Immobilized enzyme

Enzymes that catalyze in a certain space and can be used repeatedly and continuously

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Immobilized enzyme is a new technology developed in the 1960s. Immobilized enzyme refers to an enzyme that plays a catalytic role in a certain space and can be used repeatedly and continuously. Generally, enzyme catalytic reaction is carried out in aqueous solution, while immobilized enzyme is to treat water-soluble enzyme with physical or chemical methods to make it insoluble in water, but still has enzyme activity^[1] 。

Chinese name: Immobilized enzyme
Foreign name: immobilized enzyme

è Mass transfer: Limited
è Dynamic properties: undergo changes
Additional costs: è Increased immobilization

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brief introduction

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Immobilized enzyme

Enzyme immobilization After that, the general stability increases, it is easy to separate from the reaction system, and it is easy to control, and it can be used repeatedly. It is convenient for transportation and storage, and is conducive to automatic production. However, its activity and scope of use are reduced, and there is still room for technology development. Immobilized enzyme is an enzyme application technology developed in the past decade, which has an attractive application prospect in industrial production, chemical analysis and medicine.

Development history

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The research of immobilized enzyme began in 1910, and was formally studied in the 1960s. In the 1970s, it was widely carried out all over the world. Immobilization of enzymes is to use solid materials to bind or restrict the enzyme to a certain area, which can still carry out its unique catalytic reaction And can be recycled and reused. Compared with free enzyme, immobilized enzyme can maintain its high efficiency, specificity and mildness Enzyme catalysis At the same time, the reaction characteristics overcome the shortcomings of free enzyme, presenting a series of advantages such as high storage stability, easy separation and recovery, multiple reuse, continuous and controllable operation, simple process, etc. Immobilized enzyme is not only used in chemistry biology and bioengineering , medicine and life sciences, etc subject The research in this field is extremely active, which has been rapidly developed and widely applied, and because of its ecological advantages of saving resources and energy, reducing or preventing pollution environmental effect And meet the strategic requirements of sustainable development.

character

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Immobilized enzymes have various forms, including particles, lines, films and enzyme tubes according to different uses. Among them, particles account for the majority, and they and lines are mainly used for industrial fermentation production, such as packed into enzyme columns for continuous production, or batch stirring reaction in reactors; The film is mainly used in enzyme electrode and analytical chemistry; The enzyme tube has high mechanical strength and is suitable for industrial production^[2] 。

Advantages and disadvantages of immobilized enzyme compared with water-soluble enzyme

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Advantages: ① Immobilized enzyme can be reused, which improves the use efficiency of enzyme and reduces the use cost.

② The immobilized enzyme can be easily separated from the reaction system, simplifying the purification process, and the product yield is high and the quality is good.

③ In most cases, the stability of the immobilized enzyme was improved.

④ The catalytic reaction process of immobilized enzyme is easier to control.

⑤ Immobilized enzyme has a certain mechanical strength, which can be used to act on the substrate solution by agitation or column loading, so as to facilitate the continuous and automatic operation of enzyme catalytic reaction.

⑥ Compared with free enzyme, immobilized enzyme is more suitable for the use of multi enzyme system. It can not only make use of the synergistic effect in the multi enzyme system to greatly improve the enzyme catalytic reaction rate, but also control the reaction to proceed in a certain order.

Disadvantages: ① Immobilization may cause partial inactivation of enzyme and loss of enzyme activity.

② The change of enzyme catalytic microenvironment may lead to the change of reaction kinetics.

③ The use cost of immobilized enzyme increases, which increases the initial investment of the factory

④ Immobilized enzymes are generally only suitable for water-soluble small molecular substrates, not suitable for macromolecular substrates.

⑤ Compared with intact cells, immobilized enzymes are not suitable for multi enzyme reactions, especially those involving cofactors.

⑥ Enzymes must be separated and purified when immobilized^[3] 。

Preparation method

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classification

Classification diagram of preparation methods

The preparation methods of immobilized enzyme include physical method and chemical method.

Physical methods include physical adsorption Law Embedding method Etc. The advantage of physical method of enzyme immobilization is that the enzyme does not participate in chemical reaction, the overall structure remains unchanged catalytic activity It is well reserved. However, due to the embedment or Semipermeable membrane It has a certain spatial or three-dimensional blocking effect, so it is not applicable to some reactions.

