微胶囊固定化酵母培养的研究*

进行了NaCS-PDMDAAC微胶囊固定化酒精酵母和产朊假丝酵母的实验研究。考察了这两种酵母的培养规律,发现微胶囊固定化酒精酵母的产酒精情况与游离培养基本一致,在连续发酵16批后,仍具有良好的性能。同时固定化产谷胱甘肽(GSH)的产朊假丝酵母的研究也表明固定化培养GSH产量与游离细胞产量相近。     

SA/CS-CaCl2/PMCG微胶囊的生物相容性研究

介绍了一种新型多组份生物微胶囊体系－SA／CS－CaCl2/PMCG微胶囊。考察了PMCG和SA／CS－CaCl2/PMCG微胶囊体系对大肠杆菌和酿酒酵母生长的影响,并用SA／CS－CaCl2/PMCG微胶囊进行了固定化培养大肠杆菌和酿酒酵母的研究。结果表明,与其它合成聚阳离子类似,PMCG组分对细胞生长有明显的抑制作用,但是在制胶囊过程中以及在用SA／CS－CaCl2/PMCG微胶囊对大肠杆菌和酿酒酵母培养过程中,都显示了良好的生物相容性,因此作为整个体系来说,该微胶囊可用于微生物细胞的固定化培养。     

SA/CS-CaCl2/PMCG微胶囊的生物相容性研究

介绍了一种新型多组份生物微胶囊体系——SA/CS-CaCl₂/PMCG微胶囊。考察了PMCG和SA/CS-CaCl²/PMCG微胶囊体系对大肠杆菌和酿酒酵母生长的影响,并用SA/CS－CaCl²/PMCG生物微胶囊进行了固定化培养大肠杆菌和酿酒酵母的研究。结果表明,与其它合成聚阳离子类似,PMCG组分对细胞生长有明显的抑制作用,但是在制胶囊过程中以及在用SA/CS-CaCl²/PMCG微胶囊对大肠杆菌和酿酒酵母培养过程中,都显示了良好的生物相容性,因此作为整个体系来说,该微胶囊可用于微生物细胞的固定化培养。       

综述: 胞外囊泡表面糖缀合物研究进展

胞外囊泡(extracellular vesicles,EVs)是一类由细胞分泌到胞外的能够被受体细胞摄取的膜性囊泡小体,直径在20～ 1 000 nm．近年来,越来越多的研究者发现胞外囊泡在疾病诊断、预后评估以及药物递送等方面具有重要的生物学作用．胞外囊泡可以直接参与细胞间信息的传递以及物质的运输,其携带的核酸(mRNA,microRNA和lncRNA)和蛋白质可以影响受体细胞的生理状态．大量研究表明,胞外囊泡是被糖基化修饰的,胞外囊泡表面覆盖了大量的聚糖以及糖结合蛋白,而已知聚糖类物质在调控细胞黏附、细胞-细胞之间的信息传递、细胞和细胞外基质相互作用、免疫调节和肿瘤转移等方面发挥重要的作用．本文综述了近年来细胞外囊泡表面糖缀合物修饰的前沿研究,以期更好地理解聚糖在胞外囊泡的合成、释放以及运输过程及其生物学功能中的作用．     

胞外囊泡表面糖缀合物研究进展

胞外囊泡(extracellular vesicles,EVs)是一类由细胞分泌到胞外的能够被受体细胞摄取的膜性囊泡小体,直径在20~1 000 nm.近年来,越来越多的研究者发现胞外囊泡在疾病诊断、预后评估以及药物递送等方面具有重要的生物学作用.胞外囊泡可以直接参与细胞间信息的传递以及物质的运输,其携带的核酸(m RNA,micro RNA和lnc RNA)和蛋白质可以影响受体细胞的生理状态.大量研究表明,胞外囊泡是被糖基化修饰的,胞外囊泡表面覆盖了大量的聚糖以及糖结合蛋白,而已知聚糖类物质在调控细胞黏附、细胞-细胞之间的信息传递、细胞和细胞外基质相互作用、免疫调节和肿瘤转移等方面发挥重要的作用.本文综述了近年来细胞外囊泡表面糖缀合物修饰的前沿研究,以期更好地理解聚糖在胞外囊泡的合成、释放以及运输过程及其生物学功能中的作用.     

SA/NaCS-PDMDAAC微胶囊固定化酵母细胞合成胞苷三磷酸

通过海藻酸钠/纤维素硫酸钠-聚二甲基二烯丙基氯化铵(SA/NaCS-PDMDAAC)微胶囊固定化酵母细胞将胞苷一磷酸(CMP)转化为胞苷三磷酸(CTP),考察了各种因素条件对CTP转化率的影响,以提高CTP的转化率.通过考察分批补料添加葡萄糖,固定化酵母量,CMP浓度等以达到提高CTP转化率的要求.结果在250 mL锥...     

