A significant challenge remains to protect proteins (including enzymes) from inactivation against pH. This study investigated stabilization against pH denaturation of carbonic anhydrase (CA) in silk fibroin (SF)-based hydrogels. CA was immobilized into SF-based hydrogels by a Ru(II) mediated photochemical method, giving rise to ∼100% immobilization efficiency and >60% activity of the free enzyme at the optimum pH value of 8. At an unfavorable acidic pH value of 3, the immobilized CA achieved >20% of the initial activity and ∼8% in the second cycle even after 12 h storage, while the free enzyme lost its activity completely. Fourier transform infrared spectroscopy (FTIR) results suggested that the protective effect of the SF-based hydrogels on the pH stability of CA could be attributed to unique structural feature of the SF molecules, enabling strong intermolecular interactions between SF and CA, and thus increasing protein rigidity against pH denaturation. The same approach was applied to lysozyme and xylanase, and achieved protein stabilization of both enzymes at an unfavorable basic pH value of 9. The SF-based hydrogels demonstrated great potential as a promising matrix for protein immobilization against pH denaturation.

保护蛋白质（包括酶）免受pH失活仍然是一项重大挑战。这项研究调查了基于丝素蛋白（SF）的水凝胶对碳酸酐酶（CA）的pH变性的稳定作用。通过Ru（II）介导的光化学方法将CA固定在基于SF的水凝胶中，在最佳pH值为8时，游离酶的固定率约为100％，活性大于60％。如图3所示，固定的CA即使在储存12小时后仍能达到初始活性的20％以上，在第二个循环中达到约8％，而游离酶则完全丧失了活性。傅里叶变换红外光谱（FTIR）结果表明，基于SF的水凝胶对CA的pH稳定性的保护作用可能归因于SF分子的独特结构特征，可以使SF和CA之间发生强烈的分子间相互作用，从而提高蛋白质抵抗pH变性的刚性。将相同的方法应用于溶菌酶和木聚糖酶，并在不利的碱性pH值为9的情况下实现了这两种酶的蛋白质稳定化。基于SF的水凝胶显示出巨大的潜力，有望成为有前景的蛋白质固定化抗pH变性的基质。