Multi-point immobilization method based on modification of enzyme with unnatural amino acid

Abstract

The invention relates to a multi-point immobilization method based on modification of an enzyme with an unnatural amino acid. According to the method, an aldo-keto reductase is modified with an unnatural amino acid, p-azido-L-phenylalanine, the unnatural amino acid-modified aldo-keto reductase is immobilized with a cyclooctyne-modified amino-activated epoxy polymer as a solid carrier to achieve multi-point covalent linkage, and one-step purification and immobilization of the enzyme is completed by alkyne-azide cycloaddition reaction. The immobilized aldo-keto reductase modified by the unnatural amino acid prepared by the method has greatly improved enzyme activity compared with free enzyme. Through a copper-free click reaction, a traditional tedious purification method is eliminated for the aldo-keto reductase, and the five-point immobilization can achieve a purification efficiency of 60%. The method is safe and reliable, has less environmental pollution, greatly saves time and cost consumed by purification, and is suitable for industrial production.

Description

technical field [0001] The invention relates to an enzyme immobilization method, in particular to a multi-point immobilization method based on non-natural amino acid modification enzyme. Background technique [0002] Catalytic enzymes are produced from readily available renewable resources and are biodegradable, essentially harmless and non-toxic. The use of enzymes has increased dramatically over the past few decades, mainly in the areas of research methodology, pharmaceutical research, food modification, laundry in the cosmetics industry, and the paper industry. However, such practical applications are hindered by the fragility of enzymes (eg, low thermal stability, narrow pH optimum range, low tolerance to most organic solvents and many metal ions, etc.). Furthermore, enzymes lead to unavoidable purification and isolation steps. Immobilized enzymes offer a simple and elegant solution to these challenges. Methods for immobilizing enzymes can be divided into three catego...

AI Technical Summary

Problems solved by technology

However, one of the most troublesome problems in immobilization is the purification of enzymes

In general, purified proteins are widely used for immobilization on solid supports, but purification of proteins is often laborious and expensive

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