One-step purification and immobilization of his-tagged rhamnosidase for naringin hydrolysis

α-l-Rhamnosidase (EC 3.2.1.40) is an enzyme that catalyzes the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. In this study, a His-tag was genetically attached to the rhamnosidase gene ramA from Clostridium stercorarium to facilitate its purification from Escherichia coli BL21 (DE3) cells containing the pET-21d/ramA plasmid. Immobilized metal-chelate affinity chromatography (IMAC) resulted in one-step purification of N-terminally His-tagged recombinant rhamnosidase (N-His-CsRamA) which was immobilized in Ca²⁺ alginate (3%) beads. The optimum pH levels of the free and immobilized recombinant rhamnosidase were found to be 6.0 and 7.5, and the optimum temperature 55 and 60 °C respectively. At 50 °C, the free enzyme was relatively stable and exhibited a less than 50% reduction in residual activity after 180 min of incubation. The free and immobilized enzymes achieved 76% and 67% hydrolysis of the naringin in Kinnow juice respectively. Immobilization of recombinant rhamnosidase enabled its reutilization up to 9 hydrolysis batches without an appreciable loss in activity. This result indicated that the His-tagged thermostable rhamnosidase could be prepared as described and may serve to illustrate an economical and commercially viable process for industrial application.

Modulation of the Pharmacological Activities of Secretory Phospholipase A2 from Crotalus durissus cascavella Induced by Naringin

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

天然产物及相关复方配伍的质谱及液相色谱研究

质谱技术以其灵敏度高、样品需求量少、快速准确的特点在生物大分子体系和天然产物的化学成分研究中起着极为重要的作用。特别是软电离质谱技术的发展，大大简化了化合物的测定过程，提高了分析研究速度，为分析植物及食物中的结构信息提供了快速便捷的方法。HPLC-ESI-MSn是90年代发展成熟的分析技术，它集液相色谱的高分离能力与质谱的高灵敏度和高专属性于一体，已成为包括药物微量杂质、药物降解产物、药代代谢动力学研究、组合化学合成产物高通量分析以及天然产物的化学筛选在内的现代药学研究领域最强有力的分析工具之一。本文首先采用电喷雾多级串联质谱技术，系统地研究了负离子条件下黄酮普元、黄酮醇普元以及二氢黄酮普元等化合物的特征质谱行为，并对中药黄芬中三种黄酮昔化合物进行了研究。利用这些特征碎片离子，可以简便快捷的区分结构类似的黄酮类化合物及其同分异构体。通过对黄酮C一昔类化合物的电喷雾串联质谱（ESI-MSn）研究，提出黄酮C-苷类化合物的特征碎裂规律，证明〔M-H-60］-，〔M-H-90］-，〔M-H-120〕-为黄酮C-苷类化合物的特征离子。为鉴定植物粗提物中黄酮类化合物奠定了基础。之后我们进一步采用高效液相色谱与电喷雾质谱联用技术，在线区分混合物中的黄酮类化合物的同分异构体。根据黄酮类化合物的电喷雾质谱规律以及化合物的色谱保留时间分析鉴定了黄答中的七种黄酮类化合物。建立了一种有效检测黄芩（Scutellariabaicalensis Georgi）中黄酮类化合物的快速灵敏的分析方法，为建立黄答药材的质量控制标准提供了借鉴。对于黄酮类化合物灵敏、快速和准确的分析方法的建立，不仅为黄酮类化合物的结构的快速鉴定提供了一定的依据，而且对生药的鉴别和制剂的质控起到重要的作用。利用电喷雾多级串联质谱技术，对化学结构相似的黄酮营、二氢黄酮营和黄酮醇昔类化合物进行了对比研究。并且首次利用高分辨质谱FTICR-MS及SORI-CID技术对naringin进行了质谱研究。利用其超高分辩率、准确质量确证了中性碎片丢失，进一步证明了我们对其碎裂机理的推断。之后，我们通过金属离子与黄酮昔溶液混合后，采用电喷雾串联质潜法进行测定。实验表明通过人为加入某些金属离子如Li+，Na+和K+，特别是Li＋可以提高黄酮昔类化合物分析灵敏度，从而提供较多结构信息。并且在串联质谱中表现不同的碎裂行为，为黄酮类化合物结构分析提供补充信息。研究表明结合〔M-H］-，〔M＋Na〕+，〔M＋Li〕+离子串联质潜提供的信息，能够有效的鉴定黄酮普的结构。本文通过电喷雾串联质谱（ESI-MSn）和液质联机技术（HPLC-ESI-MS／MS）研究了原小聚碱型生物碱。首次利用高分辨质谱FTICR-MS和SORI-CTD技术对原小桨碱型生物碱进行了高分辨质谱研究，提出了各种碎片离子的碎裂途径，并总结了原小聚碱型生物碱的质谱碎裂规律，为鉴定此类型的生物碱提供了依据。之后通过电喷雾串联质谱研究了四种生物碱化合物质谱行为。最后我们利用液质联机技术（HPLC-ESI-MSn），通过与标准品的液相保留时间对照及原小聚碱型生物碱的质谱碎裂规律，建立了分析鉴定常用中药中原小聚碱型生物碱的快捷方法。并通过选择离子监控技术（SIM）提高了对于同分异构体的分离鉴定。通过色谱UV吸收峰定量研究了这四种药用植物中小璧碱和巴马汀的含量，为临床应用提供一定的化学基础，为建立中药质量控制标准及植物分类学提供一定的依据。采用HPLC法考察了常见的黄连药对组合煎煮对黄连中小聚碱、巴马丁煎出量的影响。为制药工艺的优化奠定了基础。之后我们以中药理论为指导，按药物性味对改良半夏泻心汤中各药进行分组，利用高效液相色谱分析了不同配伍条件下小璧碱、巴马丁煎出量的变化。为组方的合理性和科学性提供了一定的化学物质基础依据。

