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Directed evolution of Streptomyces lividans xylanase B toward enhanced thermal and alkaline pH stability

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Abstract

The thermal and alkaline pH stability of Streptomyces lividans xylanase B 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 K m value of 3SlxB6 was estimated to be similar to that of wild-type enzyme; however k cat was slightly decreased, leading to a slightly reduced value of k cat/K m, 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. Our results suggest that DNA shuffling is an effective approach for simultaneous improvement of thermal and alkaline pH stability of Streptomyces lividans xylanase B even without structural information.

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Abbreviations

SlxB:

Streptomyces lividans xylanase B

GH:

Glycoside hydrolase

LB:

Luria-Bertani

RBB-xylan:

4-O-methyl-d-glucurono-d-xylan-Remazol Brilliant Blue R

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Acknowledgements

This work was sponsored by National Natural Science Foundation of China (30770837) to Qin Wang. We thank Ms. Ying Li for the assistance with the DNA sequence analysis and mutant library construction.

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Authors and Affiliations

  1. College of Life Science, East China Normal University, 3663 Zhongshang North Rd, 200062, Shanghai, People’s Republic of China

    Tao Xia

  2. School of Life Science and Biotechnology, Key Laboratory of Microbial Metabolism, Ministry of Education, Shanghai Jiaotong University, Rm. 211, No. 3 Biopharmacology Building, 800 Dongchuan Road, 200240, Shanghai, People’s Republic of China

    Qin Wang

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  1. Tao Xia
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Correspondence to Qin Wang.

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Xia, T., Wang, Q. Directed evolution of Streptomyces lividans xylanase B toward enhanced thermal and alkaline pH stability. World J Microbiol Biotechnol 25, 93–100 (2009). https://doi.org/10.1007/s11274-008-9867-3

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  • DOI: https://doi.org/10.1007/s11274-008-9867-3

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