Cloning of complete genes for novel hydrolytic enzymes from Antarctic sea water bacteria by use of an improved genome walking technique

Juan Pablo Acevedo, Fernando Reyes, Loreto P. Parra, Oriana Salazar, Barbara A. Andrews, Juan A. Asenjo

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

The increased demand for enzymes with new properties makes indispensable the development of easy and rapid strategies to obtain complete genes of new enzymes. Here a strategy is described which includes screening by PCR of new subtilases mediated by Consensus-Degenerate Hybrid Oligonucleotide Primers (CODEHOP) and an improved genome walking method to obtain the complete sequence of the identified genes. Existing methods of genome walking have many limitations, which make them inefficient and time consuming. We have developed an improved genome walking method with novel advances to get a simple, rapid and more efficient procedure based on cassette-ligation. Improvements consist basically in the possibility of a genomic DNA digestion with any restriction enzyme, blunting and 3′ adenylation of digested DNA by Taq DNA polymerase to avoid self-circularization, followed by TA ligation of the adenine 3′ overhanging end to the same unphosphorylated oligo-cassette. The efficiency of the genome walking method was demonstrated by finding the unknown ends of all gene fragments tested, previously obtained by CODEHOP-mediated PCR, including three subtilases (P4, P6 and P7), one xylanase and one lipase, from different strains of Antarctic marine bacteria.

Original languageEnglish
Pages (from-to)277-286
Number of pages10
JournalJournal of Biotechnology
Volume133
Issue number3
DOIs
StatePublished - 1 Feb 2008
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by Conicyt (Fondef project # DO4I-1374) and Millennium Institute for Cell Dynamics and Biotechnology (project ICM # P05-001-F).

Keywords

  • Antarctic
  • Bacteria
  • Chromosome walking
  • CODEHOP
  • Genome walking
  • Protease
  • Subtilisin

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