This preview shows page 1. Sign up to view the full content.
Unformatted text preview: een t he plasmid containing the mutant subtilisin gene and
the host subtilisin gene can be avoided.
Estell et al., 1985. Engineering an Enzyme by Site-directed Mutagenesis to Be Resistant to Chemical Oxidation.
T o determine t he s. Biol. Chem. 260: 6518-6521.
Jpecific activity of each mutant enzyme,
enzymes were purified from culture supernatants, and their
concentrations were determined spectrophotometrically. T he
enzymes were assayed against t he substrate, succinyl-L-Ala- ities of codon 222 m utant subtilisins
purified a nd assayed a s described under 100 Enzyme Kinetics!
Case Study - Genetic Engineering of
Table II: Kinetic Constants for selected Codon 222 mutants
Codon 222 Kcat (S-1) Km (M) Kcat / Km (M-1 S-1) Met (wild-type)
5 1.4 x 10-4
4.8 x 10-4
6.3 x 10-4
2.6 x 10-4 36x104
2x104 Enzyme Kinetics!
Getting numbers from Reaction Curves!
Although Vmax can t be measured directly, its value can be obtained
The vo versus [S] curve is a
View Full Document
- Summer '09