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首页> 外文学位 >Hydrolases on fumed silica: Conformational stability studies to enable biocatalysis in organic solvents.
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Hydrolases on fumed silica: Conformational stability studies to enable biocatalysis in organic solvents.

机译：气相法二氧化硅上的水解酶：构象稳定性研究，可在有机溶剂中进行生物催化。

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摘要

One area of considerable importance in modern biotechnology is the preparation of highly active and selective enzyme based biocatalysts for applications in organic solvents. A major challenge is posed by the tendency of enzymes to cluster when suspended in organic solvents. Because the clusters obstruct the transport of substrates to the active site of the enzyme, the observed activity is often severely reduced. Over the past two decades, many strategies have been proposed to mitigate this problem. We have tackled this major hurdle by devising an immobilization strategy that utilizes fumed silica as carrier for the enzyme molecules. Fumed silica is a non-porous nanoparticulated fractal aggregate with unique adsorptive properties. The enzyme/fumed silica preparation is formed in two steps. The buffered enzyme molecules are physically adsorbed on the fumed silica and then lyophilized. This protocol was shown to be successful with two enzymes of industrial relevance, Candida antarctica Lipase B (CALB) and subtilisin Carlsberg. The maximum observed catalytic activity in hexane reached or even exceeded commercial immobilizates and nonbuffer salt based preparations. The results demonstrated that catalytic activity has an intricate relationship with the nominal surface coverage (%SC) of the support by the enzyme molecules. s. Carlsberg exhibited an ever increasing activity as more surface area was provided per enzyme molecule. The activity leveled off when a sparse surface population was reached. CALB showed a maximum in catalytic activity at an intermediate surface coverage with steep decreases at both lower and higher surface coverage. It was shown that this maximum results from the presence of three distinct surface loading regimes after lyophilization: (1) a low surface coverage where opportunities for multiattachment to the surface likely lead to detrimental conformational changes, (2) an intermediate surface coverage where interactions with neighboring proteins and the surface help to maintain a higher population of catalytically competent enzyme molecules, and (3) a multi-layer coverage where mass transfer limitations lead to a decrease in the apparent catalytic activity. Conformational stability analyses with both fluorescence and CD spectroscopy showed evidence that these regimes are most likely formed during the adsorption step of our protocol. A low conformational stability region was detected at low surface coverage while adsorbates with highly stable enzyme ensembles were observed at high surface coverage. Secondary structural analysis of the lyophilized nanobiocatalysts with FTIR confirmed a substantial decrease in the alpha-helical components at low surface coverage. In summary, the work presented here traces the phenomenological observation of the catalytic behavior of a nanobiocatalyst to molecular-level: enzyme-enzyme and enzyme-support interactions, which are specific to the intricate properties of the enzyme molecules.
机译：在现代生物技术中相当重要的一个领域是制备用于有机溶剂中的高活性和选择性酶基生物催化剂。当悬浮在有机溶剂中时，酶趋于聚集的趋势构成了主要挑战。由于簇阻碍了底物向酶活性位点的运输，因此观察到的活性通常会大大降低。在过去的二十年中，已经提出了许多缓解该问题的策略。我们已经通过设计一种固定策略解决了这一主要障碍，该策略利用气相二氧化硅作为酶分子的载体。气相法二氧化硅是具有独特吸附性能的无孔纳米颗粒状分形聚集体。酶/热解法二氧化硅制备分两个步骤形成。缓冲的酶分子物理吸附在气相二氧化硅上，然后冻干。该协议对两种具有工业相关性的酶，即南极假丝酵母脂肪酶B（CALB）和枯草杆菌蛋白酶Carlsberg均显示成功。在己烷中观察到的最大催化活性达到或什至超过了商用固定剂和非缓冲盐基制剂。结果表明，催化活性与酶分子对载体的标称表面覆盖率（％SC）有着复杂的关系。 s。嘉士伯展示了不断增加的活性，因为每个酶分子提供了更多的表面积。当达到稀疏的表面种群时，活动趋于平稳。 CALB在中间表面覆盖下显示出最大的催化活性，在较低和较高表面覆盖下均急剧下降。结果表明，该最大值是由于冻干后存在三种不同的表面加载方式而导致的：（1）低表面覆盖率，其中多附着到表面的机会可能导致有害的构象变化，（2）中间表面覆盖率，其中与邻近的蛋白质和表面有助于维持较高数量的催化活性酶分子，以及（3）多层覆盖，其中传质限制导致表观催化活性降低。荧光和CD光谱的构象稳定性分析表明，这些方案最有可能在我们方案的吸附步骤中形成。在低表面覆盖率下检测到低构象稳定性区域，而在高表面覆盖率下观察到具有高度稳定的酶集合的吸附物。用FTIR对冻干的纳米生物催化剂的二级结构分析证实，在低表面覆盖率下，α-螺旋组分显着降低。总而言之，此处介绍的工作追溯了纳米生物催化剂在分子水平上的催化行为的现象学观察：酶-酶和酶-载体相互作用，这对酶分子的复杂特性是特定的。

著录项

  • 作者

    Cruz Jimenez, Juan Carlos.;

  • 作者单位

    Kansas State University.;

  • 授予单位 Kansas State University.;
  • 学科 Chemistry Biochemistry.;Biophysics General.;Engineering Chemical.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 240 p.
  • 总页数 240
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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