海洋真菌Xylaria sp.(No.2508)生产xyloketal B的发酵优化

对海洋真菌Xylariasp．（No．2508）生产xyloketa]B的摇瓶发酵过程进行了初步优化。结果表明,Xylariasp．（No．2508）在培养120h时xyloketalB产量最高,当培养168h后菌体进入衰亡期且xyloketalB迅速降解,因此Xylariasp．（No．2508）生产xyloketalB的最适发酵周期为120h;Xylariasp．（No．2508）合成xyloketalB的过程及用乙酸乙酯萃取xyloketalB的过程对pH非常敏感,其中最适xyloketalB生产的发酵液初始pH为5．7,利用乙酸乙酯萃取xyloketalB时的发酵液最适pH为3。0;最适接种量为10％（v／v）;接种前发酵液中加入10g／L的大孔树脂XAD-16可以降低某些代谢产物对xyloketalB合成的抑制作用,从而使产量提高2倍左右,而菌体进入稳定期以后加入大孔树脂XAD-16对产物产量没有影响。     

Procedure for isolation of Thienamycin from fermentation broths

An improved process for the isolation of thienamycin, produced by the actinomycete Streptomyces cattleya, has been developed. The isolation procedure consists of three chromatographic steps, volume reduction by reverse osmosis between the steps, and freezedrying for obtaining the final product. The chromatographic steps are as follows: (1) ion exchange chromatography on Dowex 1 × 2 resin in the bicarbonate cycle, (2) gel chromatography on Dowex 1 × 2 resin in the chloride cycle, (3) reverse phase chromatography on XAD-2 resin. This procedure is useful for processing large volumes of fermentation broth.     

Selective adsorption of plant products

The results from this study demonstrate that neutral resins can selectively concentrate specific indole alkaloids from dilute aqueous mixtures. Adsorption was observed to provide a one to two order of magnitude improvement in concentrating these alkaloids, as compared to an equivalent single-staged extraction. Since the sorption correlates with the concentration of the neutral form of the dissolved alkaloid, the adsorption is dependent on both the pH of the medium and the pKa of the alkaloids. Also, adsorption is dependent on specific sorbent-sorbate characteristics. In this study, by exploiting differences in the acid-base properties and the sorption characteristics of specific indole alkaloids, separation factors of 20-30 were observed. Although this behavior is valuable for analytical separations, the present study considers the application to the primary recovery of alkaloids from plant cell processes. Throughout this study it was also observed that the polycarboxylic ester resin (XAD-7) behaved quite differently from the styrene divinylbenzene resin (XAD-4). Despite a lower capacity, the XAD-7 resin was considerably more selective in adsorbing indole alkaloids than the XAD-4 resin. These alkaloids could also be desorbed from the XAD-7 resin by acidifying the liquid, while organic solvents were required to desorb these compounds from the XAD-4 resin.     

Structures of trichovirins II, peptaibol antibiotics from the mold Trichoderma viride NRRL 5243.

From the culture broth of the mold Trichoderma viride NRRL 5243 a mixture of polypeptides, named trichovirins (TV), could be isolated and purified by chromatography on XAD-2 adsorber resin and Sephadex LH-20 gel. Chromatography on silica gel using chloroform/methanol 8:2 as eluent provided a mixture of peptides named TV I. Subsequent elution with chloroform/methanol 1:1 yielded a second group of peptides named TV II. That group could be separated into individual components by repetitive HPLC on an octadecylsilyl and a fluorocarbon stationary phase. The sequences of 12 peptides of TV II could be determined by electrospray ionization tandem mass spectrometry of isolated peptides and gas chromatography-mass spectrometry of methanolysates. The N-termini of the 18-mer peptides are acetylated and the C-termini consist of leucinol. Owing to the presence of alpha-aminoisobutyric acid (Aib) residues and the bactericidal and hemolytic activity, the peptides belong to the family of peptaibol antibiotics.     

Use of a Novel Sub‐2 µm Silica Hydride Vancomycin Stationary Phase in Nano‐Liquid Chromatography. II. Separation of Derivatized Amino Acid Enantiomers

A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano‐liquid chromatography (nano‐LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns‐AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74–4.17 and 1.39–1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41–6.09 and α 1.28–2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min. Chirality 27:767–772, 2015. © 2015 Wiley Periodicals, Inc.     

