Insight into permeability of protein through microcapsule membranes

The objective of this paper is to analyze the characteristics of protein permeability in alainate-polylysine-alginate (APA) and alginate-chitosan-alginate (ACA) microcapsules by mathematical models based on the balance of chemical potential. The comparison between calculated results and experimental data shows that the models can describe the process of protein diffusion from microcapsule and protein release into microcapsule successfully. The influences of membrane composition on the permeability of ACA microcapsule have been investigated and analyzed. The effect of resistance on the mass transfer is further analyzed theoretically with the aid of mathematical modeling. (c) 2005 Elsevier B.V. All rights reserved.

中药蜘蛛香的化学成分研究及以羧甲基魔芋葡苷聚糖-壳聚糖为细胞膜的天冬酰胺酶人工细胞研究

本论文由三章组成。第一章介绍了中药蜘蛛香的化学成分的研究成果，第二章为羧甲基魔芋葡苷聚糖-壳聚糖为细胞膜的天冬酰胺酶人工细胞的研究，第三章综述了人工细胞在生物医学领域的应用。 第一章报道了中药蜘蛛香（Valeriana wallichii）根部乙醇提取物的化学成分，采用正、反相硅胶层析等分离方法和MS、NMR等多种波谱手段，从中共分离鉴定出17个化合物，分别为缬草素(valtrate，1)，valechlorine（2），homobadrinal（3），baldrinal（4），乙酰缬草素(acevaltrate 5)，valeriotetrate C（6），valeriotetrate B（7），对羟基苯乙酮(4'-hydroxy-acetophenone 8)，7-hydroxy valtrate（9），8-methylvalepotriate（10），1,5-dihydroxy-3,8-epoxyvalechlorine A（11），二氢缬草素(didrovaltrate 12)，胡萝卜苷（13），橙皮苷 (hesperidin 14)，prinsepiol-4-O-β-D-glucopyranoside（15），longiflorone（16），乙基糖苷（17）。其中化合物6、7、10、和11为新化合物，化合物9、15、16为首次从该植物中得到。新化合物11为含有氯原子的刚性骨架环烯醚萜，并且确定了其绝对构型。 第二章报道了以羧甲基魔芋葡苷聚糖（CKGM）和壳聚糖（CS）为膜的固定化L-天冬酰胺酶人工细胞研究成果。利用羧甲基魔芋葡苷聚糖和壳聚糖两种生物相容性很好的天然多糖之间的静电吸引力，在非常温和的条件下制备成具有半透过性膜的人工细胞，将治疗儿童急性成淋巴细胞性白血病(ALL)的药物L-天冬酰胺酶包裹在内。通过考察温度和pH对人工细胞的影响,结果表明以CKGM- CS为膜的L-天冬酰胺酶人工细胞对温度和pH的稳定性和耐受性均高于自由酶，说明CKGM-CS对酶具有保护作用，而且小分子底物和产物可以自由进出膜内外，而包裹在膜内的生物大分子则不能泄露出来。 第三章综述了微囊化人工细胞的研究进展。 This dissertation consists of three parts. In the first part, the chemical constituents from the root of Valeriana wallichii were reported. In the second part, preparation and characteristics of L-Asparaginase Artificial cell were reported. The third part is a review on progress of microcapsule artificial cell. The first chapter is about the isolation and identification of the chemical constituents from the root of V. wallichii. Seventeen compounds were isolated from the ethanol extract of roots of V. wallichii through repeated column chromatography on normal and reversed phase silica gel. By the spectroscopic and chemical evidence, their structures were elucidated as valtrate (1), valechlorine (2), homobadrinal (3), baldrinal (4), acevaltrate (5), valeriotetrate C (6), valeriotetrate B (7), 4'-hydroxy-acetophenone (8), 7-hydroxy valtrate (9), 8-methylvalepotriate (10), 1,5-dihydroxy-3,8-epoxyvalechlorine A (11), didrovaltrate (12), daucosterol (13), hesperidin (14), prinsepiol-4-O-β-D-glucopyranoside (15), longiflorone (16), and ethyl glucoside (17). Among them, 6, 7, 10, and 11 are new compounds. 15, 16 and 9 were isolated from this plant for the first time. The absolute configuration of compound 11, an unusual iridoid bearing a C-10 chlor-group and an oxo-bridge connecting C-3 and C-8 resulting in a rigid skeleton, was confirmed. The second chapter is about the semi-permeable microcapsule of carboxymethyl konjac glucomannan-chitosan for L-asparaginase immobilization. Carboxymethyl konjac glucomannan-chitosan (CKGM-CS) microcapsules, which have good biocompatibility, prepared under very mild conditions via polyelectrostatic complexation, were used for immobilize L-asparaginase-a kind of drug for acute lymphoblastic leukemia (ALL). The activity and stability under different temperature and pH of the enzyme loaded-microcapsules were studied. The results indicated the immobilized enzyme has better stability and activity contrasting to the native enzyme. The study illustrates that the L-asparaginase could be protected in CKGM-CS microcapsules, the substrate and product could pass through the system freely.