革兰氏阳性菌细胞外囊泡的研究现状

细胞外囊泡(EVs),也称为膜小泡,是真核细胞和细菌分泌的囊泡状小体.它通过携带蛋白质、DNA、RNA和各种代谢物进行细胞间物质的交流传递.根据内容物的不同发挥不同的生理功能,如传递营养物质、参与免疫反应、治疗癌症等.目前大多数研究专注于真核细胞和革兰氏阴性菌囊泡的探索,而对革兰氏阳性菌中分泌的囊泡研究较少.这篇综述总...     

甜菊愈伤组织细胞中的液泡膜内突和液泡内囊泡

对生长在分化培养基上的甜菊愈伤组织分生区细胞的液泡膜内突和液泡内囊泡,进行了超微结构和酸性磷酸酶细胞化学研究。在不同液泡化时期的细胞中,都存在不同大小和形态的液泡膜内突,它们有的缺乏明显的内含物;有的含有许多小泡或复杂膜系;有的含有一个较大的具许多小泡或复杂膜系的膜束缚囊泡。在液泡内还存在一些游离的液泡内囊泡,它们通常具有两层紧贴的界膜或为多层同心膜,推测它们来自液泡膜内突。ＡｃＰａｓｅ定位结果显     

细胞内膜泡运输中拴系因子的功能

膜泡运输是不同细胞器间进行物质传递的基本方式,分为4个重要步骤：囊泡的出芽、转运、拴系和融合。在此过程中,有许多相关因子参与调控,如包被蛋白、Rab蛋白、拴系因子、SM蛋白和SNARE等。拴系因子在运输囊泡和靶位膜发生接触的最初阶段起重要调控作用,多数拴系因子形成大的多亚基复合体发挥功能。目前,关于拴系因子的功能已经有了一定的了解,在此,我们对酵母、哺乳动物以及植物细胞中的已知拴系因子的特点和功能进行了概述。     

植物Na^+,K^+/H^+反向转运体:pH平衡与囊泡运输

细胞内pH和囊泡运输是影响细胞功能的重要影响因子,也是决定细胞是否死亡的重要因素。植物Na^+,K^+/H^+反向转运体是位于细胞膜结构上的跨膜反向转运蛋白,介导Na^+、K^+与质子(H^+)的跨膜反向转运,影响胞内pH的动态平衡。研究表明,NHX缺失造成细胞pH失衡的同时,将影响囊泡运输,从而对生长发育产生不利影响。主要对植物NHX在pH调节、囊泡运输中的功能进展进行了概述,并对其关系进行探讨。     

Bioreactor considerations for secondary metabolite production from plant cell tissue culture: Indole alkaloids from Catharanthus roseus

Batch shake flask studies with Catharanthus roseus demonstrated that alkaloid production commenced only after growth had slowed or ceased. To obtain high alkaloid productivities for extended periods, a hormone-free production medium was used. To develop a readily scalable process, both immobilized and suspended cell systems were studied. In the immobilized cell systems, growth, glucose utilization, and alkaloid production were suppressed; for the case of membrane entrapped cells this suppression was observed to be reversible. Based on the oxygen requirements of the cells, and the oxygen transfer capabilities of a pneumatically agitated bubble column, conditions were established that allowed the growth and production dynamics observed in shake flasks to be reproduced in the air sparged column reactor. The requirement for the aseptic exchange of growth for production medium was satisfied by using a coarse cotton filter and coupling filtration to aeration. With this filtration system, media can be rapidly and completely exchanged and the filter can be quickly and effectively backwashed. By coupling filtration to aeration, a two-stage batch operation can be employed while requiring only a single bioreactor. Studies with this system demonstrated its capabilities for alkaloid production.     

