Method for catalyzing sequential enzyme reaction by using fusion protein

Abstract

The present invention discloses a method for catalyzing sequential enzyme reaction (coupling reaction) by using fusion protein. Its steps are: utilizing external gene to splice, constitute and express "enzyme-connecting peptide-enzyme" double catalytic activity fusion protein to obtain pure fusion protein product, and comparing and analyzing catalytic kinetic property of fusion protein. The connecting peptide is used for increasing molecular distance between two enzymes are reducing their steric hindrance, and making the fusion protein maximally retain its original biological activity. Said fusion protein possesses catalytic activity of two enzymes simultaneously, and in the preparation of sequential enzyme sensor and enzyme reactor implements sequential active enzyme controlled co-immobilization so as to raise the reaction rate of sequential enzyme reaction. It is suitable for making high-quality sequential enzyme sensor and immobilized enzyme reactor.

Description

technical field [0001] The invention relates to the fields of molecular enzyme engineering and analytical biotechnology, in particular to a method for catalyzing sequential enzyme reactions (also known as coupled enzyme reactions) with bifunctional fusion proteins. Background technique [0002] Due to the complexity of life metabolism, the synthesis and detection of a considerable proportion of life substances require the sequence and synergy of two or more enzymes. However, compared with single-enzyme reactions, the reaction efficiency of sequential enzyme reactions is generally lower, the reaction process is difficult to control, and the practical progress is slow. This problem is even more prominent when the reaction requires co-immobilization of sequentially acting enzymes, such as the preparation of sequential enzyme sensors and immobilized enzyme reactors. Suppose a double-enzyme reaction contains enzyme 1 (E1) and enzyme 2 (E2). Under the action of enzyme 1, the subs...

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Problems solved by technology

Suppose a double-enzyme reaction contains enzyme 1 (E1) and enzyme 2 (E2). Under the action of enzyme 1, the substrate S to be tested produces an intermediate product D, which is then catalyzed by enzyme 2 to generate the final product P and by-products. Product B (chemical substance or physical signal): S → Step I E 1 D → Srep II E 2 P + B The possible problems in this process mainly include 1) in the process of co-immobilization (Co-immobilization) of enzymes, the affinity between enzyme 1 and enzyme 2 and the cross-linker (Cross-linker) may be quite different, resulting in two The ratio of the enzymes after immobilization is different, so that the sequential catalytic efficiency deviates from the expected value; 2) The distribution and spatial distance of enzyme 1 and enzyme 2 in the immobilization medium are difficult to control and are random, which affects Step I (Step I) and Step II (Step I). II) reaction transfer, making the overall reaction rate uncertain

[0003] For a long time, many researchers have tried to solve the above problems through various improved chemical immobilization methods and controlled enzyme kinetics methods, but the results are not obvious.

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