Method for designing mutant enzyme, method for preparing mutant enzyme, and mutant enzyme
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A design method and mutation technology, applied in the field of mutant enzymes, can solve a lot of labor and other problems
Active Publication Date: 2012-07-18
AMANO ENZYME INC
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[0005] In order to change the properties of protein deamidases, it is generally necessary to prepare variants of protein deamidases, evaluate their activity, substrate specificity, etc., and identify excellent variants, but these processes require a lot of labor
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[0090] 2. Method for producing mutant enzyme The second aspect of the present invention relates to a method for producing a mutant enzyme. The preparation method of the present invention includes the following steps. (1) a step of preparing a nucleic acid encoding an amino acid sequence constructed by the design method of the present invention; (2) a step of expressing the nucleic acid, and (3) a step of recovering an expression product.
[0091] In step (1), based on the amino acid sequence constructed by the design method of the present invention, necessary mutations are applied to the gene encoding the enzyme to be mutated (that is, the amino acid at a specific position of the protein as the expression product is replaced or deleted), to obtain Nucleic acid (gene) encoding a variant enzyme. Many methods for substituting a nucleotide sequence at a specific position are known in this technical field (for example, refer to Molecular Cloning, 3rd edition, published by Cold Spr...
Embodiment
[0126] 1. X-ray crystal structure analysis of the mature body of protein glutaminase (1) preparation of mature body of protein glutaminase ., Jeens D.J.&Archer, D.B.(2001) Protein-glutaminase from Chryseobacterium proteolyticum, an enzyme that deamidates glutaminyl residues inproteins.Purification, c haracterization and gene cloning.Eur.J.Biochem., 268,1410-1421) method prepared.
[0127] (2) Crystallization
[0128] The crystallization of the mature form of protein glutaminase was carried out according to the following procedure. First, screening was performed by the sitting drop vapor diffusion method using a 24-well plate from Hampton Research. After standing at 20°C for several days, crystals in three wells were observed. Among them, using the best conditions, using the hanging drop (hanging drop) vapor diffusion method, with 5 μl of enzyme solution (40mg / ml) and 5 μl of storage solution (1.0M ammonium phosphate 0.1M sodium citrate pH5.6) A hanging drop of 10 μl was for...
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Abstract
Disclosed is a novel method for improving an enzyme capable of deamidating a protein. A mutant enzyme can be designed by the following steps (1) and (2): (1) specifying at least one amino acid residue among amino acid residues corresponding to the amino acid residues located at position-35, position-38, position-39, position-40, position-41, position-42, position-43, position-45, position-46, position-49, position-79, position-80, position-81, position-82, position-83, position-84, position-103, position-104, position-105, position-106, position-117, position-142, position-143, position-146, position-166 and position-185 in the amino acid sequence depicted in SEQ ID NO:2 which is an amino acid sequence for a protein deamidase (an enzyme to be mutated); and (2) constructing an amino acid sequence having the substitution of the amino acid residue specified in step (1) by another amino acid residue or having the deletion of the amino acid residue specified in step (1) by using the amino acid sequence for the enzyme to be mutated as a base sequence.
Description
technical field [0001] The present invention relates to a design method, a preparation method, a mutant enzyme and the like for deamidating proteins. Background technique [0002] Protein deamidase is an enzyme that hydrolyzes the amide groups of glutamine and asparagine in proteins and converts them into glutamic acid, aspartic acid, and free ammonia. Protein deamidase can be applied to improve the functionality of protein (solubility, emulsification properties, foam properties, gelation properties, etc.), improve the extensibility of wheat gluten dough (do), and reduce the induction of wheat allergens , Improving the extraction efficiency of protein from agricultural products, improving the solubility of calcium in protein solutions, etc., is an enzyme with high industrial utility. [0003] Protein deamidases exist widely in nature. The most well-known examples include protein glutaminase derived from microorganisms (Patent Documents 1 and 2). Among plants, enzymes that...
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