克鲁氏假丝酵母中甘油与海藻糖代谢关系的初步研究
文献类型：学位论文
| 作者 | 张岩 |
| 学位类别 | 硕士 |
| 答辩日期 | 2001-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 刘德华 |
| 关键词 | 克鲁氏假丝酵母 甘油代谢 海藻糖代谢 热刺激 渗透工 |
| 其他题名 | Preliminary study on the relationship of the metabolisms of glycerol and trehalose in candida krusei |
| 学位专业 | 生物化工 |
| 中文摘要 | 本文基于甘油和海藻糖在细胞抵御不利环境中所起保护作用的特性，对耐高渗克鲁氏假丝酵母在不同的环境条件下两者的代谢情况分别在摇瓶和SL搅拌罐上进行了研究，初步确定了甘油和海藻糖在保护细胞抵御高温和高渗透压的相互关系。针对所使用的实验体系确定了海藻糖和胞内甘油的提取方法。海藻糖采用三氯乙酸提取，每次用1.5mL的三氯乙酸提取15分钟，总共提取3次；酵母细胞用微波破碎2次，每次30秒，然后用蒸馏水提取30分钟得到胞内甘油。通过培养基组分的单因子和回归旋转实验发现甘油和海藻糖的代谢对营养成分有不同要求。培养基中营养不平衡会促进细胞内海藻糖的积累，而有利于甘油积累的条件同细胞生长所需要的条件基本相同。热刺激过程会促进细胞内甘油和海藻糖的合成代谢。通过正交实验发现促进甘油和海藻糖代谢的热刺激条件不同，胞内甘油对指数生长初期或中期的细胞较敏感，而且需要刺激较长的时间细胞内的积累才会有明显变化；而海藻糖在指数生长后期的细胞中积累明显。同时，由热刺激引起的海藻糖的积累会在短时间内很快消耗，而甘油一直都可以保持相对较高的含量。总体而言，克鲁氏假丝酵母细胞受到热刺激时，甘油对细胞的保护作用比海藻糖要明显。高渗透压下甘油和海藻糖代谢情况的研究表明，高渗环境会诱导细胜且速产生大量的海藻糖和胞内甘油，并且含量与渗透压正相关；随着发酵过私n,进行海藻糖作为一种贮存性碳源被很快消耗，而胞内甘油被不断运输到胞夕1也使其浓度下降；不同培养时期的酵母细胞对高渗透压的反应不同，直接转招到高渗培养基中的细胞会在细胞内迅速积累海藻糖和甘油；但指数生长初期用隐定期的细胞内只有海藻糖会出现短时间积累。此时由于培养基中已经积累丁一定浓度的甘油，胞内甘油对细胞抵御高渗透压的保护作用已不明显，主要甭细胞内海藻糖和胞外甘油起保护作用。渗透压的相关实验还发现单位重量的自胞对应的胞外甘油的浓度有着明显的规律性，同培养环境中的渗透压正相关，它可以在一定程度上反映不同条件下细胞产甘油的能力。酵母细胞原来生活王境中的渗透压越高，被转移到含有更高浓度的NaCl和乙醇培养基中时，细胞lJ生存能力越强，说明接受一种刺激的细胞可以耐受更高程度的同种刺激，同日还可以提高对其他种类的刺激的耐受 |
| 英文摘要 | In this thesis, bovine serum albumin (BSA) was selected as a model protein to elucidate the conformational change of protein in the aqueous solution and in aqueous protein/surfactant mixture. The interaction between protein and small-molecule surfactant, and the adsorption behavior of protein and surfactant at the air/water interface were studied by means of fluorescence spectroscopy, FT-Raman spectroscopy and surface tension measurement. Foaming properties of protein and protein/surfactant mixture have been evaluated by the shaking tests and the bubbling method. Enrichment of protein and ionic surfactants in mixed systems into the foam has been studied by the method of foam fractionation. Results from this work are very helpful not only to the understanding of the mechanism of interface stabilization by protein and mixed protein/surfactant solution, but also to the controlling of the foam formed during microbial fermentation and analogous industrial processes. The fluorescence spectroscopy and ultraviolet second derivative spectroscopy were used to study the changes of microenvironment of aromatic group containing amino acid residues and three-dimensional structure of BSA at various pH values. The strongest fluorescence was found at pH close to the pi of BSA, suggesting that the microenvironment of Trp residue has the weakest polarity and more compact spatial structure in BSA. The decrease of the fluorescence intensity of Trp residue and increase of the fluorescence intensity of Tyr residue were observed at pH far from pi of BSA. The results indicated that the structure of BSA underwent conformational unfolding, the distance between Trp and Tyr residues was increased, and the energy transfer from Tyr to Trp was decreased. The interaction between BSA and different surfactants has been investigated by fluorescence quenching and surface tension measurement. The results are summarized as follows: (1) The binding constant ￡ and the number of bound molecules " per BSA molecule was 316 (mol/L)"' and 0.9 respectively when the concentration of TX-100 was below its CMC (about 2.8X10"4 mol/L). K changed to 5 (mol/L)"1 and n to 0.4 when the concentration of TX-100 was above the CMC. That is, the binding constant decreased with increasing the bulk concentration of TX- 100. K was 9.18xlO5 (mol/L)'1 and n was 1.65 when the concentration of Tween 20 was below 1.5xlO"4 mol/L. It means 1 or 2 Tween 20 molecules were bound to a BSA. K was decreased to 0.09 (mol/L)"1 and n was to -0.2 when the concentration of Tween 20 was above l.SxlO"4 mol/L. It was suggested that the binding phenomenon disappeared at high bulk concentrations of Tween 20. Two break points were observed in the surface tension isotherms of SDS with the presence of BSA, which corresponded to the critical aggregation concentration (CAC) and the critical micelle concentration (CMC) of ionic surfactant. The CAC and CMC of surfactant were found to be dependent on the bulk concentration of BSA. The appearance of the plateau of CAC and CMC was irrelevant to the