📚 Immobilising Enzymes | 固定化酶
Enzymes are protein catalysts that accelerate biochemical reactions without being permanently changed. In industrial biotechnology, using free enzymes can be costly because the enzyme is often lost in the product stream or denatured after one batch. Immobilisation is a set of techniques that fix enzymes onto or inside an inert support, so they can be recovered, reused and used in continuous reactors.
酶是蛋白质催化剂，能在自身不被永久改变的情况下加速生化反应。在工业生物技术中，使用游离酶通常成本很高，因为酶往往会随产物流失，或在一个批次后变性。固定化是一类将酶固定到惰性载体表面或内部的技术，使酶能够被回收、重复使用并用于连续反应器。
1. What Are Immobilised Enzymes? | 什么是固定化酶？
An immobilised enzyme is an enzyme that has been attached to, or trapped within, an insoluble support material. The enzyme remains catalytically active, but its movement is restricted, allowing easy separation from the reaction mixture.
固定化酶是指附着在或包埋于不溶性载体材料中的酶。酶仍然具有催化活性，但其运动受到限制，因此可以很容易地从反应混合物中分离出来。
Common supports include alginate beads, silica, cellulose, glass beads, polyacrylamide and semipermeable membranes. The choice of support depends on cost, stability, pore size and the intended reactor design.
常见载体包括海藻酸盐珠、二氧化硅、纤维素、玻璃珠、聚丙烯酰胺和半透膜。载体的选择取决于成本、稳定性、孔径大小以及预期的反应器设计。
2. Why Immobilise Enzymes? | 为什么要固定化酶？
Immobilisation is used mainly for economic and practical reasons. Purified enzymes are expensive, and immobilisation allows many cycles of reuse, reducing the cost per unit of product.
固定化主要出于经济和实用原因。纯化酶价格昂贵，固定化后可多次重复使用，从而降低单位产品的成本。
Immobilised enzymes also produce a product stream that is largely free of enzyme contamination. This is especially important in food and pharmaceutical processing, where residual enzyme must be minimised.
固定化酶还能使产物流中基本不含酶污染。这在食品和制药加工中尤为重要，因为这些行业必须尽量减少残留酶。
Enzyme stability is often increased when the enzyme is held in a fixed conformation or protected by a matrix. Many immobilised enzymes tolerate higher temperatures and a wider pH range than the same enzyme free in solution.
酶被固定在特定构象或受基质保护时，稳定性通常会提高。许多固定化酶比游离状态下的同种酶更能耐受较高温度和较宽的 pH 范围。
3. Adsorption to a Solid Support | 吸附法固定
Adsorption is the simplest immobilisation method. Enzyme molecules are mixed with a solid support such as glass beads, activated charcoal, clay or cellulose, and they bind through weak reversible forces including ionic interactions, hydrogen bonds and van der Waals attractions.
吸附是最简单的固定化方法。将酶分子与玻璃珠、活性炭、黏土或纤维素等固体载体混合，酶通过离子相互作用、氢键和范德华引力等弱可逆作用力结合在载体表面。
The main advantage is that adsorption uses mild conditions, so enzyme activity is usually well preserved. However, binding is weak, and changes in pH, ionic strength or flow rate can cause the enzyme to desorb and leak into the product.
主要优点是吸附条件温和，因此酶活性通常能很好地保留。然而这种结合较弱，pH、离子强度或流速变化都可能导致酶解吸并泄漏到产品中。
4. Covalent Bonding to a Support | 共价结合法固定
Covalent bonding attaches enzymes to a support through strong chemical bonds, often using functional groups such as amino, carboxyl or hydroxyl groups on the enzyme surface. Supports may include agarose, silica or polyacrylamide that have been chemically activated.
共价结合法通过强化学键将酶连接到载体上，通常利用酶表面的氨基、羧基或羟基等官能团。载体可以是经过化学活化的琼脂糖、二氧化硅或聚丙烯酰胺。
This method minimises enzyme leakage and gives stable industrial catalysts. However, the chemistry can be harsh, may react with groups in the active site, and often destroys a significant proportion of catalytic activity.
