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Applied and Environmental Microbiology, October 2004, p. 6257-6263, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.6257-6263.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Family Shuffling of Expandase Genes To Enhance Substrate Specificity for Penicillin G

Jyh-Shing Hsu,1,2 Yunn-Bor Yang,2 Chan-Hui Deng,2 Chia-Li Wei,1 Shwu-Huey Liaw,1,3* and Ying-Chieh Tsai1*

Institute of Biochemistry,1 and Faculty of Life Science, National Yang-Ming University, Taipei,3 Synmax Biochemical Co., Ltd., Hsinchu, Taiwan2

Received 25 February 2004/ Accepted 16 June 2004

Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the kcat/Km ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.

* Corresponding author. Mailing address: Institute of Biochemistry, National Yang-Ming University, 155 Li-Nong St., Sec. 2, Pei-Tou, Taipei 11221, Taiwan. Phone: (886) 2-2826-7125. Fax: (886) 2-2826-4843. E-mail for S.-H. Liaw: shliaw{at}ym.edu.tw. E-mail for Y.-C. Tsai: tsaiyc{at}ym.edu.tw.

Applied and Environmental Microbiology, October 2004, p. 6257-6263, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.6257-6263.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Goo, K. S., Chua, C. S., Sim, T.-S. (2008). Relevant Double Mutations in Bioengineered Streptomyces clavuligerus Deacetoxycephalosporin C Synthase Result in Higher Binding Specificities Which Improve Penicillin Bioconversion. Appl. Environ. Microbiol. 74: 1167-1175 [Abstract] [Full Text]  
  • Wei, C.-L., Yang, Y.-B., Deng, C.-H., Liu, W.-C., Hsu, J.-S., Lin, Y.-C., Liaw, S.-H., Tsai, Y.-C. (2005). Directed Evolution of Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Enhancement of Penicillin G Expansion. Appl. Environ. Microbiol. 71: 8873-8880 [Abstract] [Full Text]  


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