concentration of surfactant, but was related to the surfactant-to-protein molar ratio (R). In mixed BSA/SDS solutions, the plateau of CAC appeared in the range of R from 30 to 105, and the plateau of CMC appeared at R above 300. The plateau of CMC in BSA/CTAB mixtures appeared at R above 100. The information of competitive displacement of BSA and BSA-surfactant complex from interface by surfactant can be derived from the surface tension isotherms of mixed protein/surfactant solutions. The number of molecules bound with one BSA molecule varies with the type of surfactant used. The order of binding number per BSA molecule is SDS>CTAB>Tween 20 ^TX-100. Association of surfactant with protein is an unavoidable and competitive process versus the trend of self-association of surfactant to form micelle. Study of BSA conformation by Fourier deconvolution of the amide I band of Raman spectra has been carried out at different pH in the presence of different types of surfactant. The secondary structure of BSA changed obviously and the partially ordered structure of BSA shifted into unordered structure at pH far away from pi. The content of a-helix was decreased, and the /^-structure of BSA had a tendency to increase with increasing concentration of ionic surfactant. Among three types of suilactants studied, SDS indicated the most probable chance to change the secondary structure of BSA, followed by CTAB, and the TX-100 hardly changes the BSA structure. The foaming capability of proteins has been evaluated on the thermally denatured proteins and in the presence of different surfactants by shaking flask tests and the bubbling method. The results are as follows. (I ) Foaming capability of BSA was decreased and the foam stability was increased with increasing temperature of thermal denaturing. The content of thiol groups was found to be reduced by heating in BSA molecules. Both foaming capability and foam stability of albumin were found to increase with heat-induced denaturing. The analysis of thiol group in albumin indicated that the protein was unfolded and more thiol groups were exposed to solvent. Thermal denatured of proteins can affect both surface hydrophobicity and protein-protein interactions, and these can in turn affect the foaming properties of proteins. Surface hydrophobicity is one of the most important factors to the foaming capability of proteins, and protein-protein interaction plays a key role in affecting the foam stability. It is observed that the greatest foaming capability of BSA solution corresponds to the highest content of a-helix in the BSA secondary structure. (2) The foaming property of mixed protein/surfactant solutions was obviously different from those of the protein and surfactant alone. A minimum was found in the foam volume versus surfactant concentration in mixed protein/surfactant solutions. With lower surfactant concentration, the physicochemical properties of protein-stabilized foam can be strongly modified by the surfactant-protein interactions and formation of complexes, resulting to the decrease of foaming properties of protein solution. At higher surfactant concentration, protein or protein-surfactant complexes were extensively displaced from the air/water interface by free surfactant molecules, foaming capability was increased with the increase of surfactant, and the foaming property of mixed protein/surfactant solutions was similar to that of solution with surfactant alone. Foam fractionation of BSA has been studied in the presence of ionic surfactants (SDS and CTAB) in order to check the effects of coexistence of protein and surfactants on the enrichment and the foaming properties of mixed solutions. The foam properties and enrichment in mixed solutions were found to be quite different from those with either one of two components alone. Residual ratio of protein to surfactant at the bottom of fractionation column decreased with higher surfactant concentration, which is the consequence of protein-surfactant complex formation. |
| 公开日期 | 2013-09-26 |
| 页码 | 122 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1902]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 张岩. 克鲁氏假丝酵母中甘油与海藻糖代谢关系的初步研究[D]. 北京. 中国科学院研究生院. 2001. |
入库方式： OAI收割
来源：过程工程研究所
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