖基和葡萄糖基的特异性。     

专一转化人参二醇类皂苷Rb1为Rd的真菌菌株的筛选

于2009年的7－10月间在辽宁省的桓仁,吉林省的集安、靖宇、抚松等药材产区采集人参及人参根际土壤样品45份。通过真菌分离和培养,共获得真菌菌株105株,经形态学鉴定分属于15属48种。通过活性筛选,得到具有转化人参总皂苷活性的菌株25株,其中菌株SR87和SR105对人参皂苷Rb1具有专一转化活性。通过TLC和HPLC检测,其转化产物为人参皂苷Rd。经形态学鉴定,确定阳性菌株SR87为莫勒接霉Zygorhynchus moelleri,SR105为灰绿犁头霉Absidia glauca。这两株真菌均有较高的转化潜力,可以应用于制备人参皂苷Rd。     

The diversion of 2‐C‐methyl‐d‐erythritol‐2,4‐cyclodiphosphate from the 2‐C‐methyl‐d‐erythritol 4‐phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana

2‐C‐Methyl‐d ‐erythritol‐2,4‐cyclodiphosphate (MEcDP) is an intermediate of the plastid‐localized 2‐C‐methyl‐d ‐erythritol‐4‐phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co‐factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds‐3 mutant, defective in the 1‐hydroxy‐2‐methyl‐2‐(E)‐butenyl‐4‐diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2‐C‐methyl‐d ‐erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds‐3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds‐3 mutant also showed enhanced resistance to the phloem‐feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP‐mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds‐3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.     

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

CRISPR/Cas9 technology has revolutionized biology. This prokaryotic defense system against foreign DNA has been repurposed for genome editing in a broad range of cell tissues and organisms. Trypanosomatids are flagellated protozoa belonging to the order Kinetoplastida. Some of its most representative members cause important human diseases affecting millions of people worldwide, such as Chagas disease, sleeping sickness and different forms of leishmaniases. Trypanosomatid infections represent an enormous burden for public health and there are no effective treatments for most of the diseases they cause. Since the emergence of the CRISPR/Cas9 technology, the genetic manipulation of these parasites has notably improved. As a consequence, genome editing is now playing a key role in the functional study of proteins, in the characterization of metabolic pathways, in the validation of alternative targets for antiparasitic interventions, and in the study of parasite biology and pathogenesis. In this work we review the different strategies that have been used to adapt the CRISPR/Cas9 system to Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., as well as the research progress achieved using these approaches. Thereby, we will present the state‐of‐the‐art molecular tools available for genome editing in trypanosomatids to finally point out the future perspectives in the field.     

Glucose metabolism down‐regulates the uptake of 6‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐2‐deoxyglucose (6‐NBDG) mediated by glucose transporter 1 isoform (GLUT1): theory and simulations using the symmetric four‐state carrier model

The non‐metabolizable fluorescent glucose analogue 6‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐2‐deoxyglucose (6‐NBDG) is increasingly used to study cellular transport of glucose. Intracellular accumulation of exogenously applied 6‐NBDG is assumed to reflect concurrent gradient‐driven glucose uptake by glucose transporters (GLUTs). Here, theoretical considerations are provided that put this assumption into question. In particular, depending on the microscopic parameters of the carrier proteins, theory proves that changes in glucose transport can be accompanied by opposite changes in flow of 6‐NBDG. Simulations were carried out applying the symmetric four‐state carrier model on the GLUT1 isoform, which is the only isoform whose kinetic parameters are presently available. Results show that cellular 6‐NBDG uptake decreases with increasing rate of glucose utilization under core‐model conditions, supported by literature, namely where the transporter is assumed to work in regime of slow reorientation of the free‐carrier compared with the ligand–carrier complex. To observe an increase of 6‐NBDG uptake with increasing rate of glucose utilization, and thus interpret 6‐NBDG increase as surrogate of glucose uptake, the transporter must be assumed to operate in regime of slow ligand–carrier binding, a condition that is currently not supported by literature. Our findings suggest that the interpretation of data obtained with NBDG derivatives is presently ambiguous and should be cautious because the underlying transport kinetics are not adequately established.