| Agar oligosaccharides produced from agar have important application values in foods,medicines,cosmetics and other fields.The method of agarase degradation has the advantages of high efficiency,high substrate repeatability and environmental friendliness.Therefore,it is significant to obtain agarases with good enzymatic properties.The main results are listed as follow:(1)In this study,the agar-degrading bacteria in mangrove sediments were enriched in situ by asparagus officinalis,and the 16 S r RNA gene diversity verified the enrichment of Mgv-B in the sample group.The results demonstrated that high abundance polysaccharide-degrading genus was enriched in the sediment.Subsequently,a large number of agarase genes derived from unculturable microorganisms in mangrove sediments were obtained through high-throughput sequencing.Thirty complete agarase genes with open reading frames were prokaryotically expressed in E.coli and 21 agarases showed enzymatic activity.(2)An agarase gene with the lowest similarity in amino acid sequence with known genes was selected and named as aga M1.In this study,aga M1 gene was further expressed in prokaryotic cells and recombinant Aga M1(r Aga M1)was purified.The molecular weight of the protein was approximately 70 k Da.The properties of recombinant Aga M1(r Aga M1)were studied using prokaryotic expression.The optimum temperature and p H were 50 °C and 7.0,respectively,and r Aga M1 exhibited a high adaptability to wide ranges of temperature and p H.Concretely,r Aga M1 retained 36.53% of residual activity after incubating at 50 °C for 60 h.Meanwhile,after incubation at p H 5.0 or 10.0 for 60 h,62.15% and 55.97% of the activity was remained,respectively.Ag+,Cu2+,sodium dodecyl sulfate(SDS),and Beta-mercaptoethanol(Beta-Me)negatively influenced the agarase activity,while guanidine-HCl and dithiothreitol(DTT)obviously increased the agarase activity.Besides,r Aga M1 showed a Km of 1.82 mg/ml and a Vm of 357.14 U/mg for agarose.The Km was smaller than those of most agarases reported previously.Neoagarotetraose and neoagarohexaose were the end-products from agarose after r Aga M1 degradation.(3)In this study,the effect of gene deletion on the activity of agarase was initially investigated by meathod of gene truncation.The results showed that all tested agarase totally lose their enzymatic activities after N-terminal truncation.In contrast,Aga M1-01 and r Aga M1-02 retained enzymatic activities after C-terminal truncation.However,the enzymatic activities of the C-terminal truncated proteins were decreased,and thermal stability,p H stability and tolerance to metal ions and chelating agents of truncated agarase were not as good as original full-length r Aga M1.The enzymatic property analysis showed that the optimum temperature of truncated proteins was same as original protein(50 °C),while optimum p H was significantly altered after truncation,indicating that the C-terminal sequence(480-960 bp)had important effects on the optimum p H of agarase.The end-products of r Aga M1-01 hydrolysate are neoagarotetraose ? neoagarohexaose ? neoagrooctaose ? neoagrodecaose and neoagarododecaose.The end-products of r Aga M1-02 hydrolysate are neoagrooctaose?neoagrodecaose and neoagarododecaose.The truncated region(C0-C960)may have important effects on agarase activity,stability,ionic tolerance,and substrate affinity.In addition,r Aga M1-01 and r Aga M1-02 produced high-polymer agar oligosaccharides from agarose.However,it is difficult to obtain high-polymer agar oligosaccharides by enzymatic hydrolysis.so r Aga M1-01,r Aga M1-02 and high-polymer agar oligosaccharides have important values in industrial applications. |
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