这种方法能最大限度减少酶泄漏，获得稳定的工业催化剂。但反应条件可能较剧烈，可能作用于活性位点中的基团，通常会破坏相当一部分催化活性。
5. Entrapment in a Matrix | 包埋法固定
Entrapment physically holds enzymes inside a gel or polymer network, such as calcium alginate, polyacrylamide or gelatine. The enzyme is not chemically bound to the support, and substrate and product diffuse through pores in the matrix.
包埋法将酶物理地限制在凝胶或聚合物网络中，如海藻酸钙、聚丙烯酰胺或明胶。酶不与载体发生化学结合，底物和产物通过基质中的孔隙扩散。
This gentle method usually preserves the native conformation of the enzyme. Its main drawback is diffusion limitation: large substrate molecules may penetrate slowly, reducing the apparent reaction rate.
这种温和的方法通常能保持酶的天然构象。其主要缺点是扩散限制：较大的底物分子可能扩散缓慢，从而降低表观反应速率。
6. Membrane Encapsulation | 膜封装法固定
Membrane encapsulation encloses enzyme molecules within a semipermeable membrane, such as a dialysis tube, a microcapsule or a hollow fibre. Small substrate and product molecules pass through the membrane, while the larger enzyme is retained.
膜封装法将酶分子封闭在半透膜内，如透析管、微胶囊或中空纤维。小分子底物和产物能穿过膜，而较大的酶被截留。
This method provides a clean separation and works well when products are small. However, membranes can become fouled, and diffusion of substrates and products across the membrane can be slow.
这种方法分离效果好，适用于产物分子较小的反应。但膜可能结垢，底物和产物跨膜扩散也可能较慢。
7. Alginate Bead Practical: A Model | 海藻酸钠珠实验：模型
A common school and college practical mixes yeast or lactase with sodium alginate solution. The mixture is dripped into calcium chloride solution, where Ca²⁺ ions cross-link the alginate chains to form insoluble calcium alginate beads containing the enzyme.
常见的中学或大学实验将酵母或乳糖酶与海藻酸钠溶液混合。混合物滴入氯化钙溶液中，Ca²⁺ 离子使海藻酸盐链交联，形成含有酶的不溶性海藻酸钙珠。
To test lactase activity, immobilised lactase beads can be placed in milk or lactose solution. The product glucose can be detected with glucose test strips, and the beads can be washed and reused in a fresh substrate solution.
为了检测乳糖酶活性，可将固定化乳糖酶珠放入牛奶或乳糖溶液中。产物葡萄糖可用葡萄糖试纸检测，珠子经洗涤后可在新底物溶液中重复使用。
Controls should include free enzyme, empty alginate beads and a boiled immobilised enzyme preparation. This allows comparison and shows that activity depends on the enzyme, not the support.
对照组应包括游离酶、空白海藻酸盐珠和煮沸的固定化酶制剂。这样可以进行比较，并证明活性来自酶而不是载体。
8. Effect on Enzyme Activity and Kinetics | 对酶活性与动力学的影响
Immobilisation can change the apparent kinetic parameters of an enzyme. The maximum rate Vₘₐₓ often appears lower because some enzyme molecules are inaccessible or denatured during attachment, and diffusion may limit substrate delivery.
固定化会改变酶的表观动力学参数。最大速率 Vₘₐₓ 通常看起来较低，因为部分酶分子在固定过程中不可接近或变性，而且扩散可能限制底物供应。
The Michaelis constant Kₘ often appears higher. Because substrate concentration near the immobilised enzyme is lower than in the bulk solution, a higher bulk concentration is needed to reach half Vₘₐₓ.
米氏常数 Kₘ 通常看起来较高。由于固定化酶附近的底物浓度低于主体溶液，达到半 Vₘₐₓ 需要更高的主体底物浓度。
v = Vₘₐₓ [S] / (Kₘ + [S])
where v is initial rate, Vₘₐₓ is maximum rate, [S] is substrate concentration and Kₘ is the Michaelis constant. Immobilisation mostly
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