Modelling the adsorption kinetics of erythromycin onto neutral and anionic resins

In this study the selective adsorption method was chosen to enable the recovery of erythromycin. The following sorbents were tested: neutral resins (XAD-4, XAD-7 and XAD-16) and an anionic resin (IRA-410). A mathematical kinetic model for the adsorption of erythromycin against time, on XAD-4, XAD-7 and XAD-16 resins, is proposed. Both Freundlich and Langmuir models showed a good fit for the sorbents XAD-7 and IRA-410 resins. The highest adsorption efficiency was observed when synthetic neutral resin, XAD-7 and XAD-16, were used. The estimated affinity and concentration factors show that the neutral resins tested are adequate for the selective adsorption of erythromycin. The estimated values of enthalpy and free energy of adsorption, lower than 12 kJ mol^–1 and –2 kJ mol^–1, respectively, indicate that a physiosorption process occurred.     

Separation and purification of four chromones from radix saposhnikoviae by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for the isolation and purification of 1'-O-glucosylcimifugin (1), 4'-O-beta-d-glucosyl-5-O-methylvisamminol (2), cimifugin (3) and 3'-O-glucosylhamaudol (4) from the Chinese medicinal herb radix saposhnikoviae has been successfully developed. A sample of 300 mg of crude extract was separated using ethyl acetate:n-butanol:1% aqueous acetic acid (1:4:5, v/v) as the two-phase solvent system and yielded 102.4 mg of 1 and 81.6 mg of 2. During this separation 3 and 4 remained in the stationary phase, which was collected, evaporated to dryness and separated with another two-phase solvent system involving ethyl acetate:n-butanol:1% aqueous acetic acid (5:0.5:5, v/v) to yield 31.4 mg of 3 and 12.7 mg of 4. The purities of compounds 1-4 were 98.4, 98.7, 99.3 and 98.2%, respectively, as determined by HPLC. The chemical structures of these components were established by (1)H-NMR and (13)C-NMR.     

Phenolics removal from transgenic Lemna minor extracts expressing mAb and impact on mAb production cost

Transgenic Lemna minor has been used successfully to produce several biotherapeutic proteins. For plant-produced mAbs specifically, the cost of protein A capture step is critical as the economic benefits of plant production systems could be erased if the downstream processing ends up being expensive. To avoid potential modification of mAb or fouling of expensive protein A resins, a rapid and efficient removal of phenolics from plant extracts is desirable. We identified major phenolics in Lemna extracts and evaluated their removal by adsorption to PVPP, XAD-4, IRA-402, and Q-Sepharose. Forms of apigenin, ferulic acid, and vitexin comprised ～ 75% of the total phenolics. Screening of the resins with pure ferulic acid and vitexin indicated that PVPP would not be efficient for phenolics removal. Analysis of the breakthrough fractions of phenolics adsorption to XAD-4, IRA-402, and Q-Sepharose showed differences in adsorption with pH and in the type of phenolics adsorbed. Superior dynamic binding capacities (DBC) were observed at pH 4.5 than at 7.5. To evaluate the cost impact of a phenolics removal step before protein A chromatography, a mAb purification process was simulated using SuperPro Designer 7.0. The economic analysis indicated that addition of a phenolics adsorption step would increase mAb production cost only 20% by using IRA-402 compared to 35% for XAD-4 resin. The cost of the adsorption step is offset by increasing the lifespan of protein A resin and a reduction of overall mAb production cost could be achieved by using a phenolics removal step.     

Enhanced vanillin production from recombinant E. coli using NTG mutagenesis and adsorbent resin

Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl-CoA synthase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5alpha harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin-resistant mutant of NTG-VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD-2 resin absorption. The vanillin production of NTG-VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild-type strain. When 50% (w/v) of XAD-2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG-VR1 was 2.9 g/L, which was 2-fold higher than that obtained with no use of the resin.     

Isolation and identification of S-adenosylmethionine from human urine

Procedures are described for the isolation and identification of adenosylmethionine from human urine. Previously described preliminary separative procedures using anion and cation exchange columns and an XAD-4 resin column have been extended to permit the separation of adenosylmethionine. The adenosylmethionine has been identified by conversion to methylthioadenosine followed by rechromatography of the latter compound with three different types of columns and elution systems. Mean adenosylmethionine values for urine were as follows: adults, 0.26; children, 0.36 nmole/mumole creatinine. Recovery of adenosylmethionine added to urine and determined by this separative procedure was 52%.     

Preparative isolation and purification of dicaffeoylquinic acids from the Ainsliaea fragrans champ by high-speed counter-current chromatography

Two dicaffeoylquinic acids, namely 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid, have been successfully separated by high-speed counter-current chromatography (HSCCC) from an extract of Ainsliaea fragrans Champ, followed by an initial clean-up step using AB-8 resin. A two-phase solvent system composed of chloroform:methanol:water (8:8:4) was selected for the isolation with the aqueous-rich phase as the stationary phase and the organic-rich phase as the mobile phase. The developed HSCCC method yielded 34 mg of 3,5-dicaffeoylquinic acid and 17 mg of 4,5-dicaffeoylquinic acid from 150 mg of the crude sample in a one-step separation with purities of 98 and 95%, respectively, as determined by HPLC. The structures of the two compounds were identified from ESI/MS, (1)H- and (13)C-NMR spectroscopic data.     

