Abstract
Precisely, mutagenesis can introduce mutations into the target gene by using mismatch primers which are partially complementary to the template strand of DNA using polymerase chain reaction (PCR). Oxidoreductase enzymes are generally proteins that involved in oxidation-reduction reactions in biological systems. For this study, primary sequence analyses of oxidoreductase protein from Anoxybacillus sp. SK3-4 was conducted with the aim of generating functional information and theoretically improve catalytic stability of the protein by in-silico mutagenesis. The primary sequence of a novel protein with 386 amino acid residues was analyzed using Expasy-tool for translation of the amino acid sequence into a nucleotide gene sequence. Important catalytic binding sites of the protein were predicted using 3DLigandSite program, Pheres2 and Protein Bioedit servers for generating functional information of the protein. Site-directed mutagenesis (SDM) was used against the novel protein (oxidoreductase), in which two site mutations were created based on rational design. Amino acids; leucine (L) and histidine (H), involved in substrate and metal binding sites in the protein were substituted for isoleucine (I) and arginine (R) i.e. L138I and H280R, to check for significant change in the functional stability of the protein, thereby increasing the efficiency of the enzyme to help speed up the rate of chemical reactions.
Highlights
Mutagenesis of proteins is of great importance for understanding the relation between the protein structure and function
Before sitedirected mutagenesis (SDM) has begun, the only way to obtain a mutation was to isolate naturally occurring mutants with phenotypic screening due to the reason being that the rate at which naturally occurring errors in DNA synthesis was very low, and such screening was usually done after treatment with mutagens [1]
Other important amino acid residues that form the protein binding sites predicted by 3DLigandSite program include; Leu, His, Asp, Glu, Asn, Val, Pro, Ser, Thr, Tyr, etc
Summary
Mutagenesis of proteins is of great importance for understanding the relation between the protein structure and function. The functional and structural roles of amino acid residues in a desired protein can be studied by comparing the mutant protein carrying changes in amino acid residues to the wild-type protein. Before sitedirected mutagenesis (SDM) has begun, the only way to obtain a mutation was to isolate naturally occurring mutants with phenotypic screening due to the reason being that the rate at which naturally occurring errors in DNA synthesis was very low, and such screening was usually done after treatment with mutagens [1]. Mutations can be created precisely at a specific residue with a specific codon change to produce the desired amino acid substitution (SDM), which has allowed alteration of any amino acid residue in a protein without extensive screening [4, 5]. The SDM approach is much more efficient, yielding desired mutations in 50–100% of the molecules produced, than that of phenotypic screening (
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