Abstract
Lipoxygenase from Anabaena sp. PCC 7120 (Ana-LOX) was thermally unstable. So, improving the thermostability of the enzyme was quite essential. The target site of Ana-LOX selected for site-directed mutagenesis was based on computer-aided rational design. The thermostability and specific activity of Ana-LOX were improved with replacing valine with alanine at the target site 421 and the site 40. Compared to the wild-type enzyme which has a half-life (T 1/2) of inactivation of 3.8min at 50°C, the T 1/2 of mutant enzymes with V421A and V40A substitution increased to 4.4 and 7.0min, respectively. The double mutant V421A/V40A showed a synergistic effect with a T 1/2 value of 8.3min, resulting in a 1.18-fold improvement compared to the original Ana-LOX. V421A, V40A, and V421A/V40A also obtained 4.83, 41.58, and 80.07% increase in specific activity, respectively. This study provides useful theoretical reference for enzyme molecular modification and computer-aided rational design.
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