Characterization Of Thermostable Xylanase From Deep-sea Thermophile And Gene Functions Of Deep-sea Thermophilic Bacteriophage GSVE1

The resources in the ocean are the most abundant on the earth, and become more and more attractive to people, since the terrestrial resources have been fully developed these years. Thermophiles are adapted to survive at high temperature. They are of great importance in the study of biological systematic evolution. Moreover, they can produce unique biocatalysts that function under extreme conditions comparable to those prevailing in various industrial processes. The enzymes from thermophiles, therefore, are of great interest for industrial applications. Studies on thermophilic bacteriophages suggest that bacteriophages are not only helpful for understanding the life, but also of great potential use in industry to serve as an artifical expression vector.During the isolation of thermophiles, a xylanase-producing thermophilic strain MT-1, which was assigned to Geobacillus sp. MT-1 based on 16S rDNA sequence, was isolated from a deep-sea hydrothermal field in east Pacific. Subsequently, a xylanase gene encoding a 331 amino-acid peptide from this isolate was cloned and expressed in Escherichia coli. The recombinant xylanase exhibited maximum activity at 70°C and had an optimum pH of 7.0. It was active up to 90°C and showed activity over a wide pH ranging from 5.5 to 10.0. The crude xylanase presented similar properties in temperature and pH to those of the recombinant xylanase. The recombinant xylanase was stable in 1 mM of enzyme inhibitors (PMSF, EDTA, 2-ME or DTT) and in 0.1% detergents (Tween 20, Chaps or Triton X-100), whereas, it was strongly inhibited by sodium dodecyl sulfate (SDS) (1 mM). In addition, its catalytic function was stable in the presence of Li⁺, Na⁺ or K⁺. However, it was strongly inhibited by Ni²⁺, Mn²⁺, Co²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺ and Al³⁺ (1 or 0.1 mM).Based on the complete genome sequence and ORF analysis of deep-sea thermophilic bacteriophage GSVE1, the ORF (termed as vp192 gene) that probably encoded a structural protein was identified. The gene encoding a 192 amino-acid peptide was cloned and expressed in E. coli as a fusion protein with glutathione S-transferase (GST). Specific antibody was raised using the purified VP192 protein which was digested by thrombin. Temporal analysis showed that the vp192 gene was transcripted and expressed at various infection stages, and achieved the maximum amount at 4h. After incubation with anti-VP192 IgG followed by labelling with gold-labelled secondary antibody, the gold particles could be clearly found surrounding the tail of GSVE1 virions. This provided visual evidence that the vp192 gene encoded the phage tail protein. Further study was conducted to verify the translational frameshifting. The results revealed that the sequence downstream the vp192 gene encoded two proteins with the same N-terminal end. The ratio of the two proteins seemed to be a fixed number.