C23O is catechol 2,3-dioxygenase that catalyzes ring cleavage of the catecholic compounds in Gram-positive Planococcus sp.
In one of those mutant form of catechol 2,3-dioxygenase TGC codon for cysteine at position 103 had been changed to a CGC codon for arginine and the AAG codon at position 289 for lysine had been changed to a UAG stop codon .
Amino acid sequence of mutated form and the wild-type catechol 2,3-dioxygenase was deduced using the CLC Free Workbench 4.0.1 software.
In order to determine catechol 2,3-dioxygenase activity, the formation of 2-hydroxymuconic semialdehyde (2-HMS) was measured at 375 nm ([[epsilon].sub.2-HMS at 375 nm] = 36,000/M cm) in a reaction mixture containing 20 [micro]L of catechol (50 mM), 960 [micro]L of phosphate buffer pH 7.5 (50 mM), and 20 [micro]L of crude extract in a total volume of 1mL .
The substrate specificity of mutated and wild-type catechol 2,3-dioxygenase was examined with 3-methylcatechol, 4-methylcatechol, and 4chlorocatechol.
Structural Properties of the Wild-Type and Mutant Catechol 2,3-Dioxygenase. Amino acid sequence in the polypeptide chain defines protein structure and properties.
Effect of pH on the Mutant Catechol 2,3-Dioxygenase. To verify if introduced mutations influenced C23O activity, the effect of pH on the mutated and wild-type enzyme activity was determined.
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