微生物酶法拆分消旋芳香族氨基酸的研究

通过单因子和多因子摇瓶正交试验，确定了米曲霉液态发酵产氨基酰化酶的最佳发酵条件。优化发酵培养基组成（ρ/g L-1）： 葡萄糖40,蔗糖10,可溶性淀粉20,蛋白胨2.5,马铃薯液1 000mL, pH自然。培养基装量50mL/250mL三角瓶，接种量4%。培养温度30℃，转速100 rmin-1，发酵时间42h。每50mL培养物的总酶活由优化前的2627U提高到7338U，是优化前的2.79倍。 研究了米曲霉氨基酰化酶的部分酶学性质，该酶催化反应的最适pH为7.0，最适温度为40℃，低浓度的Co2+（5×10-4mol/L）对酶活激活作用显著，催化反应过程中，底物浓度大于0.2 mol/L时，存在高浓度底物抑制酶活力现象。 初步探索了包埋法固定化米曲霉氨基酰化酶的载体，在实验的五种载体中，以海藻酸钠为载体包埋固定化米曲霉氨基酰化酶酶活保留率高，且操作简单，成本低廉。对包埋法固定化米曲霉氨基酰化酶酶学性质进行了研究，较游离米曲霉氨基酰化酶，最适温度未发生改变，最适pH向碱性范围偏移至8.0，对酸碱和热的稳定性增强，最适底物浓度增大到0.4 mol/L。 根据氨基酰化酶能立体专一水解L-氨基酰化物的特点，利用米曲霉氨基酰化酶对消旋苯丙氨酸进行了拆分。在米曲霉氨基酰化酶选择性的作用于底物N-乙酰-L-苯丙氨酸，得到L-苯丙氨酸后，通过732阳离子树脂和结晶法分别将L-苯丙氨酸和N-乙酰-D-苯丙氨酸分离，N-乙酰-D-苯丙氨酸通过酸水解脱去乙酰基得到D-苯丙氨酸，拆分得到光学纯度为98%的L-苯丙氨酸（收率84.8%）和光学纯度为92.3%的D-苯丙氨酸（收率89.5%）。 separate factors tests and orthogonal experiments,the optimum fermentation conditions of aminoacylase –producing Aspergillus oryzae were determined, as follows（ρ/g L-1）,glucose 40,sucrose 10,soluble starch 20,peptone 2.5，potato juice 1000ml, inoculation volume 4%and fermentation temperature 30℃,rotation speed 100rmin-1.The highest total enzyme activity ,7338μ,was obtained after fermentation for 42 h, increased by 279% compared with the original value of 2627μbefore optimization. We dicussed partial characteristics of aminoacylase. The optimal pH and temperature of aminoacylase were 7.0 and 40℃ respectively. Low- concentration Co2+ (5×10-4mol/L）activated the aminoacylase remarkably while high-concentration substrate lowered the aminoacylase . Five vectors has been used for immobolizing the enzyme and calcium alginate showed to be the best one for it had the slightest influence on the enzyme activity, easy to operate ,and low in price, comparing with other fours. The enzymatic charateristic study showed that its optimum temperature didn’t change, but the optimum pH and substrat concentration were higher after immobilization. The stability of immobolized enzyme to acid, alkaline and heat rised as well. The aminoacylse from Aspergillus oryzae was used to resolute racemic phenylalanine to obtain D-phenylalanine. After catalyzing process, we took two methods to separate D-phenylalanine .In end,L-phenylalanine was obtained with 98% optical purity in 84.8% yield, D-phenylalanine was obtained with 92.3% optical purity in 89.5% yield.

