High-specificity coupling enzyme YND mutant of PAP based on molecular docking, method and application

Abstract

The invention discloses a high-specificity coupling enzyme YND mutant of 3', 5'-adenosine diphosphate based on molecular docking, the gene sequence of the YND mutant is SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, and the amino acid sequence of the YND mutant is SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8. The invention designs and modifies a coupling enzyme with strong PAP specificity by taking yeast 3 ', 5'-adenosine diphosphate enzyme with a known crystal structure as a starting point. The modified enzyme is subjected to inducible expression by an escherichia coli expression system and stored in a storage solution, which can maintain activity for 3 months at-80 DEG C, modified enzyme is used by being diluted with a corresponding buffer system, the use is convenient and the reaction system can be replaced, and the problem of limitation of a system buffer substance on ST activity detection is solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a highly specific PAP (3',5'-adenosine diphosphate) coupling enzyme YND mutant, method and application based on molecular docking. Background technique [0002] Sulfotransferase (ST) is a class responsible for catalyzing the transfer of sulfonyl (SO3) groups from the universal donor molecule 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to carbohydrates, proteins and other Enzymes in various low molecular weight metabolites. Sulfonyltransferases are present in most organisms and in all human tissues and mediate the sulfation of different classes of receptors for a variety of biological functions. Sulfonyltransferases have been found to play a variety of important roles in biological systems, including detoxification, signal transduction, and modulation of receptor binding. During biological sulfation, inorganic sulfate is first activated as a high-energy cofactor, which then transfe...

AI Technical Summary

Problems solved by technology

[0008] 2. At present, the enzyme activity kits for the determination of sulfonyltransferase activity on the market are expensive

[0009] 3. The shortcomings of the reaction system, such as a single buffer, become the resistance to a large number of screening of sulfonyltransferases and corresponding substrates. In addition, the buffer system used in the reagent kit is Tris-Hcl. In some reactions, Tris may become a sulfonyl acceptor. Competitive substrates for sulfonyltransferase substrate specificity screening

[0010] 4. The existing enzyme optimization method has a certain degree of blindness, that is, the mutation is performed according to the approximate binding site, resulting in a large amount of screening experiments and not necessarily accurate

[0011] Through the search, no patent publications related to the patent application of the present invention have been found

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