Chemical method includes combination method cross-linking protocol 。 The combination method is also divided into Ions Combination method and Covalent Combination method. Is to pass the enzyme through Chemical bond It is a method of connecting to natural or synthetic polymer carrier, using coupling agent to cross link the enzyme through the groups on the enzyme surface, so as to form an immobilized enzyme with larger relative molecular weight and insolubility

Among them, adsorption method and covalent bond method can be collectively referred to as carrier binding method.

Specific methods

Adsorption method

The method of immobilizing enzyme by using various adsorbents to adsorb enzyme or enzyme containing bacteria on its surface. Usually physical adsorption Law and Ion adsorption Law.

Common adsorbents include Activated carbon 、 alumina 、 diatomite 、 Porous ceramics 、 Porous glass Etc.

The enzyme is immobilized by adsorption method, which is simple in operation, mild in condition, will not cause denaturation or inactivation of the enzyme, and the carrier is cheap and easy to obtain, and can be used repeatedly.

carrier-binding method

The most commonly used method is covalent binding, that is, the non Essential group adopt covalent bond and carrier Form an irreversible connection. Proteins that can be coupled under mild conditions Group include: amino 、 carboxyl 、 Cysteine Of Mercapto group 、 histidine Of Imidazolyl 、 Tyrosine Phenolic group serine And the hydroxyl group of threonine. Participating in covalent binding with carrier Group , cannot be the group necessary for enzyme activity. with China The bifunctional reagent "para - β - sulfate ethyl Sulfonyl Take aniline coupling carrier and enzyme as an example. The steps of carrier binding are shown in the reaction formula on the following page.

This method has been used in 3 '- Ribonuclease , 5 ′ - phosphodiesterase and Glucoamylase Immobilization of, etc. In addition, the enzyme passes physical adsorption or Ion adsorption to carrier Preparation of immobilized enzyme is also a common method.

cross-linking protocol

With the aid of bifunctional reagents, the enzyme molecules are crosslinked and coagulated into a network structure, making it insoluble in water to form an immobilized enzyme. The commonly used bifunctional reagents include glutaraldehyde Maleic anhydride Etc. Enzyme protein Dissociation of amino , phenolic group Imidazolyl And mercapto groups can participate Crosslinking reaction 。

Embedding method

The enzyme is wrapped in the fine grid of the gel or Semipermeability It is surrounded by a polymer film, and becomes lattice type and microcapsule type. Preparation of immobilized enzyme by embedding method In addition to embedding water-soluble enzyme, cells are also often embedded to make Immobilized cells For example, gelatin and glutaraldehyde can be used to embed Penicillin acylase dynamic Thallus , continuous hydrolysis N-base penicillin, industrial production 6-Aminopenicillanic acid 。

After immobilization, the enzyme is relatively resistant to changes in temperature and pH, and the optimal pH is often slightly shifted, without any change in substrate specificity, and the actual use efficiency is increased dozens of times (such as 5 '- phosphodiesterase Industrial applications) or even hundreds of times (such as Penicillin acylase Industrial applications).

characteristic	adsorbent	Covalent link	Embedding	Membrane limitation
preparation	simple	hard	hard	simple
cost	low	high	secondary	high
Binding force	Mutability	strong	weak	strong
loss	yes	nothing	yes	nothing
Applicability	wide	Selectivity	wide	Very broad
Operational problems	high	low	high	high
Matrix effect	yes	yes	yes	nothing
solubility	nothing	nothing	yes	yes
Antimicrobial properties	nothing	nothing	yes	yes

application area

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Immobilized enzymes have various forms, which can be made into particles with good mechanical properties and packed into enzyme columns for continuous production ； Or on Reactor Batch stirring reaction; Can also be made Enzyme membrane 、 Enzyme tube And other applications in analytical chemistry; It can also be made into microencapsulated enzyme, which can be used in clinic as therapeutic enzyme. Now it's used again Enzyme membrane (including membranes made of cells, tissues and microorganisms) element Form a device scale Biosensors , used to determine organic compounds, transfer information in automatic fermentation control, and detect harmful substances in environmental protection. The most commonly used is enzyme membrane and ion selective electrode Biosensors, such as urea sensors, are composed of immobilized urease , immobilized nitrifying bacteria and oxygen electrode Composition: urea is decomposed into ammonia and carbon dioxide Ammonia continues to be oxidized by nitrobacteria, and the total oxygen consumption reflects the change of current through the oxygen electrode to calculate the urea content.

Advantages: 1n is easy to separate from the reaction system, so it is easy to control the reaction time and reduce enzyme inactivation

2n reusable è reduce the cost of enzyme

3n improves enzyme stability

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