Preparation and characterization of microencapsulated hexadecane used for thermal energy storage

Polyurea microcapsules about 2.5 mum in diameter containing phase change material for thermal energy storage application were synthesized and characterized by interfacial polycondensation method with toluene-2,4-diisocyanate and ethylenediamine as monomers in an emulsion system. Hexadecane was used as a phase change material and OP, which is nonionic surfactant, and used as an emulsifier. The chemical structure and thermal behavior of the microcapsules were investigated by FTIR and thermal analysis respectively. The results show encapsulated hexadecane has a good potential as a solar energy storage material.

Microencapsulation of n-eicosane as energy storage material

For heat energy storage application, polyurea. microcapsules containing phase change material, n-eicosane, were synthesized by using interfacial polymerization method with toluene- 2,4-diisocyanate (TDI) and diethylenetriamine (DETA) as monomers in an emulsion system. Poly(ethylene glycol)octyl-phenyl ether (OP), a nonionic surfactant, was the emulsifier for the system. The experimental result indicates that TDI was reacted with DETA in a mass ratio of 3 to 1. FT-IR spectra confirm the formation of wall material, polyurea, from the two monomers, TDI and DETA. Encapsulation efficiency of n-eicosane is about 75%. Microcapsule of n-eicosane melts at a temperature close to that of n-eicosane, while its stored heat energy varies with core material n-eicosane when wall material fixed. Thermo-gravimetric analysis shows that core material n-eicosane, micro-n-eicosane and wall material polyurea can withstand temperatures up to 130, 170 and 250 degreesC, respectively.

Microencapsulation of n-hexadecane as a phase change material in polyurea

For thermal energy storage application, polyurea microcapsules about 2.5 mum in diameter containing phase change material were prepared using interfacial polycondensation method. In the system droplets in microns are first formed by emulsifying an organic phase consisting of a core material ( n-hexadecane) and an oil-soluble reactive monomer, toluene-2, 4-diisocyanate (TDI), in an aqueous phase. By adding water-soluble reactive monomer, diamine, monomers TDI and diamine react with each other at the interface of micelles to become a shell. Ethylenediamine (EDA), 1, 6-hexane diamine (HDA) and their mixture were employed as water-soluble reactive monomers. The effects of diamine type on chemical structure and thermal properties of the microcapsules were investigated by FT-IR and thermal analysis respectively. The infrared spectra indicate that polyurea microcapsules have been successfully synthesized; all the TG thermographs show microcapsules containing n-hexadecane can sustain high temperature about 300 degreesC without broken and the DSC measurements display that all samples possess a moderate heat of phase transition; thermal cyclic tests show that the encapsulated paraffin kept its energy storage capacity even after 50 cycles of operation. The results obtained from experiments show that the encapsulated n-hexadecane possesses a good potential as a thermal energy storage material.

固定化微生物降解土壤中菲和芘的研究

Immobilized with PVA,sodium alginate and activated carbon,both Zoogloea sp. and Fusarium sp.strains could degrade phenanthrene and pyrene efficiently.The optimal carrier was made of 100ρ·g -1 L PVA,5 sodium alginateρ·g -1 L and 50 activated carbon ρ·g -1 L.The degradation rates of phenanthrene and pyrene in 10 days were 87.48% and 75.34% by the immobilized bacterium,37.04% and 20.85% higher than those by the free bacterium,and the rates in 15 days were 84.36% and 74.87% by the immobilized fungus,5.35% and 11.23% higher than those by the free fungus.