Preparative liquid chromatography of 1:1 adducts derived from the reaction of malondialdehyde with amino acids

Purification of 1:1 adducts (enaminals) formed from the reaction of methyl esters of amino acids with either malondialdehyde or methylmalondialdehyde was accomplished by preparative liquid chromatography on the stationary phase Amberlite XAD-4. The enaminal was easily and rapidly separated from the by-products and starting materials by a EtOH-H₂O mobile phase and then easily and rapidly recovered from the EtOH-H₂O at good yield and high purity. Depending on column diameter, up to 50 mg of enaminal could be purified in one chromatographic step. Adducts derived from l-Arg, l-His, l-Tyr, and l-Cys were purified.     

Detection of mutagenic activity in urine samples using a new concentration procedure

A study performed with cyclophosphamide (CP) and nor-nitrogen mustard (NNM), one of its main urinary metabolites, has shown that separation on Polygosil C-18 resin is preferable to one on XAD-2 resin as a means of concentrating the mutagenic activity present in urine of rats given cytostatics such as CP. Mutagenic activity was detected, using Salmonella typhimurium tester strain TA1535. While NNM is irreversibly bound to XAD-2 resin, it can be recovered after elution with methanol on Polygosil C-18. The better efficacity of Polygosil C-18 in concentrating CP and its metabolite(s) was confirmed with experiments with urine of rats treated with increasing doses of CP.     

Permeabilization and in situ adsorption studies during growth and coumarin production in hairy root cultures of Cichorium intybus L

Effect of addition of a permeabilizing agent dimethyl sulfoxide (DMSO) and a solid adsorbent, XAD -7, on growth and coumarin production in hairy root cultures of C. intybus was studied. Continuous permeabilization of the hairy root cultures of C. intybus with DMSO has been shown to be an effective strategy for enhanced release of coumarins while preserving the root viability. DMSO at 0.2% (v/v) level showed the maximum growth and coumarin production but was less as compared to control on day 28. Treatment of cells with increasing concentrations of DMSO (0.3 - 0.6 % v/v) to hairy root cultures of C. intybus, showed an inverse relationship with growth and coumarin production. Growth and production of coumarins increased with 1% media filtrate (MF) of cultures of Phytopthora parasitica var. nicotiana treatment. It was observed that treatment with DMSO (0.2% v/v) and 1% MF of P. parasitica showed the better growth and coumarin production with an increased release of coumarins as compared to the control and other treatments. It was observed that treatment of hairy root cultures with XAD-7 resulted in lesser growth and coumarin production as compared to control during the culture period. Addition of XAD-7 along with 1% MF of P. parasitica showed enhanced growth, coumarin production and increased adsorption as compared to control and lone XAD-7 treatment. Combined addition of DMSO/XAD-7 with fungal elicitor showed synergistic response in terms of biomass and coumarin production. Excretion of coumarins in both the cases was dependent on the presence of DMSO/XAD-7. These results showed that continuous permeabilization of hairy root cultures of C. intybus by using DMSO at 0.2% (v/v) level coupled with 1% MF of P. parasitica maintained viability of tissues and produced coumarins at higher level.     

Solid phase peptide synthesis of 15N-gramicidins A, B, and C and high performance liquid chromatographic purification

Four single-site 15N-labeled molecules of gramicidin have been synthesized using the 9-fluorenylmethoxycarbonyl method of solid phase peptide synthesis. Formylvaline was coupled as the N-terminal amino acid, and the peptide was cleaved from the resin with ethanolamine. Each synthesized gramicidin was purified in one step by semipreparative reverse phase high performance liquid chromatography and obtained in overall yields as high as 86%. The peptide was characterized by comparison with natural gramicidin using amino acid analysis, u.v. spectroscopy, and analytical high performance liquid chromatography.     

