Cloning, Expression And Characterization Of β-agarase Gene YM01-3from Catenovulum Agarivorans

Agarase is a kind of glycoside hydrolase, which can catalyse the hydrolysis ofagarose into agarose-oligosaccharides. It has been recognized that agarose-oligosaccharides have potentially wide applications in many areas, because of theirantagonistic activities to bacteria, virus and tumor cells. Additionally, agarase can alsobeen used as tool enzyme to liberate DNA and other embedded molecules fromagarose, prepare protoplasts and extract bioactive or medicinal compounds from algaeand seaweed, etc. The whole genome of Catenovulum agarivorans YM01^Twassequenced and analyzed, from which one of the β-agarase genes, YM01-3, wascloned and expressed. The characterizations of the recombinant agarase were studied.This study provides a theoretical basis for new methods to prepare neo-agaroligosaccharides with specific size and for the research of novel thermostableagarases.The Blast results showed that15agarase genes could be identified in the wholegenome of YM01^T, including2α-agarase （YM01-14, YM01-15） genes and13β-agarase genes （YM01-1^YM01-13）. It was found for the first time that a bacteriumpossessed both α-and β-agarases. The molecular masses of the putative agaraseswere from25kDa （YM01-6,229aa） to137kDa （YM01-7,1245aa）, and the highestand lowest similarities of the agarases protein sequences with other known agaraseswere100%and32%, respectively. In this study, the crude enzymes were separated bySDS-PAGE after ammonium sulfate precipitation of the culture supernatant, and theresult of in situ detection showed that the protein band with highest agarase activitywas around40kDa. The β-agarases gene YM01-3（46.9kDa） was finally chosen tobe cloned and heterologously expressed according to the result of mass spectrogramanalysis.The putative β-agarase gene YM01-3is an ORF of1263bp. It can encode420amino acids with a predicted molecular mass of46.9kDa and a theoretical isoelectric point of5.66. No putative signal peptide was detected and the catalytic module wasbelong to glycoside hydrolase, family16（GH16）. After the putative β-agarase geneYM01-3was cloned, the recombinant plasmid pET24a（+）/YM01-3was transformedinto E. coli BL21（DE3） to construct the recombinant expression engineering strain E.coli BL21（DE3）/pET24a（+）/YM01-3. The supernatant of the cell lysate showed highagarase activity. Finally, the recombinant agarase was purified from supernatant of thecell lysate with a Ni-NTA agarose column. The purified recombinant agarase wasanalyzed by SDS-PAGE and the result showed a single band.The purified recombinant agarase can maintain about12%agarase activity even ifboiled for5min. The optimal temperature for this agarase was60C and after reactingfor10min at100C, it retained more than40%activity. The activity of recombinantagarase was stable within0-50C. The enzyme exhibited the maximum agaraseactivity at pH6.0. The activity of recombinant agarase was stable at a wide range of4.0-9.0for12h. Na⁺, K⁺, Ca²⁺and SDS had a slightly positive effect on the activity ofthe agarase YM01-3. But some heavy metal ions, such as Cu²⁺, Mn²⁺, Fe3+and Ni²⁺had an apparent negative effect on the agarase activity. In addition, Urea and EDTAdid not significantly affect the activity. The Km and Vmax for YM01-3were3.78mg/ml and1.14×10⁴U/mg, respectively. Finally, the enzyme hydrolyzed agarose togenerate neoagarotetraose and neoagarohexaose as the main products.