Phenolic compounds, antioxidant and antibacterial activities of three Ericaceae from Algeria

Herbs of the Ericaceae family are commonly found in Algeria and used in traditional medicine as anti- septic, diuretic, astringent, depurative, and to treat scalds and wounds. The methanolic extracts of three species, Arbutus unedo L. (A. unedo, leaves), Erica arborea L. (E. arborea, flowered aerial parts), and Erica multiflora L. (E. multiflora, flowered aerial parts), were compared regarding their content in pheno- lic compounds, their antioxidant, and antibacterial activities. A. unedo harbors the highest content in total phenolics and flavonoids, followed by E. arborea E. multiflora. The contents in total phenolics and flavonoids showed a correlation with the measured antioxidant (hydrogen-donating) activities; this was particularly the case for flavonoids content. The A. unedo extract showed antibacterial activity against all the tested strains (Staphylococcus aureus ATCC 6538, S. aureus C100459, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 9027); however, the E. arborea and E. multiflora extracts showed antibacterial activity only against Gram positive bacteria. Some polyphenols were identified in the three herbs by thin-layer chromatography and high-performance liquid chromatography coupled with diode array and mass spectrometry detection; from these, caffeic acid, p-coumaric acid, naringin, quercetin and kaempferol are reported for the first time in E. multiflora.

Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products

The metabolism of chlorogenic acid., naringin, and rutin, representative members of three common families of dietary polyphenols, the hydroxycinnamates, the flavanones, and the flavonols, respectively, was studied in an in vitro mixed culture model of the human colonic microflora. Time- and concentration-dependent degradation of all three compounds was observed, which was associated with the following metabolic events after cleavage of the ester or glycosidic bond: reduction of the aliphatic double bond of the resulting hydroxycinnamate caffeic acid residue; dehydroxylation and ring fission of the heterocyclic C-ring of the resulting deglycosylated flavanone, naringenin, and of the deglycosylated flavonol, quercetin (which differed depending on the substitution). The metabolic events, their sequences, and major phenolic end products, as identified by GC-MS or LC-MS/MS, were elucidated from the structural characteristics of the investigated compounds. The major phenolic end products identified were 3-D-hydroxyphenyl)propionic acid for chlorogenic acid, 3-(4-hydroxyphenyl)-propionic acid and 3-phenylpropionic acid for naringin, and 3-hydroxyphenylacetic acid and 3-(3-hydroxyphenyl)-propionic acid for rutin. The degree of degradation of the compounds studied was significantly influenced by the substrate concentration as well as individual variations in the composition of the fecal flora. The results support extensive metabolism of dietary polyphenols in the colon, depending on substrate concentration and residence time, with resultant formation of simple phenolics, which can be considered biomarkers of colonic metabolism if subsequently absorbed. It is also apparent that a relatively small number of phenolic degradation products are formed in the colon from the diverse group of natural polyphenols. (C) 2003 Elsevier Inc. All rights reserved.

