Feng Liping, Cao Yanping, Xu Duoxia, You Sasa, Han Fu
School of Food & Chemical Engineering, Beijing Advanced Innovation Center for Food Nutrition & Human Health (BTBU), Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University, Beijing 100048, China; Beijing Higher Institution Engineering Research Center of Food Additives and Ingredients, Beijing Key Laboratory of Flavor Chemistry, Beijing Technology & Business University, Beijing 100048, China; Beijing Laboratory for Food Quality and Safety, Beijing Technology & Business University, Beijing 100048, China.
School of Food & Chemical Engineering, Beijing Advanced Innovation Center for Food Nutrition & Human Health (BTBU), Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University, Beijing 100048, China; Beijing Higher Institution Engineering Research Center of Food Additives and Ingredients, Beijing Key Laboratory of Flavor Chemistry, Beijing Technology & Business University, Beijing 100048, China; Beijing Laboratory for Food Quality and Safety, Beijing Technology & Business University, Beijing 100048, China.
Ultrason Sonochem. 2016 Sep;32:224-230. doi: 10.1016/j.ultsonch.2016.03.015. Epub 2016 Mar 15.
The aim of the study was to investigate the impact of sodium alginate (ALG) pretreated by ultrasound on the enzyme activity, structure, conformation and molecular weight and distribution of papain. ALG solutions were pretreated with ultrasound at varying power (0.05, 0.15, 0.25, 0.35, 0.45W/cm(2)), 135kHz, 50°C for 20min. The maximum relative activity of papain increased by 10.53% when mixed with ALG pretreated by ultrasound at 0.25W/cm(2), compared with the untreated ALG. The influence of ultrasound pretreated ALG on the conformation and secondary structure of papain were assessed by fluorescence spectroscopy and circular dichroism spectroscopy. The fluorescence spectra revealed that ultrasound pretreated ALG increased the number of tryptophan on papain surface, especially at 0.25W/cm(2). It indicated that ultrasound pretreatment induced molecular unfolding, causing the exposure of more hydrophobic groups and regions from inside to the outside of the papain molecules. Furthermore, ultrasound pretreated ALG resulted in minor changes in the secondary structure of the papain. The content of α-helix was slightly increased after ultrasound pretreatment and no significant change was observed at different ultrasound powers. ALG pretreated by ultrasound enhanced the stability of the secondary structure of papain, especially at 0.25W/cm(2). The free sulfhydryl (SH) content of papain was slightly increased and then decreased with the increase of ultrasonic power. The maximum content of free SH was observed at 0.25W/cm(2), under which the content of the free SH increased by 6.36% compared with the untreated ALG. Dynamic light scattering showed that the effect of ultrasound treatment was mainly the homogenization of the ALG particles in the mixed dispersion. The gel permeation chromatography coupled with the multi-angle laser light scattering photometer analysis showed that the molecular weight (Mw) of papain/ALG was decreased and then increased with the ultrasonic pretreatment. Results demonstrated that the activity of immobilized papain improved by ultrasonic pretreatment was mainly caused by the variation of the conformation of papain and the effect of interactions between papain and ALG. This study is important to explain the intermolecular interactions of biopolymers and the mechanism of enzyme immobilization treated by ultrasound in improving the enzymatic activity. As expected, ALG pretreated by appropriate ultrasound is promising as a bioactive compound carrier in the field of immobilized enzyme.
本研究的目的是探究经超声预处理的海藻酸钠（ALG）对木瓜蛋白酶的酶活性、结构、构象以及分子量和分布的影响。将ALG溶液在135kHz、50°C下用不同功率（0.05、0.15、0.25、0.35、0.45W/cm²）的超声预处理20分钟。与未处理的ALG相比，当木瓜蛋白酶与经0.25W/cm²超声预处理的ALG混合时，其最大相对活性提高了10.53%。通过荧光光谱和圆二色光谱评估超声预处理的ALG对木瓜蛋白酶构象和二级结构的影响。荧光光谱显示，超声预处理的ALG增加了木瓜蛋白酶表面色氨酸的数量，尤其是在0.25W/cm²时。这表明超声预处理诱导了分子展开，导致更多疏水基团和区域从木瓜蛋白酶分子内部暴露到外部。此外，超声预处理的ALG导致木瓜蛋白酶的二级结构发生微小变化。超声预处理后α-螺旋含量略有增加，在不同超声功率下未观察到显著变化。超声预处理的ALG增强了木瓜蛋白酶二级结构的稳定性，尤其是在0.25W/cm²时。木瓜蛋白酶的游离巯基（SH）含量随超声功率的增加先略有增加然后下降。在0.25W/cm²时观察到游离SH的最大含量，在此条件下，游离SH的含量比未处理的ALG增加了6.36%。动态光散射表明，超声处理的效果主要是使混合分散体中ALG颗粒均匀化。凝胶渗透色谱结合多角度激光光散射光度计分析表明，木瓜蛋白酶/ALG的分子量（Mw）随超声预处理先降低后升高。结果表明，超声预处理提高固定化木瓜蛋白酶活性主要是由于木瓜蛋白酶构象的变化以及木瓜蛋白酶与ALG之间相互作用的影响。本研究对于解释生物聚合物的分子间相互作用以及超声处理酶固定化提高酶活性的机制具有重要意义。正如预期的那样，经适当超声预处理的ALG有望作为固定化酶领域的生物活性化合物载体。