Crystallization Behaviors of n-Octadecane in Confined Space: Crossover of Rotator Phase from Transient to Metastable Induced by Surface Freezing

In this paper, the confined crystallization and phase transition behaviors of n-octadecane in microcapsules with a diameter of about 3 Pm were studied with the combination of differential scanning calorimetry (DSC), temperature dependent Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD).

Construction of hollow DNA/PLL microcapsule as a dual carrier for controlled delivery of DNA and drug

DNA/poly-L-lysine (PLL) capsules were constructed through a layer-by-layer (LbL) self-assembly of DNA and PLL on CaCO3 microparticles, and then used as dual carriers for DNA and drug after dissolution of carbonate cores. The permeability of DNA/PLL microcapsules was investigated with fluorescence probes with different molecular weights by confocal microscopy. The result revealed that the fluorescence probes were able to penetrate the capsule walls even its molecular weight up to 150 kDa. The resultant capsules were used to load drug model molecules-fluorescein isothiocyanate (FITC)-dextran (4 kDa) via spontaneous deposition mechanism.

Engineering white light-emitting Eu-doped ZnO urchins by biopolymer-assisted hydrothermal method

With the presence of biopolymer-sodium alginate as additive, Eu-doped ZnO (zinc oxide) urchins consisting of nanorods were synthesized through a hydrothermal route. X-ray diffraction pattern makes evident the absence of phase other than wurtzite ZnO. Upon excited by 325 nm xenon laser, such nanostructured Eu-doped ZnO urchins emit white light, which originates from the luminescence of ZnO and the intra-4f transitions of Eu3+ ions. Besides acting as stabilizing agent, sodium alginate may also sensitize the Eu3+ ions in the nanostructures and facilitate the energy transfer from the host to Eu3+ ions. (c) 2006 American Institute of Physics.

Crystallization behaviors of n-nonadecane in confined space: Observation of metastable phase induced by surface freezing

Crystallization and phase transition behaviors of n-nonadecane in microcapsules with a diameter of about 5 mu m were studied with the combination of differential scanning calorimetry ( DSC) and synchrotron radiation X-ray diffraction ( XRD). As evident from the DSC measurement, a surface freezing monolayer, which is formed in the microcapsules before the bulk crystallization, induces a novel metastable rotator phase ( RII), which has not been reported anywhere else. We argue that the existence of the surface freezing monolayer decreases the nucleating potential barrier of the RII phase and induces its appearance, while the lower free energy in the confined geometry turns the transient RII phase to a " long- lived" metastable phase.

ZnO-based hollow microspheres: Biopolymer-assisted assemblies from ZnO nanorods

Many efforts have been made in fabricating three-dimensional (3D) ordered zinc oxide (ZnO) nanostructures due to their growing applications in separations, sensors, catalysis, bioscience, and photonics. Here, we developed a new synthetic route to 3D ZnO-based hollow microspheres by a facile solution-based method through a water-soluble biopolymer (sodium alginate) assisted assembly from ZnO nanorods. The products were characterized by X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy, selected area electron diffraction, and X-ray photoelectron spectroscopy. Raman and photoluminescence spectra of the ZnO-based hollow microspheres were obtained at room temperature to investigate their optical properties. The hollow microspheres exhibit exciting emission features with a wide band covering nearly all the visible region. The calculated CIE (Commission Internationale d'Eclairage) coordinates are 0.24 and 0.31, which fall at the edge of the white region (the 1931 CIE diagram). A possible growth mechanism of the 3D ZnO superstructures based on typical biopolymer-crystal interactions in aqueous solution is tentatively proposed, which might be really interesting because of the participation of the biopolymer.

