Cloning, functional analysis and expression of a novel gene encoding agarase from Vibrio shilonii BY

Abstract:

[Objective] The novel gene encoding agarase in a strain of Vibrio shilonii screened from marine was cloned and expressed in E. coli BL21. [Methods] Isolate an agarase producing strain BY, 16S rRNA gene sequence analysis and construct phylogenetic tree. The degenerate primer was designed according to the known agarase gene sequence, touch-down PCR and chromosome walking techniques were used to obtain agarase full-length gene sequence. After being assembled, the full-length of agarase gene was inserted into expression plasmid pET22a(+), then transformed into E. coli BL21(DE3). Activity of recombinant enzyme was determined utilizing DNS method, study the enzymatic properties of the recombinant enzyme. [Results] A new gene Vibrio sp. BY was cloned from strain BY genomic. The full length of Vibrio sp. BY consists of a 2 232 bp, encoding 744 amino acids, with a putative molecular mass of 85 kD, the amino acids sequence of agarase Vibrio sp. BY shared 86% identity with the agarase of Vibrio sp. EJY3. The activity of agarase was 71.73 U/mL, which indicated that Vibrio sp. BY was an agarase gene. The optimum temperature and pH for the recombinant activity was at 50 °C and 7.0, have a high agarase stability. [Conclusion] A new agarase gene was cloned by chromosome walking techniques, realized recombinant in E. coli BL21(DE3), which lays a good foundation for the research on application of agarase.

Key words:Agarase, Vibrio shilonii, Chromosome walking techniques, Expression