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

Acevedo, JP; Reyes, F.; Parra, LP; Salazar, O; Andrews, BA; Asenjo, JA

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. © 2007 Elsevier B.V. All rights reserved.

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Título según WOS: Cloning of complete genes for novel hydrolytic enzymes from Antarctic sea water bacteria by use of an improved genome walking technique
Título según SCOPUS: Cloning of complete genes for novel hydrolytic enzymes from Antarctic sea water bacteria by use of an improved genome walking technique
Título de la Revista: JOURNAL OF BIOTECHNOLOGY
Volumen: 133
Número: 3
Editorial: ELSEVIER SCIENCE BV
Fecha de publicación: 2008
Página de inicio: 277
Página final: 286
Idioma: English
URL: http://linkinghub.elsevier.com/retrieve/pii/S0168165607016732
DOI:

10.1016/j.jbiotec.2007.10.004

Notas: ISI, SCOPUS