Immobilized enzyme technology for debittering citrus fruit juices

There has been increased interest in the use of immobilized enzymes in fruit juice industry for debittering of citrus fruit juices due to their high efficiency to remove bitter flavonoids. The structure of naringin, responsible for immediate bitterness, and of limonin, responsible for "delayed bitterness" has been discussed. This chapter also discusses various attempts that have been made to immobilize enzymes on an appropriate support so as to enable their use in debittering of citrus fruit juices. These include physicochemical and enzyme biotechnological approaches which makes the fruit juice more acceptable and cost effective to the consumer. Despite of high volume of production of citrus fruits and fruit juices, suitable processes to produce non-bitter citrus juice by immobilized enzymes technology has not yet commercialized globally.

Covalent immobilization of naringinase for the transformation of a flavonoid

Naringinase (EC 3.2.1.40) from Penicillium sp was immobilized by covalent binding to woodchips to improve its catalytic activity. The immobilization of naringinase on glutaraldehyde-coated woodchips (600 mg woodchips, 10 U naringinase, 45 °C, pH 4.0 and 12h) through 1% glutaraldehyde cross-linking was optimized. The pH-activity curve of the immobilized enzyme shifted toward a lower pH compared with that of the soluble enzyme. The immobilization caused a marked increase in thermal stability of the enzyme. The immobilized naringinase was stable during storage at 4 °C. No loss of activity was observed when the immobilized enzyme was used for seven consecutive cycles of operations. The efficiency of immobilization was 120%, while soluble naringinase afforded 82% efficacy for the hydrolysis of standard naringin under optimal conditions. Its applicability for debittering kinnow mandarin juice afforded 76% debittering efficiency. 

Purification and characterization of naringinase from a newly isolated strain of Aspergillus niger 1344 for the transformation of flavonoids

An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg2+, SDS, p-chloromercuribenzoate, Cu2+ and Mn2+ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca2+, Co2+ and Mg2+ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg2+ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.

Optimization of process parameters for the production of naringinase by Aspergillus niger MTCC 1344

Aspergillus niger MTCC 1344 was used to produce extracellular naringinase in a complex (molasses, yeast extract and salts) medium. An initial medium pH 4.5 and cultivation temperature 30 °C were optimal for enzyme production. Among various carbon and organic nitrogen sources used, molasses and peptone were the most effective for enzyme yield. The rate of enzyme production was enhanced when metal ions were added to the medium. Fermentation conditions are described which produced a higher rate of enzyme synthesis. An increase in initial sugar concentration from 6 to 10 g l⁻¹ in the fermentation medium produced decreased naringinase synthesis while cell mass growth increased with the increase of sugar concentration. At a higher sugar level (10 g l⁻¹) the production of cell mass decreased.

Response surface optimisation of medium components for naringinase production from staphylococcus xylosus MAK 2

Response surface methodology was used to optimize the fermentation medium for enhancing naringinase production by Staphylococcus xylosus. The first step of this process involved the individual adjustment and optimization of various medium components at shake flask level. Sources of carbon (sucrose) and nitrogen (sodium nitrate), as well as an inducer (naringin) and pH levels were all found to be the important factors significantly affecting naringinase production. In the second step, a 22 full factorial central composite design was applied to determine the optimal levels of each of the significant variables. A second-order polynomial was derived by multiple regression analysis on the experimental data. Using this methodology, the optimum values for the critical components were obtained as follows: sucrose, 10.0%; sodium nitrate, 10.0%; pH 5.6; biomass concentration, 1.58%; and naringin, 0.50% (w/v), respectively. Under optimal conditions, the experimental naringinase production was 8.45 U/mL. The determination coefficients (R ²) were 0.9908 and 0.9950 for naringinase activity and biomass production, respectively, indicating an adequate degree of reliability in the model.

Molecular identification of Staphylococcus xylosus MAK2, a new α-l-rhamnosidase producer

A bacterial strain, MAK-2, was isolated as a producer of α-l-rhamnosidase from a soil sample of Dehradoon, India. The strain was identified based on morphology, physiological tests and 16S rDNA analysis. The phylogenetic analysis based on the 16S rDNA sequence, identified the isolate as Staphylococcus xylosus, a nonpathogenic member of CNS (coagulase-negative staphylococci) family. The strain was capable of producing α-l-rhamnosidase by hydrolysing flavonoids thus confirming potential application in the citrus-processing industry.

Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus MAK2 in a stirred tank reactor

Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca⁺² caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5-l reactor in 34 h. An increase in sugar concentration (15 gl^-1) in the fermentation medium further increased naringinase production (8.9 IUml^-1) in the bioreactor. Thus, availability of naringinase renders it attractive for potential biotechnological applications in citrus processing industry.