The amino acid substitution modification technique is a modification method in which one amino acid on a peptide chain of a protein molecule is replaced with another amino acid.
At a specific position of a protein molecule, the type and nature of the amino acid are specifically invariant. The amino acid substitution modification refers to changing a specific amino acid on the peptide chain to another amino acid, thereby changing the spatial configuration of the protein, thereby changing the enzyme. Certain biological properties of proteins, known as amino acid substitution modifications.
Enzyme protein amino acid substitution modification
The amino acid substitution modification mainly plays a role in increasing the enzyme activity of the enzyme protein and increasing the stability of the enzyme protein; for example, after the third isoleucine on the molecule of the T4-lysozyme enzyme protein is substituted with cysteine, Cysteine can form a disulfide bond with the cysteine at position 97, which plays an important role in maintaining the spatial configuration of the enzyme protein. The enzyme stability is increased by maintaining the enzyme activity. Times. Amino acid substitution modification methods are mainly carried out by means of genetic engineering.
Studies have shown that covalent binding of cofactors to enzymes can produce new enzyme activities. In recent years, more creative work has been to develop new coenzyme analogs and introduce them into the active site of the enzyme to achieve cofactor remodeling of the enzyme, which is to solve the problem of expensive cofactors in the catalytic reaction system. Important progress. Using the solid phase synthesis technique, the 8th residue phenylalanine in the c-peptide chain of ribonuclease s is replaced with a natural amino acid, pyridinium phosphate (vitamin B6), and the recombined enzyme catalyzes the reaction rate. Increased by 7 times.
Amino acid substitution modification methods
Chemical modification method
The most creative work of chemical modification is to make a catalyst with new catalytic properties by atomic displacement of the active site amino acid of the enzyme protein by a purely organic chemical reaction. The earliest example was the conversion of the serine active site of subtilisin to a cysteine residue, resulting in the loss of its initial aminohydrolysis activity, but still retaining the ability to catalyze the esterification reaction. It can therefore be used for the synthesis of peptides. The selenocysteine is introduced on the subtilisin to convert the aminohydrolase to an acyltransferase. They subsequently discovered that this selenium-based subtilisin has similar activity to glutathione peroxidase due to the presence of redox-active selenium atoms. Since then, other researchers have used this route for the modification of trypsin, which also has redox activity.
- Directed mutation technology
Directed evolution has evolved into a fairly mature protein and peptide modification technology in its short span of more than a decade. Its application has greatly promoted the development of other related fields such as enzyme engineering, metabolic engineering and medicine. With the joint efforts of many researchers, the field of protein directed evolution has made outstanding achievements, and there have been many successful examples of directed evolution applied to industrial biocatalyst transformation.
Directed evolution can be applied not only to the significant improvement of biocatalyst activity, thermal stability and performance of the operating conditions, but also to the vaccine and pharmaceutical fields, to modify vaccines and protein drugs, and to achieve significant evolution in various applications. Directed evolution has provided a powerful tool for biocatalysts from laboratory research to industrial applications. At present, the gratifying results of some enzymes (or proteins), arsenate detoxification pathways, radiation resistance, biosynthetic pathways, enantioselectivity, antibody libraries, and directed site directed evolution have Encouraged scientists in many related fields.
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