Immunoprotective analysis of VhhP2, a Vibrio harveyi vaccine candidate

VhhP2 is an Outer membrane protein identified in a pathogenic Vibrio harveyi strain, T4, isolated from diseased fish. When used as a Subunit Vaccine, purified recombinant VhhP2 affords high level of protection upon Japanese flounder against V harveyi challenge. Vaccination with VhhP2 induced the expression of a number of immune-related genes, especially those encoding immunoglobulin M (IgM) and major histocompatibility complex (MHC) II alpha. A VhhP2 surface display system, in the form of the fish commensal strain FIR harboring the vhhP2-expressing plasmid pJVP, was constructed. PF3/pJVP is able to produce and present recombinant VhhP2 on cell surface. Vaccination of fish with live PF3/pJVP via intraperitoneal injection elicited Strong immunoprotection. Vaccination of fish orally with live PF3/pJVP embedded in alginate microspheres also induced effective immunoprotection. In addition, a VhhP2-based surface display system was created, in which VhhP2 serves as a carrier for the Surface delivery of a heterologous Edwardsiella tarda immunogen, Et18, that is fused in-frame to VhhP2. DH5 alpha/pJVP18, which expresses and surface-displays the VhhP2-Et18 chimera, proved to be an effective vaccine that call protect fish against infections by V. harveyi and E. tarda to the extents comparable to those produced by vaccination with purified recombinant VhhP2 and Et18, respectively. These data suggest that VhhP2 may be applied as a vaccine and a vaccine carrier against infections by V. harveyi and other pathogens such as F. tarda. (C) 2009 Elsevier Ltd. All rights reserved.

Algal biotechnology industries and research activities in China

In old China there were very few people engaged in the study of the algae, but in new China, freshwater and marine algae are studied by over one hundred old and new phycologists. There is now an algal biotechnology industry consisting of an aquaculture industry, producing large amounts of the seaweeds Laminaria, Porphyra, Undaria, Gracilaria, eucheumoids, and the microalgae Dunaliella and Spirulina. There is also a phycocolloid industry, producing algin, agar and carrageenan; an industry producing chemicals and drugs, such as iodine, mannitol, phycocyanin, beta -carotene, PSS (propylene glycol alginate sulfate) and FPS (fucose-containing sulfated polysaccharides) and an industry producing food, feed and fertilizer. The Laminaria cultivation industry produces about 900,000 t dry Laminaria, probably the largest producer in the world and 13,000 t algin, undoubtedly one of the largest algin producer in the world.

Genetic structure analysis of natural Sargassum muticum (Fucales, Phaeophyta) populations using RAPD and ISSR markers

Sargassum muticum is important in maintaining the structure and function of littoral ecosystems, and is used in aquaculture and alginate production, however, little is known about its population genetic attributes. In this study, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four populations of S. muticum and one outgroup of S. fusiforme (Harv.) Setchell from Shandong peninsula of China. The selected 24 RAPD primers and 19 ISSR primers amplified 164 loci and 122 loci, respectively. Estimates of genetic diversity with different indicators (P%, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon's information index) revealed low or moderate level of genetic variations within each S. muticum population, and a high level of genetic differentiations were determined with pairwise unbiased genetic distance (D) and fixation index (F-ST ) among the populations. The Mantel test showed that two types of matrices of D and F-ST were highly correlated whether from RAPD (r = 0.9706, P = 0.009) or ISSR data (r = 0.9161, P = 0.009). Analysis of molecular variance (AMOVA) was conducted to apportion the variations among and within the S. muticum populations. It indicated that variations among populations were higher than those within populations, being 55.82% verse 44.18% by RAPD and 55.21% verse 44.79% by ISSR, respectively. Furthermore, the Mantel test suggested that genetic differentiations among populations were related to the geographical distances (r > 0.6), namely, conformed to the IBD (isolation by distance) model, as expected from UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. On the whole, the high genetic structuring among the four S. muticum populations along the distant locations was clearly indicated in RAPD and ISSR analyses (r > 0.9, P < 0.05) in our study.