Enhanced anthraquinones production from adsorbent-treated Morinda elliptica cell suspension cultures in production medium strategy

Continuous removal of anthraquinones (AQ) by Amberlite polymeric adsorbents (XAD-4, XAD-7 and XAD-16) through in situ adsorption in Morinda elliptica cell suspension cultures is studied for product recovery and improvement of the overall titre. Ethanol was the best eluting solvent for effective recovery of AQ from all adsorbents. Pre-treatment of XAD-4 with sodium acetate not only enhanced intracellular AQ, but also AQ release and subsequent recovery from the adsorbent. The addition of sodium acetate pretreated XAD-4 on day 18 for 6-day contact period, achieved comparable cell growth to control (41 g/L), but with 1.3-fold higher intracellular AQ (124 mg/g DW) and two-fold increase in extracellular AQ (14.3 mg/L). High amount of adsorbent and longer contact period for the cultures entering stationary growth phase, stimulated AQ release and recovery but at the expense of cell growth. With 5–8.3 g XAD-4 adsorbent per litre M. elliptica culture in production (P) medium, between 60 and 90% AQ was recovered from extracellular AQ after 24–26 days of culture period.     

Fmoc-Asn(Trt)-OH与Wang树脂的合成研究

研究了Fmoc-Asn(Trt)-OH与Wang树脂的酯化反应工艺。探讨了反应策略、溶剂体系、投料比、反应时间以及反应温度等条件对手工合成Fmoc-Asn(Trt)-Wang树脂反应的影响。并用微波辅助方法与常规方法进行了对比。实验结果表明了采用HBTU/HOBt/DIEA方法的连接效率最高。对反应的优化结果为:NMP/DCM体积比为1:7,体系、摩尔比为3:1,每次反应时间4 h,温度40℃。微波对本酯化反应影响不大。     

Enantioselective HPLC of potentially CNS-active acidic amino acids with a Cinchona carbamate based chiral stationary phase

A tert-butylcarbamoylquinine-based chiral stationary phase (Chiralpak QN-AX) has been employed for the enantiomer separation of underivatized chiral acidic amino acids, viz. 4-carboxyphenylalanine (4-CPHE), 1-aminoindan-1,5-dicarboxylic acid (AIDA), 2-(5-carboxy-3-methyl-2-thienyl)glycine (3-MATIDA), 2-(4-carboxy-5-methyl-2-thienyl)glycine (5-MATIDA), and 2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG). Some of the acidic amino acids have potential activity on the central nervous system and are thus of great interest. A stereoselective HPLC method that allows the baseline resolution of all the five test solutes has been developed. For that purpose the mobile phase composition (pH, organic modifier, and type) and flow rate were optimized. The final method makes use of mild elution conditions, namely methanol - 0.8 M ammonium acetate buffer (97.5:2.5; v/v) pH 5.5 which are also compatible with mass spectrometric detection.     

Synthesis of racemic and optically active glycidic ethers,precursors of liquid crystalline polyethers

A series of racemic and optically active oxiranes, bearing mesogenic groups, precursors of liquid crystalline polyethers, has been synthesized from epichlorohydrin or glycidol. The enantiomeric excess of the optically active oxiranes has been determined by chiral stationary phase HPLC. Compounds bearing 4-cyanobiphenyl mesogenic group exhibit monotropic liquid crystalline behavior. A transfer of chirality to the mesophase has been observed for the optically active oxiranes, which present a cholesteric phase. Chirality 10:779–785, 1998. © 1998 Wiley-Liss, Inc.     

A spin label study of immobilized enzyme spectral subpopulations

Electron spin resonance (ESR) spin label studies have been carried out to examine the active site conformation of alpha-chymotrypsin before and after immobilization on two types of organic polymer supports: Amberlite XAD-8 and XAD-2. alpha-Chymotryspin was first chemically modified by reaction with methyl-4-phenylbutyrimidate and then inhibited by the active site spin label 4-(2,2,6,6-tetramethyl-piperdine-1-oxyl)-m-flurosulfonylbenzamide. In general, the ESR spectra of the active site lable revealed no significant changes in conformation for most of the enzyme before or after derivatization. On the other hand, two spectral subpopulations (A and B) of spin-labeled enzyme were characterized on the basis of their ESR spectra after immobilization on Amberlite XAD-8. Spectral subpopulation A (distinguished by a highly restrained spectrum) appeared to retain its active site structure and conformation and represented a large majority of the labeled chymotrypsin on the beads. Its presence correlated with the high activity and stability of phenylbutyramidinated chymotryspin on the Amberlite XAD-8 beads. Spectral subpopulation B (distinguished by a very weakly constrained spectrum) appeared to reflect loosely bound or denatured enzyme which was removable upon washing with 40% (v/v) ethylene glycol. Two methods for examining solvent accessibility to the active site lable of the kinetics of ascorbate reduction suggested that both spectral subpopulations had identical accessibilities to the bulk solvent. Paramagnetic broadening of the signal by K(3)Fe(CN)(6) revealed differences in the spin-spin broadening of the A and B components but is deemed and inappropriate indicator of solvent accessibility.