Purification and biochemical characterization of <i>α</i>- amylase from <i>Aspergillus tamarii</i> MTCC5152

The purification and biochemical characterization of the extracellular alpha amylase from <i>A.tamarii</i> MTCC5152 were studied. The combined use of ion exchange and gel filtration chromatographic methods were used for purification studies. The specific activity was significantly increased (33 fold) and 19.41 fold purification of the enzyme <i>α</i>-amylase with 24% yield was achieved. The enzyme had an optimal pH of 6.5 and exhibited its highest activity at 55 °C. It is active over a wide range of pH 5–7 at room temperature. The enzyme is relatively stable in the temperature range of 25–35 °C for a period of 4 h hence, more suitable for industrial applications. <i>K_m</i> and <i>V</i>_max value of the enzyme was to be 5.882 mg/mL and 0.803 mg/mL/min respectively using starch as the substrate. The purified protein showed a single band on native and SDS PAGE and the molecular weight was found to be 31 kDa. Starch zymogram also revealed one clear zone of amylolytic activity which corresponded to the band obtained with native PAGE and SDS/PAGE. The characterization studies showed that the enzyme activity is inhibited by Ca²⁺, Mn²⁺, Hg²⁺, Fe²⁺.

本研究针对塔马里曲霉（*A.tamarii*）MTCC5152分泌的胞外α-淀粉酶（α-amylase）开展了纯化与生化特性表征研究。本次纯化采用离子交换层析与凝胶过滤层析联用的方案。经纯化后，该酶的比活力显著提升（提升33倍），总纯化倍数达19.41倍，酶活回收率为24%。该酶的最适pH为6.5，最适反应温度为55℃；在室温条件下，其在pH 5~7的宽范围内均保持催化活性。该酶在25~35℃的温度区间内孵育4小时后仍可保持相对稳定，因此更适用于工业应用场景。以淀粉为底物时，该酶的米氏常数（K_m）为5.882 mg/mL，最大反应速率（V_max）为0.803 mg/mL/min。经纯化的蛋白在原生聚丙烯酰胺凝胶电泳（native PAGE）与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS/PAGE）中均呈现单一条带，其分子量约为31 kDa。淀粉酶谱（starch zymogram）分析也显示出一条清晰的淀粉水解活性条带，与native PAGE及SDS/PAGE所得的蛋白条带位置一致。表征结果表明，该酶的活性可被Ca²⁺、Mn²⁺、Hg²⁺、Fe²⁺等离子抑制。

搜集汇总

背景与挑战

背景概述

该数据集聚焦于从Aspergillus tamarii MTCC5152真菌中纯化和生化表征α-淀粉酶的研究，详细描述了纯化过程（使用离子交换和凝胶过滤色谱，实现33倍活性提升和24%产率）以及酶的生化特性（如最适pH 6.5、最适温度55°C、分子量31 kDa和抑制离子）。数据集发布于2023年，涵盖生物化学、生物技术等多个学科，适用于工业应用和酶学分析，提供原始实验数据支持相关研究。