Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1.
BACKGROUND
Thermostable enzymes from thermophiles have attracted extensive studies. In this investigation, a nuclease-encoding gene (designated as GBSV1-NSN) was obtained from a thermophilic bacteriophage GBSV1 for the first time.


RESULTS
After recombinant expression in Escherichia coli, the purified GBSV1-NSN exhibited non-specific nuclease activity, being able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Based on sequence analysis, the nuclease shared no homology with any known nucleases, suggesting that it was a novel nuclease. The characterization of the recombinant GBSV1-NSN showed that its optimal temperature and pH were 60 degrees C and 7.5, respectively. The results indicated that the enzymatic activity was inhibited by enzyme inhibitors or detergents, such as ethylene diamine tetraacetic acid, citrate, dithiothreitol, beta-mercaptoethanol, guanidine hydrochloride, urea and SDS. In contrast, the nuclease activity was enhanced by TritonX-100, Tween-20 or chaps to approximately 124.5% - 141.6%. The Km of GBSV1-NSN nuclease was 231, 61 and 92 microM, while its kcat was 1278, 241 and 300 s-1 for the cleavage of dsDNA, ssDNA and RNA, respectively.


CONCLUSIONS
Our study, therefore, presented a novel thermostable non-specific nuclease from thermophilic bacteriophage and its overexpression and purification for scientific research and applications.
Song
Qing
Q
Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, The People's Republic of China. songqing12@hotmail.com
Zhang
Xiaobo
X
D017899:Caudovirales / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004720:Endonucleases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D005092:Exonucleases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D007700:Kinetics; D009696:Nucleic Acids / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000494:pharmacology*
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009696:Nucleic Acids; D011993:Recombinant Fusion Proteins; D054334:Viral Regulatory and Accessory Proteins; D004720:Endonucleases; D005092:Exonucleases