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  • Title: Enhancing the thermostability of a novel beta-agarase AgaB through directed evolution.
    Author: Shi C, Lu X, Ma C, Ma Y, Fu X, Yu W.
    Journal: Appl Biochem Biotechnol; 2008 Oct; 151(1):51-9. PubMed ID: 18785021.
    Abstract:
    To increase the thermostability of beta-agarase AgaB by directed evolution, the mutant gene libraries were generated by error-prone polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) shuffling. Mutants with high thermostability were screened by a simple method based on agarase-degrading agar to generate a clear zone on the agar plate. A mutant S2 was obtained through two rounds of error-prone PCR and a single round of DNA shuffling and selection. It has higher thermostability and slightly increased catalytic activity than wild-type AgaB. Melting temperature (T(m)) of S2, as determined by circular dichroism, is 4.6 degrees C higher than that of wild-type AgaB, and the half-life of S2 is 350 min at 40 degrees C, which is 18.4-fold longer than that of the wild-type enzyme. Saturation mutagenesis and hydrophobic cluster analysis indicated that hydrophobic interaction might be the key factor that enhances the enzyme stability.
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