人工分子伴侣辅助鱼类肌球蛋白增溶机制的研究

Aquatic protein is instability and prone to denaturation in the processing. Denaturation process involves the unfolding, refolding, aggregation and subsequent precipitation of myosin molecule, which results in loss of its solubility and related functional properties. As a result, improving the stability and solubility of aquatic protein is the biggest challenges in the processing and utilization of aquatic resources. In this research project, myosin will be prepared from Tilapia and Lizard fish, and analysis techniques, such as the fluorescence spectra and fluorescence phase diagram, circular dichroism, dynamic light scattering, non-reducing and non-denaturing PAGE, will be used to determine the dynamic changes of the acid- and alkaline-induced molten globule state of myosin. Firstly, dynamic changes of myosin molecules in pH adjustment process, including unfolding, refolding, aggregation and structural characteristics will be detected, and then effect of myosin molecule of unfolding and refolding state and extent on their solubility and mechanism will be determined. Further studies will be done on artificial chaperone system assisted refolding. To clarify the feasibility and mechanism of promoting renaturaion and solubilization by artificial chaperone assisted refolding method, and effect of artificial chaperone system composed of small molecule surfactants and cyclodextrins on refolding and aggregation behavior of acid- and alkaline-denaturated myosin will be explored. These studies can be helpful to deeply understand the relationship between the denaturation/aggregation and solubility/stability of fish myosin, and promote the value-added utilization of low-cost aquatic protein resources and development of new protein resources, and thus result in expanding the application of the aquatic protein.

水产蛋白的稳定性差，加工过程极易变性。变性过程涉及肌球蛋白分子的去折叠、重折叠、聚集和沉淀，导致其溶解性及相关功能特性损失。如何提高水产蛋白的稳定性和溶解性是水产原料加工利用中的重大难题。本项目拟以罗非鱼和蛇鲻肌球蛋白为研究对象，采用荧光光谱和荧光相图、圆二色谱、动态光散射、非还原和非变性PAGE等分析技术，从酸/碱诱导的肌球蛋白熔球态出发，研究pH值调节处理过程中肌球蛋白分子去折叠、重折叠、聚集和相关结构特征的动态变化，揭示肌球蛋白分子去折叠/重折叠状态和程度对其溶解性的影响及机制，探讨小分子表面活性剂和环糊精组成的人工伴侣体系辅助酸/碱变性肌球蛋白重折叠和聚集行为，阐明人工分子伴侣辅助重折叠促进肌球蛋白复性的可行性及增溶机制。该研究有助于深入了解鱼类肌球蛋白变性、聚集与其溶解性和稳定性之间的关系，促进低值水产蛋白资源的高值化利用以及新型蛋白资源的开发，扩大水产蛋白的应用范围。

本项目针对水产蛋白的稳定性和溶解性差的难题，探讨其稳态化和增溶行为及机制。以尼罗罗非鱼（Oreochromis niloticus）和鳄蛇鲻（Saurida Wanieso）白肉为原料，提取肌球蛋白，从广泛pH值（2.0-12.0）诱导肌球蛋白去折叠行为入手，探讨了极端酸/碱条件下肌球蛋白去折叠及变性肌球蛋白重折叠行为，获得了肌球蛋白呈现“熔球态”的极端酸/碱条件为pH 2.0-3.0以pH 11.0-12.0，此时肌球蛋白的溶解度较高（>90%），表面疏水性和巯基含量增加，而α-螺旋含量得到保留；在等电点pH范围，肌球蛋白的表面疏水性和α-螺旋含量均小；碱性条件更容易产生聚集体，且主要由肌球蛋白分子的重链通过二硫键交联产生；不同酸类型对肌球蛋白构象的影响与pH值有关；而KOH与NaOH处理对肌球蛋白构象的影响差异不明显；酸/碱变性肌球蛋白经pH值调节后，其二级和三级结构均未恢复到原有状态，分子的热稳定性下降；确定了广泛盐浓度（0-2.0 mol/L KCl）对极端酸/碱可溶性肌球蛋白去折叠行为的影响，酸性条件下，盐浓度的升高降低肌球蛋白分子的去折叠程度，中性及碱性条件盐浓度的升高促进去折叠；选取三种代表性的盐浓度，探讨了人工分子伴侣体系辅助（ACA）酸/碱变性肌球蛋白重折叠行为，由色氨酸荧光光谱及圆二色谱研究显示，在低离子（0.001 mol/L KCl）及高离子强度（0.6 mol/L KCl）条件下，SDS和β-CD组合能有效辅助变性肌球蛋白重折叠，而在生理离子强度（0.15 mol/L KCl）下，促进了肌球蛋白的变性，且碱性条件下肌球蛋白重折叠的效果优于酸性条件；进一步采用含小分子氨基酸的溶液对其进行透析处理，在低离子强度条件下，10 mmol/L的赖氨酸和精氨酸能抑制肌球蛋白纤丝形成，降低体系的浊度，增溶效果明显，增溶后α-螺旋含量增加；生理离子强度条件下，体系的浊度明显下降，溶解度增加，表面疏水性增大，α-螺旋含量下降，丝状体完全解离，体系更加分散；初步分析氨基酸诱导低离子强度下肌球蛋白增溶的原因可能与体系pH值的变化及对蛋白质分子间静电相互作用的影响有关。研究成果为pH值调节法生产鱼糜工艺提供了理论依据，对更好地控制肌球蛋白分子的稳定性提供了依据，为低盐水产蛋白食品的研发提供了新的思路。