Sunday, July 26, 2009
P97

Enhancement of the activity and alkaline pH stability of SlxB xylanase from Streptomyces lividans by directed evolution

Qin Wang, School of Life Sciences and Biotechnology, Shanghai Jiao Tong university, Rm. 211, No. 3 Biopharmacology Building, 800 Dongchuan Road, Shanghai, China

The thermal and alkaline pH stability of SlxB xylanase from Streptomyces lividans was improved greatly by random mutagenesis using DNA shuffling. Positive clones with improved thermal stability in an alkaline buffer were screened on a solid agar plate containing RBB-xylan (blue). Three rounds of directed evolution resulted in the best mutant enzyme 3SlxB6 with a significantly improved stability. The recombinant enzyme exhibited significant thermostability at 70 °C for 360 min, while the wild-type lost 50% of its activity after only 3 min. In addition, mutant enzyme 3SlxB6 shows increased stability to treatment with pH 9.0 alkaline buffer. The Km value of 3SlxB6 was estimated to be similar to that of wild-type enzyme; however kcat was slightly decreased, leading to a slightly reduced value of kcat/Km, compared with wild-type enzyme. DNA sequence analysis revealed that eight amino acid residues were changed in 3SlxB6 and substitutions included V3A, T6S, S23A, Q24P, M31L, S33P, G65A, and N93S. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. V3A, S23A, M31L and G65A were found to enhance both thermal and alkaline pH stability of the enzyme and of them, M31L showed the most significant effect. Especially the mutation G24P and S33P resulted in the highest contribution to thermal stability in the mutations found in 3SlxB6. The other two mutations, T6S and N93S, mainly gave rise to increments in alkaline pH stability rather than thermostability.

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