Improvement of Panax notoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioreactors

A Panax notoginseng cell culture was successfully scaled up from shake flask to 1.0-L bubble column reactor and concentric-tube airlift reactor. High-density bioreactor batch cultivation was carried out using a modified MS medium. The maximum cell density in batch cultures reached 20.1, 21.0 and 24.1 g/L in the shake flask, bubble column and airlift reactors, respectively, and their corresponding biomass productivity was 950, 1140 and 1350 mg/(L x d) for each. The productivity of ginseng saponin was 70, 96 and 99 mg/(L x d) in the flask, bubble column and airlift reactors, respectively; and the polysaccharide productivity reached 104, 119 and 151 mg/(L x d) for each. Furthermore, a fed-batch cultivation strategy was developed on the basis of specific oxygen uptake rate (SOUR), i.e., sucrose feeding before a sharp decrease of SOUR, and the highest cell density of 29.7 g/L was successfully achieved in the airlift bioreactor on day 17 with a very high biomass productivity of 1520 mg/(L x d). The concentrations of ginseng saponin and polysaccharide reached about 2.1 and 3.0 g/L, respectively, and their productivity was 106 (saponin) and 158 mg/(L x d) (polysaccharide). This work successfully demonstrated the high-density bioreactor cultivation of P. notoginseng cells in pneumatically agitated bioreactors and the reproduction of the shake flask culture results in bioreactors. The cell density, biomass productivity, production titer and productivity of both ginseng saponin and polysaccharide obtained here were the highest that have been reported on a reactor scale for all the ginseng species.     

Characterization of supermacroporous monolithic polyacrylamide based matrices designed for chromatography of bioparticles

Supermacroporous monolithic acrylamide (AAm)-based cryogels were prepared by radical cryo-polymerizaton (polymerization in the moderately frozen system) of AAm with functional monomers and cross-linker N,N'-methylene-bis-acrylamide (MBAAm). Electron microscopy studies revealed supermacroporous structure of the developed cryogels with pore size of 5-100 microm. Cryogel porosity depended on cryo-polymerization conditions. More than 90% of the monolithic bed volume is the interconnected supermacropores filled with water and less than 10% of the monolithic volume is pore walls. The total protein binding capacity (lysozyme in the case of immobilized metal affinity chromatography (IMAC) column and bovine serum albumin (BSA) in the case of anion-exchange (AE) column) was independent of the flow rates till 600 cm/h. Chromatographic behavior of E. coli cells when a cell suspension was applied to ion-exchange cryogel columns depended on both the density of functional ligand and the porosity of the cryogel.     

A novel approach for the extraction of herbicides and pesticides from water using liquid-core microcapsules

A novel perstraction system using liquid-core microcapsules for pesticide and herbicide removal from aqueous environments is proposed. The microcapsules contain an oil, dibutyl sebacate, surrounded by a hydrogel membrane. The extraction efficiency of the capsules was demonstrated with atrazine, methylparathion, ethylparathion, and 2,4-dichloro-phenoxyacetic acid. The results show that all of the tested compounds could be rapidly extracted, typically 75% extraction within 10 minutes using a capsule: liquid volume ratio of only 3.5% for ethylparathion, and that the rate of extraction increased with increasing hydrophobicity of the compound to be extracted. Higher rates of extraction could be achieved by changing the capsule: liquid volume ratio. The effect of different liquid core solvents, size of capsules, agitation rate, and treatment with complexing agents on the properties of the microcapsules and extraction rate were studied. Capsules of a diameter smaller than 0.800 mm show little external resistance to mass transfer. The main resistance to mass transfer of the pesticides/herbicides was found to reside in the hydrogel membrane composed of cross-linked alginate/polyacrylamide. Removal of divalent cations from the membrane by the addition of citrate, resulted in a 50% increase in the mass transfer coefficient, probably as a result of solubilization and exo-diffusion of alginate.     

Alginate concentration: a key factor in growth of temperature-sensitive baculovirus-infected insect cells in microcapsules

The desire to increase cell density and product concentration has been the primary driving force for the development of better animal cell culture processes. In the technique used in our laboratory-microencapsulation-insect cells (Spodoptera frugiperda), infected with a temperature-sensitive mutant of the Autographa californica nuclear polyhedrosis virus (AcNPV), were cultured in multiple membrane alginate-polylysine (PLL) microcapsules which had a controlled membrane molecular-weight cutoff and an intracapsular alginate concentration which was ca. 16% lower than that obtained in the commercially available single-membrane system. Cell culture experiments indicated that the intracapsular alginate concentration appears to be a key factor in achieving good cell growth. It was possible to obtain intracapsular cell densities of 8 x 10(7) cells/mL capsules and virus concentrations to 10(9) IFU/mL capsules. The virus litre in the supernatant was ca. 300 times lower, indicating that virtually all of the virus was retained within the capsules.     

