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首页> 外文期刊>Biotechnology Progress >Enzyme Activity Evaluation of Organic Solvent-Treated Phenylalanine Ammonia Lyase
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Enzyme Activity Evaluation of Organic Solvent-Treated Phenylalanine Ammonia Lyase

机译：有机溶剂处理的苯丙氨酸氨分解酶的酶活性评估

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The direct one-step synthesis of L-phenylalanine methyl ester in an organic-aqueous biphasic system using phenylalanine ammonia lyase (E.C.4.3.1.5, PAL) containing Rhodotor-ula glutinis yeast whole cells was reported earlier. We report here further optimization of this biotransformation using isolated PAL, when the lyophilized enzyme is treated with different water miscible and water immiscible organic solvents. Use of isolated PAL enzyme is advantageous in overcoming diffusion barriers encountered when using PAL containing R. glutinis whole cells, and resulted in increased product yield due to better interaction of enzyme with the substrate. Among the water miscible solvents, ethanol treated and methanol treated enzymes supported maximum PAL forward and reverse activities; respectively. In the water immiscible solvents category, heptane-treated enzyme exhibited maximal activity for both PAL forward and reverse reactions. PAL activity obtained with enzyme specimens treated with methanol, ethanol, and heptane varied in the range of 91-99% of that observed in aqueous buffer medium for the forward reaction; and 89-95% for the reverse reaction, n-butanol, acetone, and benzene were found to have a inhibitory effect on PAL enzyme, in that, it resulted in only 31-33% activity of that obtained with aqueous solution. Raman spectroscopy was used to monitor amide I and II bands which are sensitive to changes in the secondary structure of proteins. No changes in structure could be detected from the analyses of AI and AII bands of PAL spectra. This data obtained for PAL, a tetramer, could be significant in predicting how solvent interactions affect the structure and function of multimeric proteins and enzymes in nonaqueous media.
机译：早先已有报道称，使用苯丙氨酸氨裂合酶（E.C.4.3.1.5，PAL）在含有Rhodotor-ula glutinis酵母全细胞的苯丙氨酸氨裂合酶（E.C.4.3.1.5，PAL）的有机-水双相体系中直接一步合成L-苯丙氨酸甲酯。当冻干酶用不同的水混溶性和水不混溶性有机溶剂处理时，我们在这里报告了使用分离的PAL进行的这种生物转化的进一步优化。使用分离的PAL酶的优势在于克服了使用含谷氨酸棒杆菌全细胞的PAL时遇到的扩散障碍，并且由于酶与底物之间的更好相互作用而提高了产品产量。在与水混溶的溶剂中，乙醇处理和甲醇处理的酶支持最大的PAL正向和反向活性。分别。在与水不混溶的溶剂类别中，庚烷处理的酶对PAL的正向和反向反应均显示最大活性。用甲醇，乙醇和庚烷处理过的酶标本获得的PAL活性在正向反应的水性缓冲介质中观察到的PAL活性的变化范围为91-99％。在89-95％的逆反应中，发现正丁醇，丙酮和苯对PAL酶具有抑制作用，因为其仅使PAL酶的活性为31-33％。拉曼光谱法用于监测对蛋白质二级结构变化敏感的酰胺I和II谱带。通过分析PAL谱的AI和AII谱带无法检测到结构变化。从PAL（一种四聚体）获得的数据对于预测溶剂相互作用如何影响非水介质中多聚体蛋白质和酶的结构和功能可能具有重要意义。

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