Production of monoclonal antibodies against human chorionic gonadotropin by hybridoma cultures in calcium alginate capsules

A new method of making microcapsules with calcium alginate gel was developed and the cultivation of the encapsulated hybridoma cells producing monoclonal antibodies against human chorionic gonadotropin was investigated. A high cell density of 2.0×10⁸ cells/cm³ in the capsules led to a high dilution rate of 0.68 per hour and resulted in the high volumetric monoclonal antibody productivity of 652.8 mg/l/day, which was 20–30 times higher than those of traditional continuous suspension cultures. However, long-term continuous culture was not achieved with this capsule system probably because of the limitation in nutrient supply and the accumulation of waste products. Also the analysis of oxygen transfer in this system showed that oxygen supply was not enough to support such a high cell density.     

The effect of oxygen transfer on the activity of encapsulated whole cell β-galactosidase

The effect of oxygen transfer on the production of immobilized whole cell β-galactosidase has been evaluated. The encapsulated whole cell β-galactosidase was prepared by combining cell encapsulation and culture into one-step. Escherichia coli was encapsulated and cultured in the growth and production media to accumulate β-galactosidase in itself. Sunflower seed oil was coimmobilized to increase the oxygen transfer rate through the capsule membrane. The oxygen transfer rate increased 63 percent and the activity of β-galactosidase increased by 10 percent. The activity of encapsulated β-galactosidase obtained in the concentric air lift reactor was 86 percent higher than that in the shaking incubator. In the concentric air lift reactor, the accumulation of encapsulated whole cell β-galactosidase was primarily dependent on the capsule velocity. While the accumulation of specific β-galactosidase in the capsule increased with volumetric oxygen transfer coefficient, the cell biomass accumulated in the capsule decreased.     

Development of a novel membrane aerated hollow-fiber microbioreactor

A new challenge in biotechnological processes is the development of flexible bioprocessing platforms, allowing strain selection, facilitating scale-up and integrating separation steps. Miniaturization of such a cultivation system allows parallel use and the saving of resources but makes the supply of oxygen to the cells difficult. In this work we present a membrane aerated hollow-fiber microbioreactor (HFMBR) which consists of an acrylic glass module equipped with two different types of membrane fibers. Fibers of polyethersulfone and polyvinyldifluoride were used for substrate and oxygen supply, respectively. Cultivation of E. coli as model organism and production of His-tagged GFP were carried out in the extracapillary space of the membrane aerated HFMBR and compared with cultivations in shaking flask which are commonly used for screening experiments. The measurement of the oxygen transfer capacity and the online monitoring of the dissolved oxygen during the cultivation were performed using a fiber optic oxygen sensor. Online measurement of the optical density was also integrated to the bioreactor. Due to efficient oxygen transfer, a better cell growth than in the shaking flask experiments was achieved, while no negative influence on the GFP productivity was observed in the membrane aerated bioreactor. Thus the feasibility of a future integrated downstreaming could also be demonstrated.     

海藻酸钠/壳聚糖微胶囊固定化大肠杆菌的研究

本文以大肠杆菌ＤＨ5α为模型体系 ,探索了大肠杆菌ＤＨ5α用海藻酸钠 壳聚糖 (ＡＣＡ)微胶囊培养的可行性 ,并观察了微囊化大肠杆菌ＤＨ5α细胞生长与物料渗透性能 ,通过将ＡＣＡ微胶囊移植到实验组小鼠体内 ,考察了ＡＣＡ微胶囊作为口服药物载体的可能性。1　材料和方法1.1　材料壳聚糖 ,本实验室改性所得 ;海藻酸钠 ,ＫｅｌｃｏＤｉｖｏｆＭｅｒ ｃｋＣｏ .Ｉｎｃ .ＵＳＡ ;其它试剂均为国产分析纯。大肠杆菌ＤＨ5α ,长春生物制品所 ;ＬＢ培养基 ,华美生物制品公司提供。昆明系小白鼠 18～ 2 0ｇ ,解放军大连高等医学专科学校实验动物中…     

Improved production of phenylethanoid glycosides by Cistanche deserticola cells cultured in an internal loop airlift bioreactor with sifter riser

An internal loop airlift bioreactor with sifter riser (ILABSR) was composed of a bubble column and a draught-tube rolled with 40-mesh sifter that placed 5 cm above the bottom at the center of the column. A 2 L ILABSR was used for the suspension cultivation of Cistanche deserticola cells and its performance was compared with shake flask culture and a bubble column. Under the optimum culture conditions with the air flowrate of 0.075 m³/h and the inoculation size of 4.7%, about one-fifth cells were attached to the sifter draught-tube. PeG content in these cells was 16.3%, which was 104% higher than that of suspension cells. The production of phenylethanoid glycosides reached 0.85 g/L, which was 102 and 4% higher than those cultured in a 2 L bubble column and shake flasks respectively under their optimal culture conditions.